HK1206421B - Anti-cxcr1 compositions and methods - Google Patents

Description

anti-CXCR 1 compositions and methods

The application is a divisional application of PCT application with the international application date of 11/2009, 11/2009/064041, the international application number of 200980153571.6 entering the national phase and the invention name of anti-CXCR 1 composition and method.

Cross Reference to Related Applications

This application claims priority from U.S. provisional patent application No.61/113,458, filed 11/2008, the content of which is hereby incorporated by reference in its entirety.

Statement regarding federally sponsored research or development

The practice of the invention is supported by the government in accordance with the grant numbers CA66233, CA101860 and 5P30CA46592 awarded by NIH. The government has certain rights in this invention.

Technical Field

The present invention provides methods of treating cancer by administering an inhibitor of the IL8-CXCR1 pathway (e.g., an anti-CXCR 1 antibody or Repertaxin), either alone or in combination with other chemotherapeutic agents, thereby killing non-tumorigenic and tumorigenic cancer cells in a subject. The invention also provides compositions and methods for detecting the presence of and isolating solid tumor stem cells from a patient (e.g., based on the presence of CXCR1 or FBXO 21).

Background

Cancer remains the second cause of mortality in our country, resulting in over 500,000 deaths per year. Despite advances in cancer detection and treatment, cancer mortality remains high. Despite significant advances in understanding the molecular basis of cancer, this knowledge has not yet translated into an effective therapeutic strategy.

In particular, breast cancer is the most common cancer among american women, with about 1 of nine women suffering from breast cancer during their lifetime. Unfortunately, metastatic breast cancer remains an incurable disease. Most women with metastatic breast cancer die of the disease.

Traditional treatment methods (radiation therapy, chemotherapy and hormone therapy), while useful, have been limited by the emergence of resistant cancer cells. Clearly, new methods are generally needed to identify targets for the treatment of metastatic breast cancer and cancer.

Disclosure of Invention

The present invention provides methods of treating cancer by administering an inhibitor of the IL8-CXCR1 pathway (e.g., an anti-CXCR 1 antibody or reperaxin), either alone or in combination with other chemotherapeutic agents, thereby killing non-tumorigenic and tumorigenic cancer cells in a subject. The invention also provides compositions and methods for treating and diagnosing the presence of solid tumor stem cells in a patient (e.g., based on the presence of CXCR1 or FBXO 21).

In some embodiments, the present invention provides a method of treating cancer, comprising: administering to the subject an IL8-CXCR1 pathway antagonist and an additional chemotherapeutic agent. In certain embodiments, the present invention provides methods of reducing or eliminating cancer stem cells and non-tumorigenic cancer cells in a subject, comprising: administering to a subject, under certain conditions, Repertaxin or a derivative thereof, thereby killing at least a portion of the cancer stem cells and at least a portion of the non-tumorigenic cancer cells. In other embodiments, the invention provides methods of reducing or eliminating cancer stem cells and non-tumorigenic cancer cells in a subject, comprising: administering to the subject an IL8-CXCR1 pathway antagonist and an additional chemotherapeutic agent under certain conditions, thereby killing at least a portion of the cancer stem cells and at least a portion of the non-tumorigenic cancer cells. In particular embodiments, the invention provides compositions or kits comprising an IL8-CXCR1 pathway antagonist and an additional chemotherapeutic agent.

In certain embodiments, an IL8-CXCR1 pathway antagonist comprises an agent that specifically blocks IL8 binding to CXCR 1. In some embodiments, the agent binds CXCR1 (specific for CXCR 1), but does not bind CXCR 2. In other embodiments, the agent binds CXCR 1. In particular embodiments, the agent comprises an anti-CXCR 1 antibody or antibody fragment. In other embodiments, the agent comprises Repertaxin or a derivative thereof. In other embodiments, the other chemotherapeutic agent comprises an antimitotic compound. In certain embodiments, the anti-mitotic compound is selected from docetaxel (docetaxel), doxorubicin (doxorubicin), paclitaxel (paclitaxel), fluorouracil, vincristine, vinblastine, nocodazole, colchicine, podophyllotoxin (podophyllotoxin), staphylaxin (steganacin), and combretastatin (combretastatin). In other embodiments, the antimitotic compound is a vinca alkaloid (e.g., vincristine and vinblastine); or benzimidazole carbamates, such as nocodazole; or colchicine or related compounds such as podophyllotoxin, statin or combretastatin; or taxanes (taxanes), such as paclitaxel and docetaxel. In certain embodiments, the other chemotherapeutic agent comprises docetaxel.

In particular embodiments, the subject has a type of cancer and the patient has elevated levels of IL-8 production when treated with a chemotherapeutic agent (e.g., this results in an increase in the number of motile cancer stem cells). In some embodiments, the subject has a type of cancer selected from the following diseases: prostate cancer, ovarian cancer, breast cancer, melanoma, non-small cell lung cancer, and esophageal adenocarcinoma.

In other embodiments, the present invention provides methods of detecting solid tumor stem cells, comprising: a) providing: i) a sample taken from a tumor of a subject, and ii) an antibody or antibody fragment (or other binding molecule) specific for CXCR1 protein or FBXO21 protein (or other proteins of table 1); and b) contacting the tissue sample with an antibody or antibody fragment under conditions to detect the presence or absence of CXCR1+ or FBXO21+ solid tumor stem cells.

In a specific embodiment, the antibody or antibody fragment is conjugated to a signal molecule. In other embodiments, the signaling molecule comprises a fluorescent molecule. In other embodiments, the signal molecule comprises an enzyme capable of catalyzing a chromogenic reaction in the presence of a colorimetric substrate. In certain embodiments, the method further comprises contacting the sample with a secondary antibody or antibody fragment specific for the antibody or antibody fragment. In other embodiments, the secondary antibody or secondary antibody fragment comprises a signal molecule. In particular embodiments, no other proteins or nucleic acids are assayed to determine the presence or absence of the CXCR1 or FBXO21+ solid tumor stem cells. In other embodiments, the tumor is selected from the group consisting of prostate cancer tumor, ovarian cancer tumor, breast cancer tumor, melanoma, non-small cell lung cancer tumor, and esophageal adenocarcinoma tumor.

In some embodiments, the present invention provides a method of enriching a population of solid tumor stem cells, comprising: a) dissociating the solid tumor to produce dissociated cells; b) contacting the dissociated cells with an agent that binds CXCR1 or FBXO21 (or other protein of table 1); and c) selecting cells that bind the agent under certain conditions, thereby producing an enriched population of solid tumor stem cells.

In certain embodiments, no other agent is employed to generate an enriched population of solid tumor stem cells. In some embodiments, the tumor is selected from the group consisting of prostate cancer tumor, ovarian cancer tumor, breast cancer tumor, melanoma, non-small cell lung cancer tumor, and esophageal adenocarcinoma tumor. In other embodiments, the agent is an antibody or antibody fragment (e.g., a Fab fragment). In other embodiments, the agent is conjugated to a fluorescent dye or magnetic particle. In other embodiments, selecting the cells is performed by flow cytometry, fluorescence activated cell sorting, panning, affinity column separation, or magnetic selection.

In particular embodiments, the present invention provides an enriched population of solid tumor stem cells isolated by the methods described herein.

In some embodiments, the present invention provides an isolated population of cancer stem cells that are: a) tumorigenic; and b) CXCR1+ or FBXO21 +. In certain embodiments, the cancer stem cell is a cancer stem cell selected from the group consisting of: prostate cancer stem cells, ovarian cancer stem cells, breast cancer stem cells, skin cancer stem cells, non-small cell lung cancer stem cells, and esophageal adenocarcinoma stem cells. In other embodiments, the cell population comprises at least 60% cancer stem cells and less than 40% non-tumorigenic tumor cells. In other embodiments, the cancer stem cells are enriched at least two-fold (e.g., 2-fold, 3-fold, 4-fold, 5-fold,. 10-fold,. 100-fold,. 1000-fold) compared to unfractionated, non-tumorigenic tumor cells.

In some embodiments, the invention provides a method for obtaining a cell composition comprising cancer stem cells and non-tumorigenic tumor cells from a tumor, at least 60% of which are tumorigenic stem cells and 40% or less of which are non-tumorigenic tumor cells, the method comprising: a) obtaining a dissociated mixture of tumor cells from the tumor; b) separating a mixture of tumor cells into a first fraction comprising at least 60% cancer stem cells and 40% or less non-tumorigenic tumor cells and a second fraction of tumor cells lacking cancer stem cells, wherein the separation is performed by contacting the mixture with an agent directed against CXCR1 or FBXO 21; and c) demonstrating that the first component is tumorigenic by i) continuous injection into a first host animal and that the second component is non-tumorigenic by continuous injection into a second host animal. In certain embodiments, the separation is performed by flow cytometry, Fluorescence Activated Cell Sorting (FACS), panning, affinity chromatography, or magnetic selection. In some embodiments, the separation is performed by Fluorescence Activated Cell Sorter (FACS) analysis.

In a specific embodiment, the present invention provides a method for selecting a treatment for a solid tumor patient, comprising: (a) obtaining a sample from a patient; (b) determining the presence of CXCR1+ or FBXO21+ solid tumor stem cells in the sample; and (c) selecting a treatment for the patient that targets CXCR1+ or FBXO21+ solid tumor stem cells (e.g., selecting the use of an anti-CXCR 1 antibody or antibody fragment). In certain embodiments, the CXCR1+ or FBXO21+ solid tumor stem cell is a cancer stem cell selected from the group consisting of: prostate cancer stem cells, ovarian cancer stem cells, breast cancer stem cells, skin cancer stem cells, non-small cell lung cancer stem cells, and esophageal adenocarcinoma stem cells.

In some embodiments, the present invention provides methods for screening compounds comprising: a) exposing a sample comprising CXCR1+ or FBXO21+ cancer stem cells to a candidate anti-tumor compound, wherein the candidate anti-tumor compound comprises a CXCR1 or FBXO21 antagonist or an IL8-CXCR1 signal transduction pathway antagonist; and b) detecting a change in the cell in response to the compound.

In certain embodiments, the sample comprises a non-adherent mammosphere (mammosphere). In other embodiments, the CXCR1 or FBXO21 antagonist or IL8-CXCR1 signal transduction pathway antagonist comprises an antibody or antibody fragment. In some embodiments, the CXCR1 antagonist is a derivative of retataxin. In other embodiments, the detecting comprises detecting cell death of a tumorigenic breast cell. In other embodiments, the method further comprises identifying the candidate anti-neoplastic agent as being capable of killing both tumorigenic and non-tumorigenic cancer cells.

In some embodiments, the present invention provides methods for determining the ability of a test compound to inhibit tumorigenesis of solid tumor stem cells, comprising: a) obtaining enriched solid tumor stem cells, wherein the solid tumor stem cells: i) at least two-fold enriched compared to unfractionated tumor cells; and ii) expresses CXCR1 or FBXO 21; b) exposing a first, but not a second, set of solid tumor stem cells to a test compound; c) injecting a first set of solid tumor stem cells into a first host animal and a second set of solid tumor stem cells into a second host animal; and d) comparing the tumor, if present, in the first animal with the tumor formed in the second animal to determine whether the test compound inhibits tumor formation. In particular embodiments, the test compound is a CXCR1 or FBXO21 inhibitor, or an IL8-CXCR1 inhibitor pathway inhibitor.

In other embodiments, the invention provides methods for determining the ability of a test compound to inhibit tumorigenesis of solid tumor stem cells, comprising: a) obtaining a sample comprising at least 60% solid tumor stem cells, wherein the solid tumor stem cells express CXCR1 or FBXO 21; b) injecting solid tumor stem cells into the first and second host animals; c) treating the first host animal with the test compound, while treating the second host animal without the test compound; and d) comparing the tumor, if present, in the first animal with the tumor formed in the second animal to determine whether the test compound inhibits tumor formation. In other embodiments, the test compound is a CXCR1 or FBXO21 inhibitor or an IL8-CXCR1 pathway inhibitor.

Drawings

FIG. 1 shows that ALDELUOR-positive cell populations from breast cancer cell lines (MDA-MB-453, SUM159) have cancer stem cell characteristics. A-B, G-H. representative flow cytometry analyses of ALDH enzyme activity in MDA-MB-453(A-B) and SUM159 cells (G-H). The ALDEFLUOR assay was performed as described in example 1 below. The (C, I) ALDELUOR-positive population was able to generate tumors in NOD/SCID mice, which recapitulate the phenotypic heterogeneity of the original tumors. (F, L) tumor growth curves were plotted for different numbers of cells injected (50,000 cells, 5,000 cells and 500 cells for MDA-MB-453 and 100,000 cells, 10,000 cells, and 1,000 cells for SUM159) and for each population (ALDEFLUOR positive, ALLUDEFIOR negative, not isolated). Tumor growth kinetics are associated with the latency and size of tumor formation and the number of ALDELUOR-positive cells (F, L). (D, J) H & E staining of injection sites of ALDEFLUOR-positive cells, revealing the presence of tumor cells (D: MDA-MB-453 ALDEFLUOR-positive cell injection site, and J: SUM59 ALDEFLUOR-positive cell injection site). (E, K) ALDEFLUOR negative cell injection site contains only residual matrigel, apoptotic cells and mouse tissue (E: MDA-MB-453ALDEFLUOR negative cell injection site, and K: SUM59ALDEFLUOR negative cell injection site). Data represent mean ± SD.

Fig. 2 shows the classification of aldeluor-positive and aldeluor-negative populations isolated from breast cell lines based on the "cancer stem cell signature (signature)". Fig. 2A. Hierarchical clustering of 16 samples based on 413 gene expression signatures. Each row of the data matrix represents a gene, while each column represents a sample. Note that for 15 of the 16 samples, the adelutor positive (underlined name) and negative (not underlined name) samples were separated by 413 genes. Some genes contained in the signature are referred to by their HUGO abbreviations, as used in "Entrez Gene" (down-regulated genes in the aldeluor positive population are labeled in green, while up-regulated genes in the aldeluor positive population are labeled in red). 2B-C. to confirm gene expression results, expression of 5 discriminator genes (CXCR1/IL8RA, XO FB 21, NFYA, NOTCH2, and RAD51L1) that were overexpressed in the ALDELUOR positive population was measured by quantitative RT-PCR in a panel of 5 breast cancer cell lines sorted for ALDELUOR phenotype. The quantitative RT-PCR expression levels of CXCR1 and FBXO21 are presented in this figure. The results obtained using the DNA microarray were confirmed by the gene expression levels measured by quantitative RT-PCR, where CXCR1 and FBXO21mRNA levels were elevated in the aldeluor positive population compared to the aldeluor negative population (p < 0.05).

Figure 3 shows the role of IL8/CXCR1 axis in modulating breast cancer stem cells. A. Cells expressing CXCR1 are contained in an ALDEFLUOR positive population. ALDELUOR positive and negative populations from four different breast cell lines (HCC1954, SUM159, MDA-MB-453, BrCa-MZ-01) were isolated by FACS, fixed, and analyzed for CXCR1 protein expression by immunostaining and FACS analysis. ALDELUOR-positive cells are highly enriched for CXCR 1-positive cells compared to ALDELUOR-negative populations. Effect of IL8 treatment on tumor sphere formation in three different cell lines (HCC1954, SUM159, MDA-MB-453). IL8 treatment increased primary and secondary tumor sphere formation in a dose-dependent manner. Effect of il8 treatment on aldeluor positive populations of four different cell lines cultured in adherent conditions. In each of the four cell lines analyzed, IL8 increased the aldeluor positive population in a dose-dependent manner (p < 0.05/' p < 0.01, statistically significantly different from the control).

Figure 4 shows that ldefaluor positive cells exhibit increased metastatic potential. The IL8/CXCR1 axis is involved in cancer stem cell invasion. The role of the IL8/CXCR1 axis in invasion was assessed by matrigel invasion experiments using serum or IL8 as an attractant for three different cell lines (HCC1954, MDA-MB-453, SUM 159). ALDELUOR-positive cells are 6 to 20 times more aggressive (p < 0.01) than ALDELUOR-negative cells. When IL8(100ng/ml) was used as a decoy, a significant increase in aldeluor-positive cell invasion (p < 0.05) by matrigel was observed compared to serum as a decoy. In comparison, IL8 had no effect on the invasive potential of the aldeluor negative population. B-M. The ALDEFLUOR positive population showed increased metastatic potential. Quantification of normalized photon flux measured at weekly intervals after inoculation of 100,000 luciferase-infected cells from each group (ALDEFLUOR positive, ALDEFLUOR negative, unseparated). E-J. Transfer was detected using bioluminescent imaging software (E, G, I: mice face down; F, H, J: mice face up). Mice inoculated with ALDEFLUOR positive cells form several metastases located at different sites (bone, muscle, lung, soft tissue) and exhibit higher photon flux emission than mice inoculated with unseparated cells (no more than 1 metastasis per mouse). In contrast, mice inoculated with ALDEFLUOR-negative cells formed only sporadic small metastases, which were restricted to the lymph nodes. Histological confirmation of metastases in bone (K), soft tissue (L) and muscle (M) resulting from ALDEFLUOR positive cell injection by H & E staining.

Figure 5 shows the effect of CXCR1 inhibition on tumor cell viability (figure 5A) and on cancer stem cell viability (figure 5B).

Fig. 6 shows that reportaxin treatment induced bystander effects mediated by FAS/FAS ligand signaling, and clearly shows that cell growth inhibition induced by reportaxin treatment was rescued by the addition of FAS antagonist, and that cells treated with FAS agonist showed similar cell growth inhibition as cells treated with reportaxin.

Fig. 7 shows FAK, AKT and FOXOA3 activation in the absence of Repertaxin treatment (7A) and in the presence of Repertaxin (7B).

FIG. 8 shows the effect of Repetaxin, docetaxel, or a combination thereof on one breast cancer cell line (8A, SUM159) and three human breast cancer xenografts generated from three patients (8B, MC 1; 8C, UM 2; and 8D, UM 3).

Fig. 9 shows the effect of reportaxin, docetaxel, or combined treatment on cancer stem cell populations as assessed by aldeluor assays performed on various cell lines including SUM159(9A), MC1(9B), UM2(9C), UM3 (9D).

Fig. 10 shows the effect of reportaxin, docetaxel, or a combination on serial dilution of primary tumors (10a. sum159, 10b. mc1, 10c. um2, 10d. um3) implanted in mammary fat pads of secondary NOD-SCID mice.

Fig. 11 shows that reportaxin treatment reduced the metastatic potential of SUM159 cell line. Fig. 11A shows quantification of normalized photon flux measured at weekly intervals after seeding with intra-cardiac administered SUM159 cells. Bioluminescence imaging was used to monitor metastasis formation (11B: mice treated with saline solution; 11C: mice treated with Repetaxin).

Figure 12 shows an indication of overlap between the aldeluor positive subpopulation of SUM159 cells and either the CXCR1 positive subpopulation (upper) or the CXCR2 positive subpopulation (lower). SUM159 cells were cultured in adherent conditions and treated with Repetaxin (100nM) or two blocking antibodies specific for CXCR1(10 μ g/ml) or CXCR2(10 μ g/ml). After 3 days, the effect on cancer stem cell populations was analyzed using the ALDEFLUOR assay (B), and cell viability was assessed 5 days after treatment using the MTT assay (C). Significant reductions in ALDEFLUOR positive populations and cell viability were observed following treatment with reportaxin or anti-CXCR 1 antibodies. In contrast, no significant effect was observed in the case of the anti-CXCR 2 antibody. D. 4 days after treatment, apoptotic cell numbers were assessed using the TUNEL assay. 36% apoptotic cells (green staining green) were detected in Repertaxin-treated cells compared to controls where predominantly viable cells (blue staining) were present. To determine whether cell death is mediated via a bystander effect. CXCR1 positive and CXCR1 negative populations were flow sorted and each population was treated with multiple concentrations of Repertaxin (D). A decrease in cell viability was detected in CXCR1 positive and unsorted populations, whereas no effect was observed in CXCR1 negative population (E). Sorted CXCR 1-positive, CXCR 1-negative, or unsorted populations were treated with dialyzed conditioned medium (dCM) from CXCR 1-positive cells treated with reportaxin for three days. Serial dilutions of the dialyzed conditioned medium were used (controls, dCM1/4, dCM1/2, dCM3/4, dCM). After 2 days of treatment, cell viability was assessed using the MTT assay. A substantial decrease in cell viability was observed in both CXCR1 negative and non-segregating populations, while no effect was observed in the CXCR1 positive population (F).

Figure 13 shows tumorigenicity of aldeluor-positive/CXCR 1-positive and aldeluor-positive/CXCR 1-negative cell populations from SUM159 cell line. A. Tumor growth curves were plotted for different numbers of cells injected (50,000 cells, 5,000 cells, 1,000 cells and 500 cells) and for each population (aldeluor positive/CXCR 1 positive, aldeluor positive/CXCR 1 negative). Both cell populations give rise to tumors. Tumor growth kinetics are related to the latency and size of tumor formation and the number of cells injected. Tumors generated from the aldeluor positive/CXCR 1 positive population rebuild the phenotypic heterogeneity of the original tumor after serial passages, while the aldeluor positive/CXCR 1 negative population caused tumors containing only aldeluor positive/CXCR 1 negative cells. We transplanted these two cell populations for three passages.

Figure 14 shows the effect of CXCR1 blockade on tumor sphere formation. SUM159 and HCC1954 cells were cultured in adherent conditions and treated with Resertaxin (100nM), anti-CXCR 1 blocking antibody (10 μ g/ml) or anti-CXCR 2 blocking antibody (10 μ g/ml) for three days. After 3 days of treatment, the cells were separated and cultured in suspension. The number of tumor balls formed after 5 days of culture was evaluated. Similar results were observed for both cell lines, with a significant reduction in primary and secondary tumor sphere formation in Repertaxin and anti-CXCR 1 treated conditions compared to controls. In contrast, anti-CXCR 2 blocking antibodies had no effect on tumor sphere formation.

FIG. 15 shows the effect of Reertaxin treatment on cell viability of SUM159, HCC1954 and MDA-MB-453 cell lines. Three different cell lines (SUM159, HCC1954, MDA-MB-453) were cultured in adherent conditions and treated with Resertaxin (100 nM). Cell viability was assessed using the MTT assay after 1, 3 and 5 days of treatment. A decrease in cell viability was observed for SUM159 and HCC1954 cell lines 3 days after treatment. However, Resertaxin did not affect the viability of MDA-MB453 cells.

Figure 16 shows that CXCR1 blocks in vitro effects on the aldeluor positive population. A-B. HCC1954(A) and MDA-MB-453(B) cells were cultured in adherent conditions and treated with Repetaxin (100nM) or two blocking antibodies specific for CXCR1(10 μ g/ml) or CXCR2(10 μ g/ml). After 3 days, the effect on cancer stem cell populations was analyzed using the ALDEFLUOR assay. For HCC1954, significant reductions in aldeluor positive populations and cell viability were observed following treatment with Repertaxin or anti-CXCR 1 antibody. In contrast, no significant effect was observed in the case of the anti-CXCR 2 antibody (a). No effect on the ALDEFLUROR positive population was observed for MDA-MB-453 (B).

FIG. 17 shows that Reertaxin treatment induces bystander effects mediated by FAS/FAS ligand signaling. A. To determine whether the bystander killing effect induced by the reportaxin treatment was mediated by FAS ligand, the level of soluble FAS ligand in the culture medium was measured using an ELISA assay. After 4 days of treatment, a greater than 4-fold increase in soluble FAS ligand was detected in the culture medium of cells treated with Repertaxin compared to untreated controls. B. The level of FAS ligand mRNA was measured by RT-PCR and confirmed to increase FAS ligand production after treatment with reportaxin. Similar results were observed after 4 days of treatment with FAS agonist that activated FAS signaling, where FAS ligand mRNA was increased 5-fold compared to control. C. SUM159 cells were cultured in adherent conditions and treated with Reertaxin alone or in combination with an anti-FAS ligand. The addition of anti-FAS ligand partially rescues the cell growth inhibition induced by Repertaxin treatment. Cells treated with FAS agonist showed similar inhibition of cell growth as cells treated with repteraxin alone. D-e. analysis of the effect of treatment with Repertaxin alone or in combination with anti-FAS ligand and FAS agonist treatment on CXCR1 positive and ALDEFLUOR positive populations. anti-FAS ligands did not rescue the large reduction in CXCR1 positive and alfeluor positive populations induced by Repertaxin treatment, whereas treatment with FAS agonist produced 10-fold and 3-fold increases in the percentage of CXCR1 positive and alfeluor positive populations, respectively.

Figure 18 shows the effect of FAS agonists on CXCR1 positive and CXCR1 negative cells. CXCR1 positive and CXCR1 negative populations were flow sorted and each population was treated with multiple concentrations of FAS agonist. Decreased cell viability was detected for CXCR1 negative and unsorted populations, while no effect was observed in the CXCR1 positive population.

Figure 19 shows an analysis of CXCR1 protein expression in normal breast stem/progenitor cell populations and the effect of IL-8 treatment on mammosphere formation. A. Alfelulor positive and negative populations from normal mammary epithelial cells isolated from mammoplasty are isolated by FACS, fixed, and analyzed for CXCR1 protein expression by immunostaining and FACS analysis. ALDELUOR-positive cells are highly enriched for CXCR 1-positive cells compared to ALDELUOR-negative populations. Effect of il8 treatment on mammosphere formation. IL8 treatment increased the formation of primary mammaglae (B) and secondary mammaglae (C) in a dose-dependent manner.

Fig. 20 shows the effect of reportaxin treatment on normal mammary epithelial cells. A. Normal mammary epithelial cells isolated from mammoplasty were cultured in adherent conditions and treated with Resertaxin (100nM or 500nM) or FAS agonist (500 ng/ml). After 5 days of treatment, cell viability was assessed using the MTT assay. Even when high concentrations of Repertaxin (500nM) are used, the treatment with Repertaxin or FAS agonist has no effect on the viability of normal mammary epithelial cells cultured in adherent conditions. B. The level of soluble FAS ligand was assessed in the culture medium of normal mammary epithelial cells treated with reportaxin by an Elisa assay. After 4 days of treatment, an increase in soluble FAS ligand was detected in the culture medium from the treated cells. C. FAS/CD95 expression in normal breast epithelial cells was analyzed by FACS analysis. FAS/CD95 expression was not detected in normal mammary epithelial cells cultured in adherent conditions. Effect of repertaxin treatment on mammosphere formation. Normal mammary epithelial cells were cultured in adherent conditions and treated with reportaxin (100nM) over 4, 8, 11 and 15 days. After the Repertaxin treatment, the cells are separated and cultured in suspension. A significant reduction in mammosphere initiating cells was observed in the Repertaxin treated condition.

Fig. 21 shows the effect of Repertaxin treatment on FAK, AKT and FOXO3a activation. To assess the effect of Repertaxin treatment on CXCR1 downstream signaling, two different viral constructs were utilized, one knocking out PTEN expression via PTEN-siRNA and the other resulting in FAK overexpression (Ad-FAK). A. SUM159 control, SUM159PTEN-siRNA and SUM159Ad-FAK cells were cultured for 2 days in the absence or presence of 100nM Repertaxin in adherent conditions and activation of the FAK/AKT pathway was assessed by Western blotting. Reportaxin treatment resulted in a decrease in FAK Tyr397 and AKTSer473 phosphorylation, while PTEN deletion and FAK overexpression blocked the effect of reportaxin treatment on FAK and AKT activity. B. Using immunofluorescent staining of CXCR1 positive cells, we confirmed that Repertaxin treatment resulted in the disappearance of phospho-FAK (membrane staining red) and phospho-AKT expression (cytosolic staining red). Immunofluorescent staining with anti-FOXO 3A revealed cytoplasmic location of FOXO3a (in red) in untreated cells, while Repertaxin treatment induced the relocation of FOXO3A to the nucleus. In comparison, cells with PTEN deletion or FAK overexpression exhibited high levels of phospho-FAK, phospho-AKT, and cytoplasmic FOXO3A expression in both Repertaxin-treated and untreated cells. In all samples, nuclei were counterstained with DAPI (blue). C-d. the effect of reportaxin on SUM159PTEN-siRNA and SUM159Ad-FAK cell viability and on cancer stem cell populations was evaluated using MTT and ALDEFLUOR assays, respectively. After 3 days of treatment, cells with PTEN deletion or FAK overexpression developed resistance to reportaxin (C). Reertaxin treatment did not alter the proportion of ALDELUOR positive SUM159PTEN knockdown cells (D).

FIG. 22 shows the effect of Reertaxin treatment on FAK/AKT activation in HCC1954 and MDA-MB-453 cell lines. To assess the effect of Repertaxin treatment on signal transduction downstream of CXCR1, we utilized lentiviral constructs that knock out PTEN expression via PTEN-siRNA. A. HCC1954 control and HCC1954PTEN-siRNA cells were cultured in adherent conditions for 2 days in the absence or presence of 100nM reportaxin and activation of the FAK/AKT pathway was assessed by Western blot. Repertaxin treatment resulted in a decrease in FAK Tyr397 and AKT Ser473 phosphorylation, while PTEN deletion blocked the effect of Repertaxin treatment on FAK and AKT activity. Repettaxin treatment did not have any effect on cell viability of the MDA-MB-453 cell line containing a PTEN mutation. Using Western blot analysis, we confirmed that Repetaxin treatment did not interfere with the FAK/AKT pathway.

Figure 23 shows the effect of reportaxin on HCC1954PTEN-siRNA cell viability, evaluated using the MTT assay. After 3 days of treatment, cells with PTEN deletions developed resistance to reportaxin.

FIG. 24 shows FAS ligand and IL-8mRNA expression after treatment with docetaxel or Repetaxin as measured by quantitative RT-PCR. SUM159 cells cultured in adherent conditions were treated with Repetaxin (100nM), FAS agonist (500ng/ml), or docetaxel (10 nM). After 3 days of treatment, cells were harvested and RNA was extracted. Docetaxel induced FAS ligand (A) and IL-8(B) mRNA in SUM159 cells. A4-fold increase in IL-8mRNA levels (B) was detected following FAS agonist or docetaxel treatment.

Figure 25 shows the assessment of PTEN/FAK/AKT activation in three different breast cancer xenografts. Western blot analysis revealed that these two xenografts exhibited expression of PTEN and activation of the FAK/AKT pathway, as shown by FAK Tyr397 and AKT Ser473 phosphorylation.

Fig. 26 shows the in vivo effect of reportaxin treatment on a population of breast cancer stem cells. To evaluate the in vivo effect of Repertaxin treatment on tumor growth and cancer stem cell populations, a breast cancer cell line (SUM159) and three human breast cancer xenografts (MC1, UM2, UM3) generated from three patients were utilized. A. For each sample, 50,000 cells were injected into the humanized mammary fat pad of NOD/SCID mice and tumor size was monitored. At a tumor of about 4mm, two s.c. injections of repteraxin (15mg/Kg) per day were started for 28 days or one i.p. injection of docetaxel (10mg/Kg) or combination (repteraxin/docetaxel) per week. The figure shows the tumor size before and during each designated treatment procedure (arrows, start treatment). Similar results were observed for each sample, where tumor size was statistically significantly reduced in the group treated with docetaxel alone or the Repertaxin/docetaxel combination compared to the control, while no difference was observed between the growth of the control tumor and the tumor treated with Repertaxin alone. Reportaxin, docetaxel, or combination treatment assessment of cancer stem cell populations as assessed by ALDEFLUOR assay (B) and re-transplantation into secondary mice (C). Docetaxel treated tumor xenografts showed similar or increased percentages of ALDEFLUOR-positive cells compared to controls, while Repertaxin treated alone or in combination with docetaxel treatment produced statistically significant reductions of ALDEFLUOR-positive cells, with cancer stem cells reduced by 65% to 85% (p < 0.01) (B) compared to controls. Serial dilutions of cells obtained from primary tumors, untreated (control) and treated mice were transplanted into mammary fat pads of secondary NOD/SCID mice that received no further treatment. Control and docetaxel treated primary tumors formed secondary tumors at all dilutions, while only a higher number of cells obtained from primary tumors treated with reportaxin or in combination with docetaxel were able to form tumors. Furthermore, tumor growth was significantly delayed and the resulting tumors were significantly smaller in size than control or docetaxel-treated tumors (C). D. Xenografts from each group were collected and immunohistochemical staining was performed to detect the expression of phospho-FAK, phospho-AKT, FOXO3A and ALDH 1. Membrane phospho-FAK expression and cytosolic phospho-AKT expression (arrows) were detected in control and docetaxel treated tumors, whereas no expression was detected in tumors treated with Repertaxin alone or in combination with docetaxel. Nuclear FOXO3A expression (brown) was detected in cells treated with docetaxel or Repertaxin alone or in combination. A decrease in ALDH1 expression was detected in tumors treated with reperaxin alone or in combination (arrow) compared to control and docetaxel treated tumors.

Fig. 27 shows the in vivo effect of reportaxin treatment on a population of breast cancer stem cells. To evaluate the in vivo effect of Repertaxin treatment on tumor growth and cancer stem cell populations, a breast cancer cell line (SUM159, a) and three human breast cancer xenografts generated from different patients were utilized. For each sample, 50,000 cells were injected into the humanized mammary fat pad of NOD/SCID mice and tumor size was monitored. At a tumor of about 4mm, two s.c. injections of repteraxin (15mg/Kg) per day were started for 28 days or one i.p. injection of docetaxel (10mg/Kg) or combination (repteraxin/docetaxel) per week. The figure shows the tumor size before and during each designated treatment procedure (arrows, start treatment). Similar results were observed for each sample, where tumor size was statistically significantly reduced in the group treated with docetaxel alone or the Repertaxin/docetaxel combination compared to the control, while no difference was observed between the growth of the control tumor and the tumor treated with Repertaxin alone. The effect of Repertaxin, docetaxel or combination treatment on cancer stem cell populations was assessed by ALDEFLUOR assay and re-transplantation into secondary mice. Docetaxel treated tumor xenografts showed similar or increased percentages of aldeluor-positive cells compared to controls, whereas treatment with repertixin alone or in combination with docetaxel resulted in a statistically significant reduction of aldeluor-positive cells, with cancer stem cells reduced by 65% to 85% (p < 0.01) compared to controls. Serial dilutions of cells obtained from primary tumors, untreated (control) and treated mice were transplanted into mammary fat pads of secondary NOD/SCID mice that received no further treatment. Control and docetaxel treated primary tumors formed secondary tumors at all dilutions, while only a higher number of cells obtained from primary tumors treated with reportaxin or in combination with docetaxel were able to form tumors. Tumor growth was significantly delayed and the resulting tumors were significantly smaller in size than control or docetaxel-treated tumors.

FIG. 28 shows the effect of Repetaxin treatment on breast cancer stem cell populations as assessed by the CD44+/CD 24-phenotype. A-b. the effect of Repertaxin, docetaxel or combination treatment on cancer stem cell populations was assessed by the presence of CD44+/CD 24-cells. In the residual tumors treated with docetaxel alone, we consistently observed an unchanged or increased percentage of CD44+/CD 24-cells, whereas treatment with Repertaxin alone or in combination with docetaxel resulted in a reduction of the CD44+/CD 24-cell population. A. A flowchart analysis of UM3 xenografts is presented. B. Similar results were observed for MC1, UM2, and UM 3. Almost all SUM159 cells were CD44+/CD 24-under all treatment conditions.

Fig. 29 shows that reportaxin treatment reduced the formation of systemic metastases. To evaluate the effect of Reertaxin treatment on metastasis formation, HCC1954(A), SUM159(B), MDA-MB-453(C) breast cancer cell line was infected with luciferase-expressing lentiviruses, and 250,000 luciferase-infected cells were inoculated into NOD/SCID mice via intracardiac injection. Mice were treated 12 hours after intracardiac injection by s.c. injection of saline solution or s.c. injection of Repertaxin (15mg/kg) twice a day during 28 days. Transfer formation was monitored using bioluminescent imaging. Quantification of the normalized photon flux measured at weekly intervals after inoculation revealed a statistically significant reduction in metastasis formation for the reportaxin treatment compared to the saline control for mice inoculated with HCC1954 or SUM159 cells (a-B). In contrast, for mice injected with MDA-MB-453 cells, Reertaxin treatment had no effect on metastasis formation. (C) Histology confirmed by H & E staining for metastasis in bone and soft tissue of mice not treated with reportaxin (D).

Figure 30 shows IL-8/CXCR1 signaling in cancer stem cells treated with chemotherapy alone or in combination with reportaxin. A. Representing potential IL-8/CXCR1 cell signaling in cancer stem cells. Activation of CXCR1 following IL-8 binding induces phosphorylation of Focal Adhesion Kinase (FAK). Active FAK phosphorylates AKT and activates WNT pathway that regulates stem cell self-renewal and FOXO3A that regulates cell survival. FAK activation protects cancer stem cells from FAS ligand/FAS-mediated bystander effects by inhibiting FADD, a downstream effector of FAST signaling. In the presence of chemotherapy, only a large number of tumor cells are sensitive to treatment and release high levels of IL-8 and FAS ligand protein during apoptosis. Breast cancer stem cells are stimulated via IL-8 mediated bystander effects and are resistant to bystander killing effects mediated via FAST ligands. Repetlaxin treatment blocks IL-8/CXCR1 signaling and inhibits breast cancer stem cell self-renewal and survival. When combined with chemotherapy, cancer stem cells are sensitized to bystander killing effects mediated by FAS ligand.

Definition of

To facilitate an understanding of the present invention, a number of terms and phrases are defined below:

as used herein, the term "anti-cancer agent," "conventional anti-cancer agent," or "cancer treatment drug" refers to any therapeutic agent (e.g., chemotherapeutic compounds and/or molecular therapeutic compounds), radiation therapy, or surgical intervention used in the treatment of cancer (e.g., in a mammal).

As used herein, the terms "drug" and "chemotherapeutic agent" refer to pharmacologically active molecules useful for the diagnosis, treatment or prevention of a disease or pathological condition in a physiological system (e.g., a subject, or in vivo, in vitro or ex vivo cells, tissues and organs). Drugs act by altering the physiology of a living organism, tissue, cell, or in vitro system that has received administration of the drug. The terms "drug" and "chemotherapeutic agent" are intended to encompass anti-hyperproliferative and anti-neoplastic compounds, as well as other biotherapeutic compounds.

An "effective amount" refers to an amount sufficient to achieve a beneficial or desired result. An effective amount may be administered in one or more administrations.

As used herein, the term "administering" refers to the act of administering a drug, prodrug, antibody or other agent, or therapeutic treatment to a physiological system (e.g., a subject or in vivo, in vitro, or ex vivo cells, tissues, and organs). Exemplary routes of administration to the human body can be via the eye (ocular), mouth (oral), skin (transdermal), nose (nasal), lung (inhaled), oral mucosa (buccal), ear, by injection (e.g., intravenous, subcutaneous, intratumoral, intraperitoneal, etc.), and the like.

"Co-administration" refers to the administration of more than one chemical agent or therapeutic treatment (e.g., radiation therapy) to a physiological system (e.g., a subject or in vivo, in vitro, or ex vivo cells, tissues, and organs). "co-administration" of various chemical agents (e.g., IL8-CXCR1 signal transduction pathway antagonist and other chemotherapeutic agents) can be concurrent, or in any temporal order or physical combination.

As used herein, the term "regression" refers to a return of a diseased subject, cell, tissue or organ to a non-pathological, or less pathological, state as compared to the underlying non-pathogenic exemplary subject, cell, tissue or organ. For example, regression of a tumor includes a decrease in tumor mass and complete disappearance of one or more tumors.

As used herein, the term "in vitro" refers to an artificial environment and processes or reactions occurring within an artificial environment. In vitro environments may include, but are not limited to, test tubes and cell cultures. The term "in vivo" refers to the natural environment (e.g., an animal or cell) and processes or reactions occurring within the natural environment.

As used herein, the term "cell culture" refers to any in vitro culture of cells. This term includes continuous cell lines (e.g., with an immortal phenotype), primary cell cultures, finite cell lines (e.g., untransformed cells), and any other cell population maintained in vitro, including oocytes and embryos.

As used herein, the term "subject" or "patient" refers to an organism to be treated by the methods of the present invention. Such organisms include, but are not limited to, humans and veterinary animals (dogs, cats, horses, pigs, cattle, sheep, goats, etc.). In the context of the present invention, the term "subject" or "patient" generally refers to an individual who is about to receive or has received treatment.

As used herein, the term "diagnosis" refers to the identification of a disease based on signs and symptoms of the disease or genetic analysis, pathological analysis, histological analysis, and the like.

As used herein, the term "antisense" is used in reference to a nucleic acid sequence (e.g., RNA, phosphorothioate DNA) that is complementary to a particular RNA sequence (e.g., mRNA). This definition includes natural or synthetic antisense RNA molecules, including molecules that modulate gene expression, such as small interfering RNAs or microRNAs. One class of antisense sequences that can be employed in the present invention is of the type specific for CXCR1 mRNA.

The term "test compound" or "candidate compound" refers to any chemical entity, agent, drug, etc., that can be used to treat or prevent a disease, disorder, condition, or bodily dysfunction, or otherwise alter the physiological or cellular state of a sample. The test compounds comprise both known and potential therapeutic compounds. Test compounds can be determined to be therapeutic by using the screening methods of the present invention. "known therapeutic compound" refers to a therapeutic compound that has been shown (e.g., via animal testing or prior experience with administration to humans) to be effective in such treatment or prevention. In a preferred embodiment, the "test compound" is an anti-cancer agent. In a particularly preferred embodiment, the "test compound" is an anti-cancer agent that induces apoptosis.

As used herein, the term "antigen binding protein" refers to a protein that binds a particular antigen. "antigen binding proteins" include, but are not limited to, immunoglobulins, including polyclonal, monoclonal, chimeric, single chain and humanized antibodies, Fab fragments, F (ab') 2 fragments, and Fab expression libraries. Polyclonal antibodies are prepared using a variety of methods known in the art. To produce antibodies, a variety of host animals including, but not limited to, rabbits, mice, rats, sheep, goats, etc., can be immunized by injection with a peptide corresponding to the desired epitope. In a preferred embodiment, the peptide is conjugated to an immunogenic carrier, such as diphtheria toxoid, Bovine Serum Albumin (BSA), or Keyhole Limpet Hemocyanin (KLH). Depending on the host species, a variety of adjuvants are used to enhance the immune response, including but not limited to Freund (complete and incomplete), mineral gels (such as aluminum hydroxide), surface active substances (such as lysolecithin), Pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum (Corynebacterium parvum).

For the preparation of monoclonal Antibodies, any technique that provides for the production of antibody molecules by continuous cell lines in culture may be used (see, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). These include, but are not limited to, the compounds originally made byAnd the hybridoma technology developed by Milstein: (And Milstein, Nature, 256: 495-497(1975)), and the trioma (trioma) technique, the human B-cell hybridoma technique (see, e.g., Kozbor et al, immun 0. today, 4: 72(1983)) and the EBV hybridoma technique for the production of human Monoclonal Antibodies (Cole et al, Monoclonal Antibodies and cancer Therapy, Alan R.Liss, Inc., p.77-p.96 (1985)).

In accordance with the present invention, the described techniques for making single chain antibodies (U.S.4,946,778; incorporated herein by reference) can be adapted to make specific single chain antibodies as desired. Other embodiments of the invention utilize techniques known in the art for constructing Fab expression libraries (Huse et al, Science, 246: 1275-.

Antibody fragments containing the idiotype (antigen-binding region) of an antibody molecule can be generated by known techniques. For example, such fragments include, but are not limited to: f (ab') 2 fragments which can be generated by pepsin digestion of an antibody molecule; fab 'fragments which can be generated by reducing the disulfide bond of the F (ab') 2 fragment; and Fab fragments that can be generated by treating the antibody molecule with papain and a reducing agent.

The gene encoding the antigen binding protein may be isolated by methods known in the art. In antibody preparation, screening for the desired antibody can be accomplished by techniques known in the art (e.g., radioimmunoassays, ELISAs (enzyme linked immunosorbent assays), "sandwich" immunoassays, immunoradiometric assays, gel diffusion precipitin reactions, immunodiffusion assays, in situ immunoassays (e.g., using colloidal gold, enzyme, or radioisotope labels), Western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays, etc.), complement fixation assays, immunofluorescence assays, protein a assays, and immunoelectrophoresis assays, etc.), and the like.

As used herein, the term "modulate" refers to an activity of a compound that affects (e.g., promotes or delays) an aspect of a cell function (including, but not limited to, cell growth, proliferation, invasion, angiogenesis, apoptosis, etc.).

Detailed Description

The present invention provides methods of treating cancer by administering an inhibitor of the IL8-CXCR1 pathway (e.g., an anti-CXCR 1 antibody or Repertaxin), either alone or in combination with other chemotherapeutic agents, thereby killing non-tumorigenic and tumorigenic cancer cells in a subject. The invention also provides compositions and methods for treating and diagnosing the presence of solid tumor stem cells in a patient (e.g., based on the presence of CXCR1 or FBXO 21).

I. Tumorigenic cancer cells, ALDH, CXCR1 and CXCR1 inhibition

The progression of normal cells into fully transformed cells requires counter-regulation of multiple cellular processes (1, 2). These events can occur in any cell according to classical carcinogenesis models. In contrast, the "cancer stem cell hypothesis" considers that the preferential target for oncogenic transformation is tissue stem cells or early progenitor cells that have acquired self-renewal potential (3-6). These "tumor initiating cells" or "cancer stem cells" (CSCs) are then characterized by their ability to undergo self-renewal, a process that directs tumorigenesis and differentiation, which contributes to tumor cell heterogeneity. Recent evidence supporting the cancer stem cell hypothesis has been generated using xenografts of primary human tumors. These studies have shown that tumors are composed of a hierarchy of cells that are directed by the components of cancer stem cells to form. In addition, recent data indicate that immortalized cell lines derived from both murine and human tissues may also contain populations of cells that exhibit stem cell characteristics. Most of these studies are based on in vitro properties including clonogenic potential, sphere formation and multilineage differentiation potential (7-10). More limited studies using functional transplantation of immortalized cell lines in xenografts have also shown the existence of such layers. These studies generally use Hoechst dye exclusion to identify so-called "side population" (SP) (7, 9, 11). In addition, the use of primary tumor xenograft-defined cell surface markers such as CD44 and CD133 to identify similar populations in established cell lines was also used (7, 8).

As described in the examples below, the expression of the stem cell marker aldehyde dehydrogenase (ALDH) was studied in a series of 33 cell lines derived from human breast cancer and untransformed breast cells. ALDH is a detoxifying enzyme responsible for oxidizing intracellular aldehydes and is thought to play a role in stem cell differentiation by metabolizing retinal to retinoic acid (12, 13). ALDH activity assessed by fluorescent ALDEFLUOR assays has been successfully used to isolate cancer stem cells in multiple myeloma and Acute Myeloid Leukemia (AML) and to isolate cancer stem cells from brain tumors (14-16). It has recently been demonstrated that ALDH activity can be used to isolate subpopulations of cells that exhibit stem cell characteristics from normal human breast tissue and breast cancer (17). The ALDELUOR positive population isolated from breast reduction angioplasty (reduction mammoplasty) tissues was able to reconstitute ductal alveolar-like structures in the mammary fat pad of humanized NOD/SCID mice. Furthermore, ALDELFUOR-positive cells isolated from human breast cancer have stem cell characteristics as indicated by their ability to reconstitute tumors and produce phenotypic heterogeneity of the original tumor in NOD/SCID mice after serial passage (17). In the examples below, it was demonstrated that most breast cancer cell lines contain an aldeluor positive population with a unique molecular profile that displays characteristics of cancer stem cells.

As described in the examples below, work carried out during the development of embodiments of the present invention identified CXCR1 (which is a receptor for the inflammatory chemokine IL 8) as a cancer stem cell marker. Only cells within the Aldefluor positive population expressed CXCR 1. Furthermore, it was demonstrated that this receptor exerts a functional effect, since recombinant IL8 was able to increase the proportion of stem cells in the cell line, as determined by Aldefluor and spheroidimetric analysis. Although IL8 has been reported to be associated with invasive breast cancer and higher in the serum of women with metastatic disease, it is believed that the present invention for the first time shows a functional link between IL8 and the receptor CXCR 1in their stem cells.

As further described in the examples below, it was demonstrated that cancer stem cells can be selectively targeted by blocking CXCR1 receptors in these cells. In one method described in the examples, breast cancer cells are treated with a monoclonal antibody directed against CXCR1 but not against the other IL8 receptor CXCR 2. Such treatments selectively target cancer stem cells as indicated by a reduction in Aldefluor positive population. Notably, it was found that although CXCR1 is expressed only in a very small proportion of cells (e.g., less than 1%), the blockade of CXCR1 receptors induces cell death in most other cancer cells, despite their lack of CXCR1 receptors. Molecular pathways have been elucidated that mediate the effects of IL on cancer stem cells and cause this so-called "bystander effect" of killing other cells. IL8 stimulates stem cell self-renewal by binding to CXCR1, CXCR 1in turn activates the focal adhesion kinase Fak pathway. This results in activation of Akt, which drives stem cell self-renewal. Upon blocking this pathway in cancer stem cells, a decrease in Akt signaling causes cytoplasmic sequestration of the Foxo transcription factor, resulting in increased Fas ligand synthesis. Fas ligand is secreted by cancer stem cells and induces cell death in surrounding cells containing the Fas receptor.

Although the present invention is not limited to any particular mechanism, and an understanding of the mechanism is not necessary to practice the present invention, it is believed that CXCR1 mediates cancer stem cell self-renewal via pathways including Fak and Akt, and blocking this pathway induces cell death in cancer stem cells and surrounding tumor cells. Thus, in certain embodiments, the present invention provides compositions and methods for disrupting the IL8-CXCR1 pathway (e.g., with an anti-CXCR 1 antibody, an anti-FAK antibody, or other agent) to treat cancer.

Since IL8 is a chemokine involved in tissue inflammation, there has been prior interest in the development of inhibitors of IL8 signalling. The small molecule inhibitor, Repartaxin, has been developed as an anti-inflammatory agent to potentially reduce complications of myocardial infarction and stroke. Repeataxin has been introduced in phase I and phase II clinical trials and has been shown to have little toxicity. As shown in the examples below, retrievaxin (as well as anti-CXCR 1 antibody) is capable of targeting cancer stem cells and inducing Fas ligand Fas-mediated apoptosis in peripheral cells through bystander effects. Importantly, in tumor xenografts, Repartaxin potentiates the effects of chemotherapy. Furthermore, the method is simple. Unlike chemotherapy, which preferentially destroys differentiated cells in tumors and avoids tumor stem cells, retataxin is able to target tumor stem cells. As shown in the examples, this is evidenced by a reduction in Aldefluor population in the tumor treated with retaxin and a reduction in the ability of these treated tumor cells to form secondary tumors in mice. The effect of Repartaxin on the ability to block metastasis was also tested. Tumor cells were labeled with luciferase and injected into the experimental metastasis model. 1 day after introduction of tumor cells, one group of animals was placed on Repeataxin alone, while the other group was untreated. Repeataxin significantly reduces the formation of metastases.

The present invention identifies the IL8 receptor CXCR1 as a target for the treatment of cancer stem cells. The small molecule inhibitor, Repartaxin, inhibits CXCR1 and CXCR 2. The examples demonstrate that the most important receptor in cancer stem cells is CXCR 1. Furthermore, the examples demonstrate that the inability of cytotoxic chemotherapy to effectively treat established cancers may not only be due to the inability of this therapy to target cancer stem cells, but also due to the demonstrated elevated secretion of IL8 following treatment with tumor cytotoxic chemotherapy. This example demonstrates that the use of CXCR1 inhibitors has beneficial effects in being able to specifically target cancer stem cells and block IL8 stimulation of these cells induced by cytotoxic chemotherapy.

The targeted IL8-CXCR1 pathway is not limited to breast cancer, but may be employed in any type of cancer. Preferably, the type of cancer treated is one in which there is evidence of increased production of IL8 (e.g., associated with chemotherapy). Chemotherapeutic agents have been shown to directly modulate IL8 transcription in cancer cells. Paclitaxel increases transcription and secretion of IL8 in ovarian, breast and lung cancer cell lines (Uslu et al, 2005, int.j.gynecol.cancer, 15: 240-. Also, administration of doxorubicin and 5-fluoro-2' -deoxyuridine (Delorco et al, 2001, Can. Res.61: 2857-2861, incorporated herein by reference), 5-FU (Tamatani et al, 2004, int., J.Can., 108: 912: 921, incorporated herein by reference) to oral cancer cells, doxorubicin (Shibakura et al, 2003, int. J.Can., 103: 380-386, incorporated herein by reference) to small cell lung cancer cells, and dacarbazine (dacarbazine) to melanoma cells (Lev et al, 2003, mol., Can. Ther., 2: 753-763, incorporated herein by reference) all resulted in increased CXCL8 expression. Thus, in certain embodiments, the invention provides agents for targeting IL-CXCR 1in combination with chemotherapeutic agents (e.g., such as those mentioned in the above references) for use in treating a subject having one cancer including, but not limited to, prostate cancer, ovarian cancer, breast cancer, melanoma, non-small cell lung cancer, and esophageal adenocarcinoma.

The present invention is not limited to the type of cancer being treated, but includes, but is not limited to, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, liver cancer, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, wilms' tumor, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, sarcoma, myxoma, pancreatic carcinoma, chordoma, adenocarcinomas, sebaceous carcinoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

Detection of solid tumor Stem cell cancer markers

In some embodiments, the invention provides methods for detecting expression of stem cell cancer markers (e.g., CXCR1, FBXO21, NFYA, NOTCH2, RAD51L1, TBP, and other proteins of table 1). In some embodiments, expression is measured directly (e.g., at the RNA or protein level). In some embodiments, expression is detected in a tissue sample (e.g., biopsy). In other embodiments, expression is detected in a bodily fluid (e.g., including, but not limited to, plasma, serum, whole blood, mucus, and urine).

The invention further provides panels (panels) and kits for detecting markers. In some embodiments, the presence of a stem cell cancer marker is used to provide a prognosis to a subject. The provided information is also used to guide the course of treatment. For example, if a subject is found to have markers indicative of solid tumor stem cells (e.g., CXCR1, FBXO21, NFYA, NOTCH2, RAD51L1, TBP, and other proteins of table 1), then other therapies (e.g., radiation therapy) can be initiated at an early time point when they are likely to be effective (e.g., prior to metastasis). In addition, if a subject is found to have a tumor that is not responsive to certain therapies, the expense and inconvenience of such therapies can be avoided.

In some embodiments, the present invention provides panels for analyzing multiple markers (e.g., CXCR1 or FBXO21 in combination with at least one of CD44, CD24, and ESA). The panel allows simultaneous analysis of multiple markers associated with carcinogenesis and/or metastasis. Depending on the subject, the panels can be analyzed individually or in combination to provide the best diagnosis and prognosis. Markers included on the panels are selected by screening their predictive value using any suitable method, including but not limited to those described in the illustrative examples below.

Detection of RNA

In some embodiments, the solid tumor stem cell cancer marker is detected by measuring the expression of the corresponding mRNA in a tissue sample. mRNA expression can be determined by any suitable method, including but not limited to those disclosed below. The accession number for human CXCR1 nucleic acid is NM _000634 (incorporated by reference herein), while the accession number for human FBXO21 is NM _033624 (incorporated by reference herein). These sequences can be used to design primers and probes (as well as siRNA sequences).

In some embodiments, RNA is detected by Northern blot analysis. Northern blot analysis involves the isolation of RNA and hybridization of complementary labeled probes.

In still other embodiments, the RNA (or corresponding cDNA) is detected by hybridization to an oligonucleotide probe. A variety of hybridization assays are available using a variety of techniques for hybridization and detection. For example, in some embodiments, TaqMan assays (PE Biosystems, Foster City, Calif.; see, e.g., U.S. Pat. Nos. 5,962,233 and 5,538,848, each of which is incorporated herein by reference) are utilized. The assay is performed during the PCR reaction. TaqMan assays utilize the 5 '-3' exonuclease activity of AMPLITAQ GOLD DNA polymerase. The PCR reaction contains a probe consisting of an oligonucleotide with a 5 '-reporter dye (e.g., a fluorescent dye) and a 3' -quencher dye. During PCR, the 5 '-3' nucleolytic activity of the AMPLITAQ GOLD polymerase cleaves the probe between the reporter dye and the quencher dye if the probe binds to its target. The separation of the reporter dye from the quencher dye results in an increase in fluorescence. The signal accumulates with each PCR cycle and can be monitored with a fluorometer.

In yet other embodiments, reverse transcriptase PCR (RT-PCR) is used to detect expression of RNA. In RT-PCR, RNA is enzymatically converted to complementary DNA or "cDNA" using reverse transcriptase enzymes. The cDNA was then used as a template for the PCR reaction. PCR products can be detected by any suitable method, including but not limited to gel electrophoresis and staining with DNA-specific dyes or hybridization to labeled probes. In some embodiments, quantitative reverse transcriptase PCR is utilized with a standardized competitive template mixture method, which is described in U.S. patents 5,639,606, 5,643,765, and 5,876,978 (each of which is incorporated herein by reference).

2. Detection of proteins

In other embodiments, gene expression of a stem cell cancer marker is detected by measuring expression of the corresponding protein or polypeptide (e.g., CXCR1, FBXO21, NFYA, NOTCH2, RAD51L1, TBP, and other proteins of table 1). Protein expression can be detected by any suitable method. In some embodiments, the protein is detected by immunohistochemistry. In other embodiments, the protein is detected by its binding to an antibody directed against the protein. The accession number for human CXCR1 protein is NP _000625 (incorporated herein by reference), while the accession number for human FBXO21 is NP _296373 (incorporated herein by reference). The production of antibodies is described below.

Antibody binding is detected by techniques known in the art (e.g., radioimmunoassays, ELISAs (enzyme linked immunosorbent assays), "sandwich" immunoassays, immunoradiometric assays, gel diffusion precipitation reactions, immunodiffusion assays, in situ immunoassays (e.g., using colloidal gold, enzyme, or radioisotope labels), Western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays, etc.), complement binding assays, immunofluorescence assays, protein a assays, and immunoelectrophoresis assays, etc.).

In one embodiment, antibody binding is detected by detecting a label on the primary antibody. In another embodiment, the primary antibody is detected by detecting binding of a secondary antibody or agent to the primary antibody. In yet another embodiment, the secondary antibody is labeled. Many methods for detecting binding in immunoassays are known in the art and are within the scope of the present invention.

In some embodiments, automated detection assays are utilized. Automated methods for immunoassays include those described in U.S. patents 5,885,530, 4,981,785, 6,159,750, and 5,358,691, each of which is incorporated herein by reference. In some embodiments, the analysis and presentation of results is also automated. For example, in some embodiments, software is utilized that generates a prognosis based on the presence or absence of a series of proteins corresponding to a cancer marker.

In other embodiments, immunoassays are described in U.S. patents 5,599,677 and 5,672,480; each of these patents is incorporated herein by reference.

cDNA microarray technology

cDNA microarrays consist of a plurality (usually thousands) of different cdnas spotted (usually using a robotic positioning device) onto a solid support (such as a glass microscope slide) at known locations. Typically, cDNA is obtained by PCR amplification of plasmid library inserts using primers that are complementary to portions of the plasmid vector backbone or to the gene itself of known sequence. The length of PCR products suitable for generating microarrays is typically between 0.5 and 2.5 kB. Full-length cDNAs, Expressed Sequence Tags (ESTs) or randomly selected cDNAs from any library of interest may be selected. For example, as described by Hillier et al, 1996, 6: 807-828 ESTs are partially sequenced cDNAs. Although some ESTs correspond to known genes, very little or no information is often available about any particular EST, except for a small amount of 3 'and/or 5' sequences and the tissue of origin of the mRNA from which the EST may be derived. As one of ordinary skill in the art will appreciate, in general, cdnas contain sufficient sequence information to uniquely identify genes within the human genome. In addition, in general, the cDNA is of sufficient length to selectively, specifically, or uniquely hybridize under the hybridization conditions of the experiment to cDNA obtained from mRNA derived from a single gene.

In a typical microarray experiment, a microarray is hybridized with a population of differentially labeled RNA, DNA, or cDNA derived from two different samples. Most commonly, RNA (total or poly a + RNA) is isolated from the target cell or tissue and reverse transcribed to produce cDNA. Labeling is typically performed during reverse transcription by incorporating labeled nucleotides into the reaction mixture. Most commonly, nucleotides are conjugated to fluorescent dyes Cy3 or Cy5, although various labels can be used. For example, Cy5-dUTP and Cy3-dUTP can be used. cDNA derived from one sample (representing, for example, a particular cell type, tissue type, or growth condition) is labeled with one fluorophore, while cDNA derived from a second sample (representing, for example, a different cell type, tissue type, or growth condition) is labeled with a second fluorophore. Similar amounts of labeled material from both samples were co-hybridized to the microarray. In the case of microarray experiments in which the samples were labeled with Cy5 (which fluoresces red) and Cy3 (which fluoresces green), the raw data (obtained by scanning the microarray using a detector capable of quantitatively detecting the fluorescence intensity) was the ratio of the fluorescence intensities (red/green, R/G). These ratios represent the relative concentration of cDNA molecules that hybridize to the cDNA presented on the microarray, and thus reflect the relative expression levels of mRNA corresponding to each cDNA/gene presented on the microarray.

Each microarray experiment can provide thousands of data points, each representing the relative expression of a particular gene in two samples. Proper organization and analysis of data is crucial, and a variety of computer programs incorporating standard statistical tools have been developed to facilitate data analysis. One basis for organizing gene expression data is to cluster genes with similar expression patterns together. Methods for hierarchical cluster analysis (hierarchical cluster analysis) and display of data derived from microarray experiments are described in Eisen et al, 1998, PNAS 95: 14863-14868. As described therein, clustering may be combined with a graphical representation of the raw data, where each data point is represented by a color that quantitatively and qualitatively represents the data point. This process facilitates intuitive analysis of data by transforming data from a large digital table into a visible form. Additional information and details about the mathematical tools and/or clustering methods themselves can be found in, for example, Sokal and Sneath, Principles of numerical taxonomy, xvi, 359, w.h.freeman, SanFrancisco, 1963; hartigan, Clustering algorithms, xiii, 351, Wiley, New York, 1975; paull et al, 1989, J.Natl.cancer Inst.81: 1088-92; weinstein et al 1992, Science 258: 447-51; van Osdol et al, 1994, j.natl.cancer inst.86: 1853-9; and Weinstein et al, 1997, Science, 275: 343-9.

For more details of the experimental methods used in the present invention, see the examples below. Additional information describing methods for making and using microarrays is found in U.S. Pat. No.5,807,522, which is incorporated herein by reference. Instructions for building microarray hardware (e.g., arrayer and scanner) using commercially available components. Additional discussion of microarray technology and protocols for preparing samples and performing microarray experiments are described, for example, in DNA arrays for analysis of gene expression, Methods Enzymol, 303: 179-205, 1999; fluoro-based decompression monitoring using microarrays, Methods Enzymol, 306: 3-18, 1999; schena (eds), DNA microarray: a Practical Approach, Oxford University Press, Oxford, UK, 1999.

4. Data analysis

In some embodiments, a computer-based analysis program is used to convert the raw data (e.g., the presence, absence, or amount of a given marker or markers) generated by the detection assay into predictive valuable data for the clinician. The clinician may evaluate the predictive data using any suitable means. Thus, in some embodiments, the invention provides the additional benefit that clinicians who are not likely to be genetically or molecularly trained do not need to understand the raw data. The data is presented directly to the clinician in its most useful form. The clinician can then immediately utilize the information to optimize the care of the subject.

The present invention contemplates any method capable of accepting, processing, and communicating information between the laboratory, information provider, medical personnel, and subject in which the assay is performed. For example, in some embodiments of the invention, a sample (e.g., a biopsy tissue or serum or urine sample) is obtained from a subject and submitted to a profiling service (e.g., a clinical laboratory of a medical facility, a genomic profiling enterprise, etc.) anywhere in the world (e.g., in a country different from the country in which the subject resides or the information is ultimately used) to generate raw data. In the case where the sample comprises tissue or other biological sample, the subject may visit a medical center to have the sample obtained and sent to a spectral analysis center, or the subject may collect the sample by itself and send it directly to the spectral analysis center. In the case where the sample contains previously determined biological information, the subject can send the information directly to the spectral analysis service (e.g., an information card containing the information can be scanned by a computer and the data transferred to the computer of the spectral analysis center using an electronic communication system). Once received by the profiling service, the sample is processed and a profile (e.g., expression data) is generated that is specific to the diagnostic or prognostic information desired by the subject.

The treatment clinician then prepares the spectral pattern data in a form suitable for interpretation. For example, a prepared form may represent a diagnosis or risk assessment for a subject along with a recommendation of a particular treatment option, rather than providing raw expression data (e.g., examining a number of markers). The data may be displayed to the clinician by any suitable method. For example, in some embodiments, the spectral analysis service generates a report that a clinician can print (e.g., at a point of care) or can display to the clinician on a computer monitor.

In some embodiments, the information is first analyzed at the point of care or at the regional facility. The raw data is then sent to a central processing facility for further analysis and/or conversion into information useful to the clinician or patient. The central processing facility provides the advantages of privacy (all data stored at the central facility with a uniform security scheme), speed, and consistency of data analysis. The central processing facility may then control the final processing of the data after treatment of the subject. For example, using an electronic communication system, a central facility may provide data to a clinician, subject, or researcher.

In some embodiments, the subject is able to directly evaluate the data using an electronic communication system. The subject may select further intervention or counseling based on the results. In some embodiments, the data is used for research purposes. For example, the data can be used to further optimize the inclusion or exclusion of markers that are useful indicators of a particular state or stage of a disease.

5. Reagent kit

In still other embodiments, the invention provides kits for detecting and characterizing cancer (e.g., for detecting one or more markers, or for modulating the activity of a peptide expressed by one or more markers). In some embodiments, the kit contains an antibody specific for a cancer marker in addition to the detection agent and buffer. In other embodiments, the kit contains reagents (e.g., oligonucleotide probes or primers) specific for detecting mRNA or cDNA. In some embodiments, the kit contains all of the components necessary and/or sufficient to perform a detection assay, including all controls, instructions for performing the assay, and any necessary software for analyzing and presenting the results.

Another embodiment of the invention includes a kit for testing for the presence of a polynucleotide or protein. The kit may comprise, for example, an antibody for detecting a polypeptide or a probe for detecting a polynucleotide. Additionally, the kit may comprise a reference or control sample; instructions for processing the sample, performing the test, and interpreting the results; and buffers and other reagents necessary to perform the test. In other embodiments, the kit comprises a primer pair for detecting expression of one or more genes of the solid tumor stem cell gene signature. In other embodiments, the kit comprises a cDNA or oligonucleotide array for detecting expression of one or more genes of the solid tumor stem cell gene signature.

6. In vivo imaging

In some embodiments, in vivo imaging techniques are used to visualize expression of a cancer marker in an animal (e.g., a human or non-human mammal). For example, in some embodiments, a cancer marker mRNA (e.g., CXCR1 or FBXO21mRNA) or protein (e.g., CXCR1 or FBXO21 protein) is labeled with a labeled antibody specific for the cancer marker. The specifically bound and labeled antibody may be detected in the individual using in vivo imaging methods including, but not limited to, radionuclide imaging, positron emission tomography, computerized axial tomography, X-ray or magnetic resonance imaging, fluorescence detection, and chemiluminescence detection. Methods for generating antibodies against the cancer markers of the invention are described below.

The in vivo imaging methods of the invention are useful in diagnosing cancers that express the solid tumor stem cell cancer markers of the invention. In vivo imaging is used to visualize the presence of markers indicative of cancer. Such techniques allow diagnosis without the use of unpleasant biopsies. The in vivo imaging methods of the invention are also useful for providing a prognosis to a cancer patient. For example, the presence of a marker indicative of cancer stem cells may be detected. The in vivo imaging method of the present invention may be further used to detect metastatic cancer in other parts of the body.

In some embodiments, an agent (e.g., an antibody) specific for CXCR1 or FBXO21 is fluorescently labeled. The labeled antibody is introduced into the subject (e.g., orally or parenterally). The fluorescently labeled antibody is detected using any suitable method (e.g., using the apparatus described in U.S. patent No.6,198,107, which is incorporated herein by reference).

In other embodiments, the antibody is radiolabeled. The use of antibodies for in vivo diagnostics is well known in the art. Sumerdon et al, (Nucl. Med. biol 17: 247-. Griffin et al, (JClin Onc 9: 631-640[1991]) have described the use of this agent for the detection of tumors in patients suspected of having pancreatic cancer. The use of similar agents with paramagnetic ions as markers for Magnetic Resonance imaging is known in the art (Lauffer, Magnetic Resonance in Medicine 22: 339-. The label used will depend on the imaging modality selected. Planar scanning or Single Photon Emission Computed Tomography (SPECT) can be performed using radioactive labels such as indium-111, technetium-99 m, or iodine-131. Positron emission markers such as fluorine-19 can also be used for Positron Emission Tomography (PET). For MRI, paramagnetic ions, such as gadolinium (III) or manganese (II), may also be used.

Radiometals with half-lives of 1 hour to 3.5 days may be used for conjugation to antibodies, such as scandium-47 (3.5 days), gallium-67 (2.8 days), gallium-68 (68 minutes), technetium-99 m (6 hours), and indium-111 (3.2 days), with gallium-67, technetium-99 m, and indium-111 being preferred for gamma camera imaging and gallium-68 being preferred for positron imaging.

A useful method of labelling antibodies with such radiometals is by means of bifunctional chelators, such as diethylenetriaminepentaacetic acid (DTPA), as described, for example, for In-111 and Tc-99m, by Khaw et al (Science 209: 295[1980]), and by Scheinberg et al (Science 215: 1511[1982 ]). Other chelating agents may also be used, but the carboxycarbonic anhydrides of 1- (p-carboxymethoxybenzyl) EDTA and DTPA are advantageous because their use allows conjugation without substantially affecting the immunoreactivity of the antibody.

Another method for coupling DPTA to proteins utilizes the cyclic anhydride of DTPA, labeled with In-111 albumin as described by Hnatowich et al (int.J.appl.radial.Isot.33: 327[1982]), but can be adapted for use In labeling antibodies. One suitable method for labeling antibodies with Tc-99m is the pre-stannation method of Crockford et al, (U.S. Pat. No.4,323,546, incorporated herein by reference), which does not use chelation with DPTA.

The method for labeling immunoglobulins with Tc-99m is the method described by Wong et al (int.J.appl.radial.Isot., 29: 251[1978]) for plasma proteins, and recently was successfully applied to labeled antibodies by Wong et al (J.Nucl.Med., 23: 229[1981 ]).

In the case of radiometals conjugated with specific antibodies, it is also desirable to introduce as high a proportion of radiolabel as possible into the antibody molecule without destroying its immunospecificity. Further improvements can be achieved by effecting radiolabelling in the presence of the specific stem cell cancer markers of the invention to ensure that the antigen binding site on the antibody will be protected.

In still other embodiments, in vivo imaging is performed using in vivo biophotonic imaging (Xenogen, Almeda, CA). This real-time in vivo imaging utilizes luciferase. Luciferase genes are incorporated into cells, microorganisms, and animals (e.g., as fusion proteins with the cancer markers of the invention). When active, it causes a light-emitting reaction. Images were captured using a CCD camera and software and analyzed.

Antibodies and antibody fragments

The present invention provides isolated antibodies and antibody fragments directed to CXCR1, FBXO21, NFYA, NOTCH2, RAD51L1, TBP, and other proteins of table 1. The antibody or antibody fragment may be any monoclonal or polyclonal antibody that specifically recognizes these proteins. In some embodiments, the invention provides monoclonal antibodies or fragments thereof that specifically bind CXCR1, FBXO21, NFYA, NOTCH2, RAD51L1, TBP, and other proteins of table 1. In some embodiments, the monoclonal antibodies or fragments thereof are chimeric or humanized antibodies that specifically bind to these proteins. In other embodiments, the monoclonal antibodies or fragments thereof are human antibodies that specifically bind to these proteins.

Antibodies directed to CXCR1, FBXO21, NFYA, NOTCH2, RAD51L1, TBP and other proteins of table 1 find use in the experimental, diagnostic and therapeutic methods described herein. In certain embodiments, antibodies of the invention are used to detect expression of a cancer stem cell marker protein in a biological sample (such as, for example, a patient biopsy, pleural effusion, or blood sample). The biopsy tissue may be sectioned and the proteins detected using, for example, immunofluorescence or immunohistochemistry. Alternatively, single cells from the sample are isolated and protein expression is detected on fixed or living cells by FACS analysis. In addition, antibodies can be used on protein arrays to detect expression of cancer stem cell markers, e.g., on tumor cells, in cell lysates, or in other protein samples. In other embodiments, the antibodies of the invention are used to inhibit tumor cell growth by contacting the antibody with a tumor cell in a cell-based in vitro assay or in an in vivo animal model. In yet other embodiments, the antibodies are used to treat cancer in a human patient by administering a therapeutically effective amount of an antibody against a cancer stem cell marker (e.g., from table 1).

Polyclonal antibodies can be prepared by any known method. Animals (e.g., rabbits, rats, mice, donkeys, etc.) can be immunized by multiple subcutaneous or intraperitoneal injections of a relevant antigen (purified peptide fragment, full-length recombinant protein, fusion protein, etc.), optionally conjugated to Keyhole Limpet Hemocyanin (KLH), serum albumin, etc., to generate polyclonal antibodies, which are diluted in sterile saline and combined with an adjuvant (e.g., complete or incomplete freund's adjuvant) to form a stable emulsion. Then, the polyclonal antibody is recovered from blood, ascites, and the like of the animal thus immunized. The collected blood was allowed to clot and the serum was decanted, clarified by centrifugation and assayed for antibody titer. Polyclonal antibodies can be purified from serum or ascites fluid according to standard methods in the art, including affinity chromatography, ion exchange chromatography, gel electrophoresis, dialysis, and the like.

Methods such as those described by Kohler and Milstein (1975) Nature 256: 495 to produce monoclonal antibodies. Using the hybridoma method, a mouse, hamster, or other suitable host animal, is immunized as described above to elicit lymphocytes that produce antibodies that will specifically bind to the immunizing antigen. Alternatively, lymphocytes can be immunized in vitro. Following immunization, the lymphocytes are isolated and fused with a suitable myeloma cell line using, for example, polyethylene glycol to form hybridoma cells, which can then be selected from unfused lymphocytes and myeloma cells. Hybridoma cells that produce Monoclonal Antibodies specific for a selected antigen, as determined by immunoprecipitation, immunoblotting, or by an in vitro binding assay such as Radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA), can then be propagated in vitro in culture or in vivo in animals using standard methods (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, 1986).

Alternatively, monoclonal antibodies can also be generated using recombinant DNA methods, as described in U.S. patent 4,816,567. Polynucleotides encoding monoclonal antibodies are isolated, such as by RT-PCR, from, for example, mature B cells or hybridoma cells using oligonucleotide primers that specifically amplify the genes encoding the heavy and light chains of the antibody, and their sequences determined using conventional methods. The isolated polynucleotides encoding the heavy and light chains are then cloned into suitable expression vectors that, when transfected into a host cell, such as an escherichia coli cell, a simian COS cell, a Chinese Hamster Ovary (CHO) cell, or a myeloma cell that does not otherwise produce immunoglobulin protein, produce monoclonal antibodies. Also, recombinant monoclonal antibodies or fragments thereof of a desired species can be isolated from phage display libraries as described (McCafferty et al, 1990, Nature, 348: 552- > 554; Clackson et al, 1991, Nature, 352: 624-.

One or more polynucleotides encoding a monoclonal antibody can be further modified in a number of different ways using recombinant DNA techniques to generate alternative antibodies. In one embodiment, the constant domains of the light and heavy chains of, for example, a mouse monoclonal antibody, can be substituted 1) with those of, for example, a human antibody to produce a chimeric antibody, or 2) with a non-immunoglobulin polypeptide to produce a fusion antibody. In other embodiments, the constant region is truncated or removed to generate the desired antibody fragment of the monoclonal antibody. In addition, site-directed or high-density mutagenesis of the variable regions can be used to optimize the specificity, affinity, etc., of the monoclonal antibody.

In some embodiments of the invention, the monoclonal antibody directed against a cancer stem cell marker is a humanized antibody. Humanized antibodies are antibodies that contain minimal sequences from non-human (e.g., murine) antibodies in the variable region. Such antibodies are used therapeutically to reduce antigenicity and HAMA (human anti-mouse antibody) responses when administered to a human subject. In practice, humanized antibodies are typically human antibodies with minimal to no non-human sequences. A human antibody is an antibody produced by a human or an antibody having an amino acid sequence corresponding to an antibody produced by a human.

Humanized antibodies can be generated using a variety of techniques known in the art. Antibodies can be humanized by substituting the CDRs of a human antibody with the CDRs of a non-human antibody (e.g., mouse, rat, rabbit, hamster, etc.) of the desired specificity, affinity, and performance (capability) (Jones et al, 1986, Nature, 321: 522-92; Riechmann et al, 1988, Nature, 332: 323-327; Verhoeyen et al, 1988, Science, 239: 1534-1536). Humanized antibodies can be further modified by substitution of other residues within the Fv framework regions and/or within the substituted non-human residues to improve and optimize the specificity, affinity, and/or performance of the antibody.

Human antibodies can be made directly using a variety of techniques known in the art. Immortalized human B lymphocytes can be generated that are immunized in vitro or isolated from an immunized individual that produces Antibodies to a target antigen (see, e.g., Cole et al, Monoclonal Antibodies and Cancer Therapy, Alan R.Liss, page 77 (1985); Boemer et al, 1991, J.Immunol., 147 (1): 86-95; and U.S. Pat. No.5,750,373). Also, human antibodies can be selected from phage libraries that express human antibodies (Vaughan et al, 1996, Nature Biotechnology, 14; 309-. Humanized antibodies can also be produced in transgenic mice containing human immunoglobulin loci that are capable of producing fully human antibodies after immunization without endogenous immunoglobulin production. This method is described in U.S. patents 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425 and 5,661,016.

The invention also includes bispecific antibodies that specifically recognize cancer stem cell markers. Bispecific antibodies are antibodies that are capable of specifically recognizing and binding at least two different epitopes.

Bispecific antibodies can be intact antibodies or antibody fragments. Techniques for the preparation of bispecific antibodies are common in the art (Millstein et al, 1983, Nature 305: 537-.

In certain embodiments of the invention, for example, it may be desirable to use antibody fragments, rather than whole antibodies, to enhance tumor penetration. Various techniques are known for generating antibody fragments. Traditionally, these fragments have been generated by proteolytic digestion of intact antibodies (e.g., Morimoto et al, 1993, Journal of Biochemical and biophysical methods 24: 107-117 and Brennan et al, 1985, Science, 229: 81). However, these fragments are now typically produced directly by recombinant host cells, as described above. Thus, Fab, Fv and scFv antibody fragments can all be expressed in and secreted from E.coli or other host cells, thus allowing for the production of large quantities of these fragments. Alternatively, such antibody fragments may be isolated from an antibody phage library as discussed above. Antibody fragments may also be linear antibodies, such as described in U.S. Pat. No.5,641,870, and may be monospecific or bispecific. Other techniques for generating antibody fragments will be apparent to the skilled practitioner.

Particularly in the case of antibody fragments, it may be desirable to further modify the antibody to extend its serum half-life. This can be achieved by introducing the salvage receptor binding epitope into the antibody fragment, for example by mutation of an appropriate region in the antibody fragment, or by introducing the epitope into a peptide tag which is then fused to the antibody fragment at either end or in between (e.g., by DNA or peptide synthesis).

The invention further includes variants and equivalents that are substantially homologous to the chimeric, humanized and human antibodies or antibody fragments thereof set forth herein. These may contain, for example, conservative substitution mutations, i.e., substitution of one or more amino acids with similar amino acids. For example, a conservative substitution refers to the substitution of one amino acid with another within the same general class, such as, for example, the substitution of one acidic amino acid with another acidic amino acid, the substitution of one basic amino acid with another basic amino acid, or the substitution of one neutral amino acid with another neutral amino acid. The intent of conservative amino acid substitutions is well known in the art.

The invention also pertains to immunoconjugates comprising an antibody conjugated to a cytotoxic agent. Cytotoxic agents include chemotherapeutic agents, growth inhibitory agents, toxins (e.g., enzymatically active toxins of bacterial, fungal, plant or animal origin or fragments thereof), radioisotopes (i.e., radioconjugates), and the like. Chemotherapeutic agents useful in generating such immunoconjugates include, for example, methotrexate, doxorubicin, melphalan (melphalan), mitomycin C, chlorambucil (chlorembucil), daunorubicin, or other chimeric agents. Enzymatically active toxins and fragments thereof that may be used include diphtheria a chain, non-binding active fragments of diphtheria toxin, exotoxin a chain, ricin a chain, abrin a chain, balanophyllin (modecin) a chain, alpha-sarcin, Aleurites fordii protein, dianthin (dianthin) protein, phytolacca americana (PAPI, PAPII, and PAP-S) protein, momordica charantia (momordia) inhibitor, curcin, crotin, saponaria officinalis (sapaonaria officinalis) inhibitor, gelonin (gelonin), mitogellin (mitogellin), restrictocin (tricotocin), phenomycin (enomycin), enomycin (neomycin), and trichothecene (tricothecene). A variety of radionuclides are useful for generating radioconjugated antibodies, including 212Bi, 131I, 131In, 90Y, and 186 Re. Conjugates of the antibody with cytotoxic agents are generated using a variety of bifunctional protein coupling agents such as N-hydroxysuccinimide 3- (2-pyridinedimercapto) propionate (SPDP), Iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), diazide compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-nitrogen derivatives (such as bis- (p-diazoniumbenzoyl) -ethylenediamine), diisocyanates (such as toluene 2, 6-diisocyanate), and bis-active fluorine compounds (such as 1, 5-difluoro-2, 4-dinitrobenzene). Conjugates of the antibody with one or more small molecule toxins, such as calicheamicin (calicheamicin), maytansinoids (maytansinoids), trichothecenes (trichothenes), and CC1065, and derivatives of these toxins having toxin activity, may also be used.

In some embodiments, the antibodies of the invention contain a human Fc region modified to enhance effector functions, such as antigen-dependent cell-mediated cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC). This can be achieved by introducing one or more amino acid substitutions in the Fc region of the antibody. For example, one or more cysteine residues may be introduced into the Fc region to allow interchain disulfide bonds to form in this region to improve complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC) (Caron et al, 1992, J.exp Med.176: 1191-1195; shop, 1992, Immunol.148: 2918-2922). Heterodimeric cross-linking agents can also be used to prepare homodimeric antibodies with enhanced anti-tumor activity, as described by Wolff et al, 1993, Cancer Research 53: 2560 and 2565. Alternatively, antibodies with dual Fc regions can be designed (Stevenson et al, 1989, Anti-Cancer Drug Design 3: 219-230).

Screening of drugs

In some embodiments, the invention provides drug screening assays (e.g., to screen for anti-cancer drugs). The screening methods of the invention utilize stem cell cancer markers (e.g., CXCR1, FBXO21, NFYA, NOTCH2, RAD51L1, TBP, and other proteins of table 1) that are identified using the methods of the invention. For example, in some embodiments, the invention provides methods of screening for compounds that have altered (e.g., increased or decreased) CXCR1 or FBXO21 expression or activity. In some embodiments, the candidate compound is an antisense or siRNA formulation (e.g., an oligonucleotide) directed against a cancer marker. In other embodiments, the candidate compound is an antibody that specifically binds to a stem cell cancer marker of the invention. In certain embodiments, a library of small molecule compounds is screened using the methods described herein.

In one screening method, a candidate compound is evaluated for its ability to alter expression of a stem cell cancer marker by contacting the compound with a cell expressing a stem cell cancer marker and then determining the effect of the candidate compound on expression. In some embodiments, the effect of a candidate compound on cancer marker gene expression is determined by detecting the level of cancer marker mRNA expressed by the cell. mRNA expression can be detected by any suitable method. In other embodiments, the effect of a candidate compound on cancer marker gene expression is determined by measuring the level of polypeptide encoded by the cancer marker. Any suitable method may be used to measure the level of the expressed polypeptide, including but not limited to those disclosed herein. In some embodiments, other changes in cell biology (e.g., apoptosis) are detected.

In particular, the invention provides methods for identifying modulators, i.e., candidate or test compounds or agents (e.g., proteins, peptides, peptidomimetics, peptoids, small molecules, or other drugs), that bind to or alter signal transduction or function associated with the cancer markers of the invention, have an inhibitory (or stimulatory) effect on, for example, stem cell cancer marker expression or cancer marker activity, or have a stimulatory or inhibitory effect on, for example, expression or activity of a cancer marker substrate. The compounds so identified can be used to directly or indirectly modulate the activity of a target gene product (e.g., a stem cell cancer marker gene such as CXCR1 or FBXO21), to improve the biological function of a target gene product, or to identify compounds that disrupt normal target gene interactions in a therapeutic regimen. Compounds that inhibit the activity or expression of cancer markers are useful for treating proliferative disorders, such as cancer, particularly metastatic cancer, or for eliminating or controlling tumor stem cells to prevent or reduce the risk of cancer.

In one embodiment, the invention provides an assay for screening candidate or test compounds as substrates for cancer marker proteins or polypeptides or biologically active portions thereof. In another embodiment, the invention provides an assay for screening for binding to or modulating the activity of a cancer marker protein or polypeptide or biologically active portion thereof.

The test compounds of the present invention can be obtained using any of a variety of methods known in the art for combinatorial library approaches, including biological libraries; peptoid libraries (libraries of molecules with peptide functionality but with a new, non-peptide backbone, which are resistant to enzymatic degradation but which nevertheless retain biological activity; see e.g., Zuckennan et al, J.Med.chem.37: 2678-85[1994 ]); spatially addressable parallel solid or liquid phase libraries; synthetic library methods requiring deconvolution; "one-bead-one-compound" library approach; and synthetic library methods using affinity chromatography selection. The biological library and peptoid library methods are preferably used with peptide libraries, while the other four methods are applicable to peptide, non-peptide oligomer of compounds or small molecule libraries of compounds (Lam (1997) Anticancer Drug Des.12: 145).

Examples of methods for synthesizing libraries of molecules can be found in the art, for example: DeWitt et al, Proc.Natl.Acad.Sci.U.S.A.90: 6909[1993 ]; erb et al, proc.nad.acad.sci.usa 91: 11422[1994 ]; zuckermann et al, j.med.chem.37: 2678[1994 ]; cho et al, Science 261: 1303[1993 ]; carrell et al, Angew.chem.int.Ed.Engl.33.2059[1994 ]; carell et al, angelw.chem.int.ed.engl.33: 2061[1994 ]; and Gallop et al, j.med.chem.37: 1233[1994].

Libraries of compounds may be present in solution (e.g., Houghten, Biotechniques 13: 412- & 421[1992]), or on beads (Lam, Nature 354: 82-84[1991]), on-chip (Fodor, Nature 364: 555- & 556[1993]), on bacteria or spores (U.S. Pat. No.5,223,409; incorporated herein by reference), on plasmids (Cull et al, Proc. Nad. Acad. Sci. USA 89: 18651869[1992]), or on phages (Scott and Smith, Science 249: 386- & 390[1990 ]; Devrin Science 249: 404- & 406: 1990; Cwirla et al, Proc. NatI. Acad. Sci. 87: 1990 8- & 6382[1990 ]; Felici, J.biol. biol. 222: 301[1991] 301.1991..

In one embodiment, the assay is a cell-based assay in which a cell expressing a stem cell cancer marker protein or a biologically active portion thereof is contacted with a test compound and the ability of the test compound to modulate the activity of a cancer marker is determined. Determination of the ability of a test compound to modulate the activity of a stem cell cancer marker can be accomplished by monitoring, for example, changes in enzyme activity. For example, the cell may be of mammalian origin.

The ability of a test compound to modulate a cancer marker that binds to the compound (e.g., a stem cell cancer marker substrate) can also be assessed. This can be accomplished, for example, by coupling a compound (e.g., a substrate) to a radioisotope or enzymatic label, such that binding of the compound (e.g., substrate) to the cancer marker can be determined by detecting the labeled compound (e.g., substrate) in the complex.

Alternatively, stem cell cancer markers are conjugated to a radioisotope or enzyme label to monitor the ability of the test compound to modulate the cancer marker binding to the cancer marker substrate in the complex. For example, a compound (e.g., a substrate) can be labeled with 125I, 35S, 14C, or 3H, either directly or indirectly, and the radioisotope detected by direct counting of radioactive emissions or by scintillation counting. Alternatively, the compounds may be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase or luciferase, and the enzymatic label detected by measuring the conversion of an appropriate substrate to product.

The ability of a compound (e.g., a stem cell cancer marker substrate) to interact with a stem cell cancer marker with or without labeling any of the interactors can be assessed. For example, a microphysiometer can be used to detect the interaction of a compound with a cancer marker in the absence of labeled compound or cancer marker (McConnell et al Science 257: 1906-. As used herein, a "microphysiometer" (e.g., a cell sensor) is an analytical instrument that uses a light-addressable potentiometric sensor (LAPS) to measure the rate at which cells acidify their environment. This change in acidification rate can be used as an indicator of the interaction between the compound and a cancer marker.

In yet another embodiment, a cell-free assay is provided in which a cancer marker protein or biologically active portion thereof is contacted with a test compound and the ability of the test compound to bind to the stem cell cancer marker protein or biologically active portion thereof is assessed. Biologically active portions of the cancer marker proteins to be used in the assays of the invention include fragments involved in interactions with substrates or other proteins, e.g., fragments with a high surface probability score.

Cell-free assays involve preparing a reaction mixture of the target gene protein and the test compound under conditions and for a time sufficient to allow the two components to interact and bind, thus forming a complex that can be removed and/or detected.

Interactions between two molecules can also be detected, for example, using fluorescence energy transfer (FRET) (see, e.g., Lakowicz et al, U.S. Pat. No.5,631,169; Stavrianopoulos et al, U.S. Pat. No.4,968,103; each of these patents is incorporated herein by reference). The fluorophore labels are selected such that the fluorescent energy emitted by the first donor molecule will be absorbed by the fluorescent label on the second "acceptor" molecule, which in turn is capable of fluorescing due to the absorbed energy.

Alternatively, the "donor" protein molecule may utilize only the natural fluorescence energy of tryptophan residues. The labels are selected to emit light at different wavelengths such that the "acceptor" molecular label is distinguishable from the "donor" molecular label. Since the efficiency of energy transfer between labels is related to the distance separating the molecules, the spatial relationship between the molecules can be assessed. In the case of intermolecular binding, the fluorescence emission of the "receptor" molecular marker in the assay should be maximal. FRET binding events may be conveniently measured via standard fluorometric detection means well known in the art (e.g., using a fluorometer).

In another embodiment, the determination of the ability of a stem cell cancer marker protein to bind to a target molecule can be accomplished using a real-time Biomolecule Interaction Assay (BIA) (see, e.g., Sjolander and Urbaniczky, anal. chem.63: 2338-Struct 2345[1991] and Szabo et al, curr. Opin. struct. biol.5: 699-705[1995 ]). "surface plasmon resonance" or "BIA" detects biospecific interactions in real time without labeling any interactors (e.g., BlAcore). Mass changes on the binding surface (indicative of binding events) result in changes in the optical refractive index of light near the surface (an optical phenomenon of Surface Plasmon Resonance (SPR)), which produces a detectable signal that can be an indicator of real-time reactions between biomolecules.

In one embodiment, the target gene product or test substrate is anchored to a solid phase. The target gene product/test compound complex anchored to the solid phase can be detected at the end of the reaction. The target gene product can be anchored to a solid surface and the test compound (which is not anchored) can be labeled, either directly or indirectly, with a detectable label as discussed herein.

Immobilization of stem cell cancer markers, anti-cancer marker antibodies, or their target molecules may be required to facilitate separation of complexed and uncomplexed forms of one or both proteins, as well as to accommodate automation of the assay. Binding of the test compound to the stem cell cancer marker protein, or interaction of the cancer marker protein with the target molecule in the presence and absence of the candidate compound, can be achieved in any vessel suitable for holding reagents. Examples of such containers include microtiter plates, test tubes, and microcentrifuge tubes. In one embodiment, fusion proteins can be provided that add domains that allow one or both proteins to bind to a matrix. For example, glutathione-S-transferase-cancer marker fusion protein or glutathione-S-transferase/target fusion protein can be adsorbed onto glutathione Sepharose beads (Sigma Chemical, st. louis, MO) or microtiter plates derived from glutathione, which are then combined with a test compound or test compound and unadsorbed target protein or cancer marker protein and the mixture incubated under conditions conducive to complex formation (e.g., physiological conditions in terms of salt and pH). After incubation, the beads or microtiter plate wells are washed to remove any unbound components, in the case of beads, the matrix is immobilized, and the complexes are determined directly or indirectly, e.g., as described above.

Alternatively, the complex can be dissociated from the matrix and the level of cancer marker binding or activity determined using standard techniques. Other techniques for immobilizing cancer marker proteins or target molecules on a substrate include the use of conjugation of biotin to streptavidin. Biotinylated cancer marker proteins or target molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques known in the art (e.g., biotinylation kit, Pierce chemicals, Rockford, IL) and immobilized in wells of a streptavidin-coated 96-well plate (Pierce Chemical).

For the assay, the non-immobilized component is added to the coated surface containing the anchoring component. After the reaction is complete, unreacted components are removed (e.g., by washing) under various conditions so that any complexes formed will remain immobilized on the solid surface. Detection of complexes anchored to a solid surface can be achieved in a variety of ways. In the case of pre-labelling of previously non-immobilised components, detection of immobilised label on the surface forms a complex. In the case where the previously non-immobilized components are not pre-labeled, indirect labels can be used to detect the complexes anchored on the surface; for example, a labeled antibody specific for the immobilized component is used (in turn, the antibody can be directly labeled or indirectly labeled with, for example, a labeled anti-IgG antibody).

This assay is performed using an antibody that is reactive with the stem cell cancer marker protein or target molecule, but does not interfere with the binding of the stem cell cancer marker protein to its target molecule. Such antibodies can be derivatized to the wells of the plate and unbound target or cancer marker protein immobilized within the wells by antibody conjugation. In addition to the methods described above for GST-immobilized complexes, methods for detecting such complexes include immunodetection of the complexes using antibodies reactive with cancer marker proteins or target molecules, and enzyme-linked assays that rely on detecting enzyme activity associated with cancer marker proteins or target molecules.

Alternatively, cell-free assays can be performed in liquid phase. In such assays, the reaction products are separated from unreacted components by any of a number of standard techniques, including but not limited to: differential centrifugation (see, e.g., Rivas and Minton, Trends Biochem Sci 18: 284-7[1993 ]); chromatography (gel filtration chromatography, ion exchange chromatography); electrophoresis (see, e.g., Current Protocols in Molecular Biology 1999, J.Wiley: New York, et al, Ausubel et al); and immunoprecipitation (see, e.g., Current Protocols in molecular Biology 1999, J.Wiley: New York, authored by Ausubel et al). Such resins and chromatographic techniques are known to those skilled in the art (see, e.g., Heegaard J.mol.Recognit 11: 141-8[1998 ]; Hagel and Tween J.Chromatogr.biomed.Sci.Appl 699: 499-525[1997 ]). Furthermore, as described herein, fluorescence energy transfer can also be conveniently used to detect binding without further purification of the complex from solution.

The assay can include contacting a stem cell cancer marker protein, or a biologically active portion thereof, with a known compound that binds to a cancer marker to form an assay mixture, contacting the assay mixture with a test compound, and assaying the ability of the test compound to interact with the cancer marker protein, wherein assaying the ability of the test compound to interact with the cancer marker protein includes assaying the ability of the test compound to preferentially bind to the cancer marker, or the biologically active portion thereof, or modulate the activity of a target molecule as compared to the known compound.

Inhibitors of such interactions are useful in the sense that a cellular cancer marker can interact with one or more cells or extracellular macromolecules (such as proteins) in vivo. The inhibitors can be identified using the same assay.

For example, a preformed complex of the target gene product and the interacting cellular or extracellular binding partner product is prepared, thereby labeling the target gene product or its binding partner, but the signal generated by the label is quenched due to complex formation (see, e.g., U.S. Pat. No.4,109,496, incorporated herein by reference, which utilizes this method for immunoassays). Addition of a test substance that competes with and displaces one of the species from the preformed complex results in a signal above background. In this way, test substances that disrupt the target gene product-binding partner interaction can be identified. Alternatively, the cancer marker protein may be used as a "decoy protein" in a two-hybrid assay or a three-hybrid assay (see, e.g., U.S. Pat. No.5,283,317; Zervos et al, Cell 72: 223-232[1993 ]; Madura et al, J.biol. chem.268.12046-12054[1993 ]; Bartel et al, Biotechniques 14: 920-924[1993 ]; Iwabuchi et al, Oncogene 8: 1693-1696[1993 ]; and Brent WO 94/10300; each of which is incorporated herein by reference) to identify other proteins that bind to a cancer marker or interact with a cancer marker ("cancer marker-binding protein" or "cancer marker-bp") and participate in cancer marker activity. Such cancer marker-bp may be a signal activator or inhibitor, e.g., as a downstream element of a cancer marker-mediated signal transduction pathway, by a cancer marker protein or target.

Modulators of cancer marker expression may also be identified. For example, a cell or cell-free mixture is contacted with a candidate compound and expression of the cancer marker mRNA or protein is assessed relative to the level of expression of the stem cell cancer marker mRNA or protein in the absence of the candidate compound. Identifying the candidate compound as a stimulator of cancer marker mRNA or protein expression when cancer marker mRNA or protein expression is greater than its deficiency in the presence of the candidate compound. Alternatively, a candidate compound is identified as an inhibitor of cancer marker mRNA or protein expression when the expression of the cancer marker mRNA or protein is less (i.e., statistically significantly less) than its deficiency in the presence of the candidate compound. The level of cancer marker mRNA or protein expression can be determined by the methods described herein for detecting cancer marker mRNA or protein.

The invention further pertains to novel agents identified by the screening assays described above (see, e.g., the description below for cancer therapies). Thus, it is within the scope of the present invention to further use agents identified as described herein (e.g., cancer marker modulators, antisense cancer marker nucleic acid molecules, siRNA molecules, cancer marker-specific antibodies, or cancer marker binding partners) in suitable animal models, such as those described herein, to determine the efficacy, toxicity, side effects, or mechanism of action of treatment with such agents. In addition, new agents identified by the screening assays described above can be used, for example, in treatment as described herein (e.g., to treat a human patient suffering from cancer).

In certain embodiments, the present invention employs non-adherent mammospheres for each screening step, including methods for screening for CXCR1 or FBXO21 signal transduction pathway antagonists. Non-adherent mammospheres are in vitro culture systems that allow proliferation of primary human mammary epithelial stem and progenitor cells in an undifferentiated state, based on their ability to proliferate as spherical structures in suspension. Non-adherent mammospheres were previously described in Dontu et al Genes dev.2003, 5 months and 15 days; 17(10): 1253-70, and Dontu et al, Breast Cancer Res.2004; 6(6): r605-15, both incorporated herein by reference. The entire contents of these references are incorporated by reference, particularly for teaching the construction and use of non-adherent mammospheres. As described by Dontu et al, mammospheres are characterized as being composed of stem and progenitor cells capable of self-renewal and multipotent differentiation. Dontu et al also describe that the mammosphere contains cells capable of asexually propagating in a reconstituted 3-D culture system in matrigel to produce complex functional ductal-alveolar-like structures.

In certain embodiments, the following exemplary screening methods are employed. For in vitro studies, cells can be treated with either an adenovirus construct expressing control or CXCR1 or FBXO21 candidate siRNA (m.o.i. 10 to 100) for 3 days or with a small molecule candidate (e.g., PHA665752 derivative) (0.1-0.5uM) for 3 days and the ability of CXCR1+ or FBXO21+ cells to form tumor spheres is compared in untreated and treated cells. For in vivo studies, human breast cancer cells can be infected with luciferase-expressing lentiviruses to monitor tumor growth. Luciferase-expressing cancer cells can be injected into breast tissue and tumors of about 0.5-0.7cm in size can be established, with 5 animals per group. Animals with established tumors can then be treated with candidate CXCR1 or FBXO21 inhibitors (i.v.30mg/kg/day for 7 days per day) or vehicle controls. Infection with adenovirus expressing control or candidate CXCR1 or FBXO21 siRNA (m.o.i. 100 or 500 for 7 days) can be used to perform parallel studies. Animals can be imaged at day 7,14, 21 and 28 to assess tumor size and then sacrificed. Tumor size can be further assessed at necropsy and a portion of the tumor stained to assess tumor histology. The remaining tumors can be collected and classified to assess the percentage of CXCR1 or FBXO21 positive and CXCR1 or FBXO21 negative cells. To verify that administration of candidate CXCR1 or FBXO21 inhibitors and candidate CXCR1 or FBXO21 siRNA adenovirus infection inhibits CXCR1 or FBXO21 signaling function, phosphorylation of downstream mediators such as Gab-1 and ERK can be examined (see Chistensen et al, Cancer Res., 2003; 63: 7345-7355, incorporated herein by reference).

Cancer therapy

In some embodiments, the invention provides cancer therapies. In some embodiments, the therapy targets cancer markers (e.g., including, but not limited to CXCR1 or FBXO21 and CXCR1 or proteins in the FBXO21 signal transduction pathway). In some embodiments, any known or later developed cancer stem cell therapy may be used. For example, cancer stem cell therapeutics are described in U.S. Pat. Nos. 6, 984, 522 and 7, 115, 360 and applications WO03/050502, WO05/074633 and WO05/005601, which are incorporated herein by reference in their entirety. Antibody therapy

In some embodiments, the invention provides antibodies that target tumors expressing the stem cell cancer markers of the invention. Any suitable antibody (e.g., monoclonal, polyclonal, or synthetic) can be utilized in the therapeutic methods disclosed herein. In some embodiments, the antibody used in cancer therapy is a humanized antibody. Methods for humanizing antibodies are well known in the art (see, e.g., U.S. Pat. Nos. 6,180,370, 5,585,089, 6,054,297, and 5,565,332; each of which is incorporated herein by reference).

In some embodiments, the therapeutic antibody comprises an antibody generated against a stem cell cancer marker of the invention, wherein the antibody is conjugated to a cytotoxic agent. In such embodiments, tumor-specific therapeutic agents are generated that do not target normal cells, thus reducing many of the deleterious side effects of traditional chemotherapy. For certain applications, it is envisioned that a therapeutic agent will be a pharmacological agent that will act as a useful agent for the attachment of antibodies, particularly a cytotoxic or in other cases an anti-cellular agent, that has the ability to kill or inhibit endothelial cell growth or cell division. The present invention contemplates the use of any pharmacological agent that can be conjugated to an antibody and delivered in an active form. Exemplary anti-cellular agents include chemotherapeutic agents, radioisotopes, and cytotoxins. Therapeutic antibodies of the invention can include a variety of cytotoxic moieties, including, but not limited to, radioisotopes (e.g., iodine-131, iodine-123, technetium-99 m, indium-111, rhenium-188, rhenium-186, gallium-67, copper-67, yttrium-90, iodine-125, or astatine-211); hormones, such as steroids; antimetabolites such as cytosine (e.g., arabinoside, fluorouracil, methotrexate or aminopterin; anthracyclines; mitomycin C); vinca alkaloids (e.g., colchicine; etoposide; mithramycin); and antineoplastic alkylating agents, such as chlorambucil or melphalan. Other embodiments may include agents such as clotting agents, cytokines, growth factors, bacterial endotoxins, or lipid a moieties of bacterial endotoxins. For example, in some embodiments, the therapeutic agent will include a plant, fungal or bacterial derived toxin, such as an a-chain toxin, ribosome inactivating protein, α -sarcin, aspergillin, restrictocin, ribonuclease, diphtheria toxin or pseudomonas exotoxin, to name a few. In some embodiments, deglycosylated ricin a chain is utilized.

In any case, it is proposed that agents such as these may be successfully conjugated to antibodies, if desired, in a manner that will allow them to be targeted, internalized, released or presented to blood components at the site of the target tumor cell, using known conjugation techniques as needed (see, e.g., Ghose et al, Methods enzymol., 93: 280[1983 ]).

For example, in some embodiments, the invention provides immunotoxins that target the stem cell cancer markers of the invention. Immunotoxins are conjugates of a specific targeting agent (typically an antibody or fragment directed against a tumor) and a cytotoxic agent (such as a toxin moiety). The targeting agent directs the toxin to the cells carrying the target antigen and thereby selectively kills the cells. In some embodiments, therapeutic antibodies employ cross-linking agents that provide high in vivo stability (Thorpe et al, Cancer Res., 48: 6396[1988 ]).

In other embodiments, particularly those directed to the treatment of solid tumors, the antibodies are designed to have a cytotoxic or anti-cellular effect against the tumor vasculature by inhibiting the growth or cell division of vascular endothelial cells. This attack is intended to cause local vascular collapse (vascular collapse) of the tumor, which deprives the tumor cells, particularly those far from the vascular system, of oxygen and nutrients, ultimately leading to cell death and tumor necrosis.

In some embodiments, the antibody-based therapeutic agent is formulated as a pharmaceutical composition as described below. In some embodiments, administration of an antibody composition of the invention results in a measurable reduction in cancer (e.g., tumor shrinkage or elimination).

Therapeutic compositions and administration

The pharmaceutical compositions containing the modulators of tumorigenesis according to the invention can be administered by any effective method. For example, an IL8-CXCR1 signal transduction pathway antagonist, or other therapeutic agent that acts as an antagonist of a protein in the IL8-CXCR1 signal transduction/response pathway, can be administered by any effective method. In certain embodiments of the invention, the therapeutic agent comprises repteraxin or a derivative thereof.

In certain embodiments, a physiologically appropriate solution containing an effective concentration of an IL8-CXCR1 signal transduction pathway antagonist can be administered topically, intraocularly, parenterally, orally, intranasally, intravenously, intramuscularly, subcutaneously, or by any other effective means. In particular, an IL8-CXCR1 signal transduction pathway antagonist agent can be injected directly into a target cancer or tumor (e.g., into breast tissue) through a needle in an amount effective to treat tumor cells of the target tissue. Alternatively, cancers or tumors present in body cavities such as the eye, gastrointestinal tract, genitourinary tract (e.g., bladder), lung and bronchial system, etc., can receive a physiologically suitable composition (e.g., a sterile solution, such as saline or phosphate buffer; suspension or emulsion) containing an effective concentration of an IL8-CXCR1 signal transduction pathway antagonist via direct injection with a needle or via a catheter or other delivery tube placed in a hollow organ with the cancer or tumor. Any effective imaging device, such as X-ray, sonogram, or fiber optic visualization systems, may be used to locate the target tissue and guide the needle or catheter. In another alternative, a physiologically suitable solution system containing an effective concentration of an IL8-CXCR1 signal transduction pathway antagonist can be administered into the systemic blood circulation to treat cancers or tumors that cannot be directly reached or structurally segregated.

The common goal of such manipulations is to bring the IL8-CXCR1 signaling pathway antagonist into sufficient contact with the target tumor to allow the antagonist to contact, transduce, or transfect the tumor cells (depending on the nature of the agent). In one embodiment, solid tumors present in the epidermal lining (lining) of a hollow organ may be treated by infusing a suspension into the hollow organ filled with a fluid, or by spraying or nebulizing the suspension into the hollow organ filled with air. Thus, tumor cells (such as solid tumor stem cells) may be present in or between the lining of the bronchopulmonary tree, the lining of the gastrointestinal tract, the lining of the female reproductive tract, the genitourinary tract, the bladder, epithelial tissue in the gallbladder and any other organ tissue susceptible to contact with IL8-CXCR1 signal transduction pathway antagonists. In another embodiment, the solid tumor may be located in or on the lining of the central nervous system, such as, for example, the spinal cord, spinal root, or brain, such that perfused IL8-CXCR1 signaling pathway antagonist in cerebrospinal fluid contacts and transduces solid tumor cells in the space. One skilled in the art of oncology will appreciate that an antagonist can be administered to a solid tumor by direct injection into the tumor, such that the antagonist contacts and affects tumor cells within the tumor.

The tumorigenic cells identified by the present invention may also be used to generate anti-cancer cell antibodies. In one embodiment, the method involves obtaining an enriched population of tumorigenic cells or isolated tumorigenic cells; treating the population to prevent cell replication (e.g., by irradiation); and administering the treated cells to a human or animal subject in an amount effective to induce an immune response to the solid tumor stem cells. For guidance on effective dosages of cells to be injected or orally administered, see U.S. Pat. Nos. 6,218,166, 6,207,147, and 6,156,305, which are incorporated herein by reference. In another embodiment, the method involves obtaining an enriched population of solid tumor hepatocytes or isolated solid tumor hepatocytes; mixing the tumor stem cells in vitro culture with immune effector cells from a human subject or host animal to be antibody-raised (according to immunological methods known in the art); removing immune effector cells from the culture; and transplanting the immune effector cells into the host animal at a dose sufficient to stimulate an immune response in the animal.

In some embodiments of the invention, the anti-tumorigenic therapeutic agents of the invention (e.g., IL8-CXCR1 signal transduction pathway antagonists) are co-administered with other anti-tumor therapies. A wide variety of therapeutic agents may be used with the present invention. Any therapeutic agent that can be co-administered with, or combined with, an agent of the invention is suitable for use in the methods of the invention.

A variety of anti-tumor, e.g., anti-cancer) agents are contemplated for use in certain embodiments of the invention. Anticancer agents suitable for use with the present invention include, but are not limited to, agents that induce apoptosis, agents that inhibit adenosine deaminase function, inhibit pyrimidine biosynthesis, inhibit purine ring biosynthesis, inhibit nucleotide interconversions, inhibit ribonucleotide reductase, inhibit Thymidine Monophosphate (TMP) synthesis, inhibit dihydrofolate reduction, inhibit DNA synthesis, adduct formation with DNA, damage DNA, inhibit DNA repair, insert DNA, deaminate asparagine, inhibit RNA synthesis, inhibit protein synthesis or stability, inhibit microtubule formation or function, and the like.

In some embodiments, exemplary anti-cancer agents suitable for use in the compositions and methods of the invention include, but are not limited to: 1) alkaloids including microtubule inhibitors (e.g., vincristine, vinblastine, and vindesine, etc.), microtubule stabilizers (e.g., paclitaxel (TAXOL), and docetaxel, etc.), and chromatin function inhibitors, including topoisomerase inhibitors such as epipodophyllotoxins (e.g., etoposide (VP-16), and teniposide (VM-26), etc.) and agents targeting topoisomerase I (e.g., camptothecin and irinotecan (isirinotecan) (CPT-11), etc.); 2) covalent DNA binding agents (alkylating agents) including nitrogen mustards (e.g., dichloromethyldiethylamine, chlorambucil, cyclophosphamide, ifosfamide, and busulfan (myrenr), etc.), nitrosoureas (e.g., carmustine, lomustine, and semustine, etc.), and other alkylating agents (e.g., dacarbazine, methylolmelamine, thiotepa, and mitomycin, etc.); 3) non-covalent DNA binding agents (antitumor antibiotics) including nucleic acid inhibitors (e.g., dactinomycin (actinomycin D) and the like), anthracyclines (e.g., daunorubicin (daunorubicin and Cerubidine), doxorubicin (adriamycin) and idarubicin (idarubicin) (demethoxydaunorubicin) and the like), anthracenediones (e.g., anthracycline analogs such as mitoxantrone and the like), Bleomycin (BLENOXANE) and the like, and plicamycin (mithramycin) and the like; 4) antimetabolites including antifolates (e.g., methotrexate, FOLEX, and MEXATE, etc.), purine antimetabolites (e.g., 6-mercaptopurine (6-MP, PURINETHOL), 6-thioguanine (6-TG), azathioprine, acyclovir, ganciclovir, chlorodeoxyadenosine, 2-chlorodeoxyadenosine (CdA), and 2' -deoxysynomycin (pentostatin), etc.), pyrimidine antagonists (e.g., fluoropyrimidines (e.g., 5-fluorouracil (ADRUCIL), 5-fluorodeoxyuridine (FdUrd) (floxuridine)), etc.), and cytarabine (e.g., CYTOSAR (ara-C) and fludarabine (fludarabine), etc.); 5) enzymes including L-asparaginase, hydroxyurea, and the like; 6) hormones, including glucocorticoids, antiestrogens (e.g., tamoxifen (tamoxifen), etc.), nonsteroidal antiandrogens (e.g., fludarabine (flutamide), etc.), and aromatase inhibitors (e.g., Anastrozole (ARIMIDEX), etc.); 7) platinum compounds (e.g., cisplatin and carboplatin), and the like); 8) monoclonal antibodies conjugated with anticancer drugs, toxins, and/or radionuclides, and the like; 9) biological response modifiers (e.g., interferons (e.g., IFN-. alpha., etc.) and interleukins (e.g., IL-2, etc.); 10) adoptive immunotherapy; 11) a hematopoietic growth factor; 12) agents that induce tumor cell differentiation (e.g., all-trans retinoic acid, etc.); 13) gene therapy techniques; 14) antisense therapy techniques; 15) a tumor vaccine; 16) therapies directed at tumor metastasis (e.g., batimastat, etc.); 17) an angiogenesis inhibitor; 18) proteosome inhibitors (e.g., VELCADE); 19) inhibitors of acetylation and/or methylation (e.g., HDAC inhibitors); 20) modulators of NF κ B; 21) inhibitors of cell cycle regulation (e.g., CDK inhibitors); 22) modulators of p53 protein function; and 23) radiation.

Any oncolytic agent (oncolytic agent) conventionally used in the context of cancer therapy finds use in the compositions and methods of the invention. For example, the U.S. food and drug administration (u.s.food and drug administration) maintains a prescribed set of oncolytic agents approved for use in the united states. The international corresponding office of u.s.f.d.a. maintains a similar set of prescriptions. Table 3 provides a list of exemplary antineoplastic agents approved for use in the united states. It will be appreciated by those skilled in the art that the "product label" required on all U.S. approved chemotherapeutic agents describes approved indications, dosage information, toxicity data, etc. regarding exemplary agents.

TABLE 3

In the present invention, an antibacterial therapeutic agent can also be used as a therapeutic agent. Any agent capable of killing, inhibiting, or otherwise impairing the function of a microbial organism and any agent expected to have such activity may be used. Antibacterial agents include, but are not limited to, natural and synthetic antibiotics, antibodies, inhibitory proteins (e.g., defensins), antisense nucleic acids, membrane disruptive agents, and the like, used alone or in combination. Virtually any type of antibiotic may be used, including, but not limited to, antibacterial agents, antiviral agents, antifungal agents, and the like.

In still other embodiments, the invention provides compounds of the invention (and any other chemotherapeutic agents) conjugated to a targeting agent capable of specifically targeting a particular cell type (e.g., tumor cell). Generally, a therapeutic compound bound to a targeting agent targets cancer cells via interaction of the targeting agent with a cell surface moiety that is taken into the cell via receptor-mediated endocytosis.

Any moiety known to be located on the surface of a target cell (e.g., a tumor cell) finds use in aspects of the invention. For example, an antibody directed against this moiety targets the composition of the invention to the cell surface containing the moiety. Alternatively, the targeting moiety may be a ligand for a receptor present on the surface of the cell or vice versa. Similarly, vitamins can also be used to target the therapeutic agents of the present invention to specific cells.

As used herein, the term "targeting molecule" refers to a chemical group and portions thereof that can be used to target a therapeutic compound to a target cell, tissue, and organ. Various types of targeting molecules are contemplated for use with the present invention, including but not limited to signal peptides, antibodies, nucleic acids, toxins, and the like. In addition, the targeting moiety can additionally facilitate binding of bound chemical compounds (e.g., small molecules) or facilitate entry of the compounds into target cells, tissues, and organs. Preferably, the targeting moiety is selected for its specificity, affinity and efficacy in selectively delivering the bound compound to a target site within a subject, tissue or cell (including specific subcellular locations and organelles).

Various efficiency issues affect the administration of all drugs and in particular highly cytotoxic drugs (e.g., anticancer drugs). One issue of particular importance is ensuring that the administered agent affects only the target cells (e.g., cancer cells), tissues, or organs. Non-specific or unintended delivery of highly cytotoxic agents to non-targeted cells can cause serious toxicity problems.

Many attempts have been made to design drug targeting protocols to address the problems associated with non-specific drug delivery. (see, e.g., K.N.Syrigos and A.A.Epenetos Anticancer Res., 19: 606-. Conjugation of targeting moieties such as antibodies and ligand peptides (e.g., RDG against endothelial cells) to drug molecules has been used to alleviate some of the attendant toxicity issues associated with particular drugs.

The compound and anticancer agent may be administered in any sterile biocompatible pharmaceutical carrier including, but not limited to, saline, buffered saline, dextrose, and water. In some embodiments, a pharmaceutical composition of the invention may contain an agent (e.g., an antibody). In other embodiments, the pharmaceutical composition contains a mixture of at least two agents (e.g., an antibody and one or more conventional anti-cancer agents). In still other embodiments, the pharmaceutical compositions of the present invention comprise at least two agents that are administered to a patient under one or more of the following conditions: at different periodicities, at different durations, at different concentrations, by different routes of administration, and the like. In some embodiments, the IL8-CXCR1 signaling pathway antagonist is administered prior to the second anticancer agent, e.g., 0.5, 1, 2, 3,4, 5, 10, 12, or 18 hours, 1, 2, 3,4, 5, or 6 days, 1, 2, 3, or 4 weeks prior to administration of the anticancer agent. In some embodiments, the IL8-CXCR1 signaling pathway antagonist is administered after the second anticancer agent, e.g., 0.5, 1, 2, 3,4, 5, 10, 12, or 18 hours, 1, 2, 3,4, 5, or 6 days, 1, 2, 3, or 4 weeks after administration of the anticancer agent. In some embodiments, the IL8-CXCR1 signaling pathway antagonist and the second anticancer agent are administered simultaneously but on a different schedule, e.g., the IL8-CXCR1 signaling pathway antagonist compound is administered daily and the second anticancer agent is administered once a week, once every two weeks, once every three weeks, or once every four weeks. In other embodiments, the IL8-CXCR1 signaling pathway antagonist is administered once a week, while the second anticancer agent is administered daily, once a week, once every two weeks, once every three weeks, or once every four weeks.

Preferred embodiments of the present pharmaceutical compositions are formulated and administered systemically or locally, depending on the condition being treated. Techniques for formulation and administration can be found in the recent version of Remington's Pharmaceutical Sciences (MackPublishing Co, Easton Pa.). Suitable routes may include, for example, oral or transmucosal administration and parenteral delivery (e.g., intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal or intranasal administration).

The present invention contemplates administration of therapeutic agents and, in some embodiments, one or more conventional anti-cancer agents in accordance with acceptable methods of drug delivery and manufacturing techniques. For example, a therapeutic compound and a suitable anti-cancer agent in a pharmaceutically acceptable carrier (such as physiological saline) can be administered intravenously to a subject. Standard methods for intracellular delivery of agents (e.g., via liposome delivery) are contemplated. Such methods are well known to those of ordinary skill in the art.

In some embodiments, the formulations of the present invention may be used for parenteral administration (e.g., intravenous, subcutaneous, intramuscular, intramedullary, and intraperitoneal). Therapeutic co-administration of some contemplated anti-cancer agents (e.g., therapeutic polypeptides) can also be achieved using gene therapy agents and techniques.

In some embodiments of the invention, a therapeutic compound is administered to a subject alone, or in combination with one or more conventional anti-cancer agents (e.g., nucleotide sequences, drugs, hormones, etc.), or as a pharmaceutical composition of the components optionally mixed with excipients or other pharmaceutically acceptable carriers. In a preferred embodiment of the invention, the pharmaceutically acceptable carrier is biologically inert. In a preferred embodiment, the pharmaceutical compositions of the present invention are formulated in dosages suitable for oral administration using pharmaceutically acceptable carriers well known in the art. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, capsules, dragees, liquids, gels, syrups, slurries, solutions, suspensions and the like, for oral or nasal ingestion by a subject, respectively.

Pharmaceutical preparations for oral use can be obtained by combining the active compounds with solid excipients, optionally grinding a resulting mixture, and processing the mixture, after adding suitable auxiliaries, if desired, into granules, to obtain tablets or dragee cores. Suitable excipients are carbohydrate or protein fillers such as sugars, including lactose, sucrose, mannitol or sorbitol; starches derived from corn, wheat, rice, potato, and the like; cellulose such as methyl cellulose, hydroxypropyl methyl cellulose, or sodium carboxymethyl cellulose; gums, including arabic and tragacanth; and proteins such as gelatin and collagen. If desired, disintegrating or solubilizing agents may be added, such as cross-linked polyvinylpyrrolidone, agar, alginic acid or a salt thereof, such as sodium alginate.

In preferred embodiments, the dosage and administration regimen are adapted by the clinician or other person skilled in the art of pharmacology according to well-known pharmacological and therapeutic considerations, including, but not limited to, the level of therapeutic effect desired and the actual level of therapeutic effect achievable. Generally, it is desirable to follow well-known pharmacological principles for administration of chemotherapeutic agents (e.g., it is generally desirable to not change the dose more than 50% over time and not more than every 3-4 agent half-lives). For compositions with relatively little or no dose-related toxicity considerations, and where maximum efficacy (e.g., destruction of cancer cells) is desired, it is not uncommon for the dose to exceed the average required dose. This method for dosing is commonly referred to as a "maximum dose" strategy. In certain embodiments, the subject is administered an IL8-CXCR1 signaling pathway antagonist at a dose of 1-40mg per day (e.g., for 4-6 weeks). In certain embodiments, a 15-70mg loading dose of an IL8-CXCR1 signaling pathway antagonist is administered to a subject. In certain embodiments, a loading dose of about 35-45mg of an IL8-CXCR1 signal transduction pathway antagonist (e.g., subcutaneously) is administered to the subject followed by a daily dose of about 10mg (e.g., subcutaneously) for about 4-6 weeks.

Other dosage considerations relate to calculating an appropriate target level for the agent administered, accumulation and potential toxicity of the agent, irritation of resistance, lack of efficacy, and a range of therapeutic indices describing the agent.

In certain embodiments, the present invention contemplates the use of conventional methods of titrating agent administration. One common administration strategy is to set a reasonable target level for the agent in the subject. In some preferred embodiments, the level of agent is measured in the plasma of the subject. The appropriate dose level and frequency is then designed to achieve the desired steady state target level of the agent. The actual or average level of the agent in the subject is monitored (e.g., hourly, daily, weekly, etc.), so that the dosage level or frequency can be adjusted to maintain the target level. Of course, the pharmacokinetics and pharmacodynamics (e.g., bioavailability, clearance or bioaccumulation, biodistribution, drug interactions, etc.) of the particular agent or agents administered can potentially affect what is considered a reasonable target level, and as such, dose level or frequency.

The target level dosing method generally relies on establishing a reasonable therapeutic objective, which is defined according to the desired range (or therapeutic range) of the agent in the subject. Generally, the lower end of the therapeutic range is approximately equal to the concentration of the agent that provides about 50% of the maximum possible therapeutic effect. The upper limit of the therapeutic range is generally determined by the toxicity of the agent rather than its efficacy. The present invention contemplates that the upper limit of the therapeutic range for a particular agent will be a concentration at which less than 5 or 10% of the subjects exhibit toxic side effects. In some embodiments, the upper limit of the therapeutic range is about two times or less the lower limit. It will be understood by those skilled in the art that these dosage considerations are highly variable and, to some extent, individualized (e.g., based on genetic predisposition, immunological considerations, tolerance, resistance, etc.). Thus, in some embodiments, the effective target dose level of an agent in a particular subject may be 1,. 5,. 10,. 15,. 20,. 50,. 75,. 100,. 200,. X% greater than the optimal value in another subject. Conversely, some subjects may experience significant side effects and toxicity related health problems at dosage levels or frequencies that are much less than those that (1,. 5,. 10.. 15.. 20.. 50.. 75.. 100.. 200.. X%) typically produce optimal therapeutic levels in some or most subjects. In the absence of more specific information, the target administration level is often set in the middle of the treatment range.

In a preferred embodiment, the clinician rationally designs the individualized dosing regimen based on known pharmacological principles and equations. Generally, clinicians design individualized dosing regimens based on knowledge of various pharmacological and pharmacokinetic properties of the agent, including but not limited to F (fractional bioavailability of the dose), Cp (concentration in plasma), CL (clearance/clearance), Vss (volume of drug distribution at steady state), Css (concentration at steady state), and t1/2 (drug half-life), and information about the rate of absorption and distribution of the agent. For further explanation of these variables and the overall equations illustrating the calculation of individualized dosage regimens, those skilled in the art refer to a number of well-known pharmacological textbooks (e.g., Goodman and Gilman, Pharmaceutical Basis of Therapeutics, 10 th edition, Hardman et al, 2001). One skilled in the art would also be able to anticipate possible variations in these variables in individual subjects. For example, standard deviations of the observed values for F, CL and Vss are typically about 20%, 50%, and 30%, respectively. The actual effect of the widely varying parameters possible in individual subjects was that 95% of the subjects reached Css and between 35 and 270% of the target level. For drugs with low therapeutic indices, this is an undesirably wide range. However, it will be appreciated by those skilled in the art that once the Cp (concentration in plasma) of the agent is measured, it is possible to directly estimate the values of F, CL and Vss. This allows the clinician to effectively fine-tune the dosage regimen for a particular subject.

In still other embodiments, the present invention contemplates using continuous therapeutic drug monitoring techniques to further adjust the dosage methods and regimens of an individual. For example, in one embodiment, the Css data is used to further refine the estimation of CL/F, and then the individual's maintenance dose is adjusted using known pharmacological principles and equations to achieve the desired target level of the agent. In fact, the therapeutic monitoring can be performed at any time during the dosage schedule. In preferred embodiments, the detection is performed at multiple time points during the dosage process, and in particular when intermittent doses are administered. For example, regardless of the dosage method employed, drug monitoring (e.g., intermittent dose, loading dose, maintenance dose, random dose, or any other dosage method) can be concomitantly performed within a fraction of a second, seconds, minutes, hours, days, weeks, months, etc., of administration of the agent. However, one skilled in the art will appreciate that changes in the effectiveness and kinetics of a pharmaceutical agent may not be readily observed when a sample is taken quickly after administration of the agent, as changes in plasma concentration of the agent may be delayed (e.g., due to a slow rate of distribution or other pharmacodynamic factors). Thus, a subject sample obtained immediately after administration of the agent may obtain limited or reduced values.

The primary purpose of collecting a biological sample from a subject during a predicted steady state target level of administration is to modify the dosage regimen of the individual based on subsequently calculating a revised estimate of the CL/F ratio of the agent. However, it will be appreciated by those skilled in the art that the drug concentration does not generally reflect drug clearance after early absorption. The early post-absorption drug concentration is primarily dictated by the rate of absorption of the agent, the central (rather than steady-state) volume of agent distribution, and the rate of distribution. Each of these pharmacokinetic profiles has limited values when calculating a therapeutic long-term maintenance dosage regimen.

Thus, in some embodiments, when the objective is a therapeutic long-term maintenance dose, a biological sample is obtained from the target subject, cell or tissue long after a previous dose has been administered, and even more preferably shortly before the next planned dose is administered.

In yet another embodiment, where the therapeutic agent is almost completely cleared by the subject in the time interval between doses, then the present invention contemplates taking biological samples from the subject at various time points after the last administration, and more preferably shortly after administration of the dose.

Resetaxin and other small molecule CXCR1 inhibitors

In certain embodiments, the methods, kits, and compositions of the present invention employ small molecule inhibitors of CXCR 1. One exemplary agent is Repartaxin. In certain embodiments, the in vivo dose of retataxin is between 3 and 60mg per kilogram (e.g., 3.. 30.. 50.. 60 mg/kg). In a specific embodiment, the dose of Repeataxin is about 30mg per kilogram. The formula of Repartaxin is shown below:

in other embodiments, derivatives of reportaxin are employed. Other small molecule CXCR1 antagonists include SB265610(Glaxo SmithKline Beecham; Benson et al 2000, 151: 196-) 197) and SCH 527123 (2-hydroxy-N, N-dimethyl-3- {2- [ [ (R) -1- (5-methylfuran-2-yl) propyl ] amino ] -3, 4-dioxan-1-enylamino } benzamide (SCH 527123), orally bioavailable CXCR2/CXCR1 receptor antagonists (ScheringPlough)). Other small molecule inhibitors can be identified by the screening methods described above.

Examples

The following examples are provided to demonstrate and further illustrate certain preferred embodiments and aspects of the present invention and should not be construed as limiting the scope thereof.

Example 1

Identification of cancer stem cells by CXCR1

This example describes the identification of CXCR1 and other proteins (e.g., FBXO21) as cancer stem cell markers.

And (5) culturing the cells. Mammary cell lines (BCL) were obtained from ATCC ("http:// WWW. lgcpromochem-ATCC. com/common/category/cell biology index. cf. m") or from S.Ethier (now available on "http:// WWW. asterand. com/asterand/BIORSITORY/hbrastencearcelllines. aspx"), SUM44, SUM52, SUM149, SUM159, SUM185, SUM190, SUM225, SUM229), V.J.(BrCa-MZ-01) and V.Catros (S68) the set developed in the laboratory by PhD. All BCL tested were derived from carcinoma, except MCF10A (which was derived from fibrocystic disease) and HMEC-derived 184a1 (which was derived from normal breast tissue). The cell lines were cultured using the recommended culture conditions. All experiments were performed with confluent cells in exponential growth phase.

ALDEFLUOR assay and ALDH positive populations were isolated by FACS. ALDH activity was assessed in 33 BCL representing the major molecular subtype of human breast cancer. The ALDEFLUOR kit (StemCell technologies, Durham, NC, USA) was used to isolate populations with high ALDH enzyme activity (17). Cells obtained from trypsinized subconfluent cell lines or from freshly dissociated xenografts were plated in a medium containing ALDH substrate (BAAA, per 1 × 10)⁶1. mu. mol/l per cell) in ALDELUOR assay buffer and incubated at 37 ℃ for 40 minutes. In each experiment, cell samples were stained under the same conditions with 50mmol/L Diethylaminobenzaldehyde (DEAB), a specific ALDH inhibitor, as a negative control. Flow cytometry sorting was performed using facstar plus (becton dickinson). ALDELUOR fluorescence was excited at 488nm and detected using a standard Fluorescein Isothiocyanate (FITC)530/30 band pass filter. For xenografted tumors, cells of mouse origin were eliminated by incubation with anti-H2 Kd antibody (BD biosciences, 1/200, 20 min on ice) followed by incubation with a secondary antibody labeled with phycoerythrin (Jackson labs, 1/250, 20 min on ice). Classification gates were established using PI-stained cells, ALDEFLUOR-stained cells treated with DEAB, and those stained with secondary antibody only, in terms of viability. The purity of the sorted population was checked using a double sort of 10,000 ALDELUOR positive and negative cells in BrCa-MZ-01 and SUM159 cell lines prior to RNA profiling or NOD/SCID mouse injection. The sorted aldeluor positive population contained more than 98% aldeluor positive cells for both cell lines, whereas aldeluor positive cells were not detected in the aldeluor negative population.

Tumorigenicity in NOD/SCID mice. ALDELFUOR positive, negative and unseparated SUM159, MDA-MB-453 and BrCa-MZ-01 cells were evaluated for tumorigenicity in NOD/SCID mice. Fat pads were de-epithelialized at 3 weeks of early puberty and humanized by injection of human fibroblasts (1: 1 irradiated: non-irradiated, 50,000 cells/100 μ Ι matrigel/fat pad) as described (17). When the tumor had a maximum diameter of 1.2cm, the animals were euthanized according to the regulations for use of vertebrate animal research in research. A portion of each fat pad was fixed in formalin and embedded in paraffin for histological analysis. Another fraction was evaluated by ALDEFLUOR assay followed by sorting and serial transplantation.

Anchorage independent culture. ALDELUOR positive, negative, and non-detached cells from 184A1, SUM149, and SUM159 were plated as single cells at low density (5000 viable cells/ml) in ultra-low attachment plates (Corning, Acton, MA). Cells were cultured for 3-7 days in serum-free mammary epithelial basal medium (Cambrex Bio Science, walker ville, MD) as described (18). The capacity of cells to form spheres was quantified after treatment with different doses of IL8(GenWay Biotech, san diego, CA) added to the medium.

And (4) extracting RNA. Total RNA was extracted from frozen ALDEFLUOR positive and negative cells using The DNA/RNA All Prep Maxi kit according to The manufacturer's instructions (Qiagen, Sampleand Assay technologies, The Netherlands). Transcription analysis was performed using 8 BCL: 184A1, BrCa-MZ-01, HCC1954, MDA-MB-231, MDA-MB-453, SK-BR-7, SUM149, and SUM 159. RNA integrity was controlled by denaturing formaldehyde agarose gel electrophoresis and microanalysis (agilent bioanalyzer, Palo Alto, CA).

Gene expression profiling with DNA microarrays. Gene expression analysis using Affymetrix U133 plus2.0 human oligonucleotide microarrays containing over 47,000 transcripts and variants, including 38,500 well-characterized human genes. Preparation, hybridization, washing and detection of cRNA were performed as recommended by the supplier (http:// WWW. affymetrix. com/index. affx). Expression data was analyzed by RMA (robust multi-chip Average) method in R using Bioconductor and related package (19) as described (20, 21). RMA performed background adjustment, quantile normalization and a summary of 11 oligonucleotides per gene.

Prior to analysis, the filtering method removed genes from the dataset that had low and poorly measured expression, as defined by expression values next to 100 units in all 16 samples, which retained 25,285 genes/EST. Unsupervised analysis was performed using a second filter, which was based on the intensity of Standard Deviation (SD) to exclude genes that showed low expression changes between analyses. SD was calculated for log2 transformed data, with 13,550 genes/EST remaining with SD better than 0.5, first pressing the minimum value down to 100 units of the minimum value, i.e. background intensity. Unsupervised analysis was performed on 16 ALDEFLUOR positive, negative cells on 13,550 genes. Prior to hierarchical clustering, the filtered data was log2 transformed and submitted to a clustering program (22) which performed using median-centered data on genes, Pearson correlations as similarity measures and centroid linkage clustering. The results are presented using the TreeView program (22). To identify and rank the genes that distinguish the ALDELUOR positive and negative groups, the Mann and Whitney U tests were applied to 25,285 genes/ESTs and the multiple test hypotheses were corrected using false discovery rate (FDR, (23). samples were classified by hierarchical clustering to account for the classifier's label's classification rights (classificationpower). LOOCV was applied to assess the predictive accuracy of the identified molecular labels and the effectiveness of the supervised analysis, each sample was excluded one by one and classified using the linear discriminant analysis (LDA, (24) using the model defined on the non-excluded samples.

Real-time RT-PCR. After sorting ALDELUOR positive and ALDELUOR negative populations from different cell lines, total RNA was isolated using RNeasy Mini kit (QIAGEN) and used in ABI with 384 well panels and automated adjuncts7900 the real-time quantitative RT-PCR (qRT-PCR) assay was performed in the HT sequence detection System (Applied Biosystems). Primers and probes for the Taqman system were selected from the Applied Biosystems website. The sequences of PCR primer pairs and fluorogenic probes for CXCR1, FBXO21, NFYA, NOTCH2, RAD51L1 and TBP were available on the applied biosystems website (CXCR1 assay ID: Hs _00174146_ mi; FBXO21 assay ID: Hs _00372141_ mi; NFYA assay ID: Hs _00953589_ mi; NOTCH2 assay ID: Hs _01050719_ mi; RAD51L1 assay ID: hs00172522_ mi, TBP assay ID: hs _00427620_ mi). Relative expressed mRNA levels of CXCR1, FBXO21, NFYA, NOTCH2, RAD51L1 were calculated relative to internal standard TBP genes to normalize for changes in RNA quality and input cDNA amount as previously described (25).

And (4) determining invasion. Assays were performed in triplicate in a Transwell chamber with 8um well polycarbonate filter inserts for 12 well plates (Corning, NY). The filters were coated with 30ul ice cold 1: 6 basement membrane extract (matrigel, BD-Bioscience) in DMEM/F12, which was incubated at 37 ℃ for 1 hour. Cells were added to the upper chamber in 200ul serum-free medium. For the invasion assay, 5000 cells were seeded on matrigel-coated filters and the lower chamber was filled with 600ul of medium supplemented with 10% human serum (Cambrex) or 600ul of serum-free medium supplemented with IL8(100 ng/mL). After 48 hours of incubation, the cells on the underside of the filter were counted using light microscopy. Under serum conditions, relative invasion was normalized to the corresponding cell line without isolation.

Lentivirus infection. For luciferase gene transduction, 70% confluent cells from HCC1954, MDA-MB-453 and SUM159 were incubated overnight with a 1: 3 precipitated lentivirus supernatant Lenti-LUC-VSVG (Vector Core, AnnArbor, MI) mixture in culture medium. The following day, cells were harvested by trypsin/EDTA and subcultured at a 1: 6 ratio. After 1 week of incubation, cells were sorted according to the ALDEFLUOR phenotype and luciferase expression was verified in each sort population (ALDEFLUOR positive and ALDEFLUOR negative) by adding 2ml of D-luciferin 0.0003% to the culture medium (Promega, Madison, WI) and calculating the photon flux by a device camera system (Xenogen, Alameda, CA).

Intracardiac vaccination. 6-week-old NOD/SCID mice were anesthetized with a 2% isoflurane/air mixture and injected with 100 μ L of Ca-deficient mice in the left ventricle of the heart²⁺And Mg²⁺100,000 cells in sterile Dulbecco's PBS. For each of the three cell lines (HCC1954, MDA-MB-453, SUM159) and for each population (ALDELUOR positive, ALDELUOR negative and unsorted), notes were madeThree animals were injected.

And (4) detecting bioluminescence. Initial bioluminescence was assessed before and weekly after inoculation. Mice were anesthetized with a 2% isoflurane/air mixture and given i.p. a single dose of 150 mg/kgD-luciferin (Promega, Madison, WI) in PBS. The animals were then anesthetized again 6 minutes after D-luciferin administration. For photon flux counting, a charge coupled device camera system (Xenogen, Alameda, CA) was used with a nose cone isoflurane delivery system and a heating stage for maintaining body temperature. Results were analyzed after 2 to 12 minutes exposure using the Living Image software with Xenogen imaging system. The signal strength is quantified as the sum of all detected photon flux counts within a target coincidence zone manually placed during data post-processing. The normalized photon flux represents the ratio of the photon flux detected weekly after inoculation to the photon flux detected before inoculation.

And (5) carrying out statistical analysis. Results are presented as mean ± SD of individual experiments for at least three replicates of each group. Statistical analysis used SPSS software (version 10.0.5). Correlations between the sample set and the molecular parameters were calculated using Fisher's exact test or one-way ANOVA on independent samples. The p value 0.05 was considered significant.

Most breast cell lines contain an ALDELUOR positive population. CSCs were isolated from 33 BCLs representing diverse molecular subtypes and characteristics of breast cancer using the ALDEFLUOR assay (17) (20). It was found that 23 of the 33 cell lines contained an ALDEFLUOR positive population ranging from 0.2 to almost 100%. All 16 basal/mesenchymal BCLs contained ALDEFLUOR positive populations, while 7 of 12 luminal BCLs did not contain any detectable ALDEFLUOR positive cells (p ═ 0.0006, Fischer exact test).

ALDEFLUROR-positive cells have tumor sphere-forming ability. It has previously been reported that mammary epithelial stem and progenitor cells are able to survive and proliferate in anchorage-independent conditions and form floating spherical colonies called mammospheres (18). Data from breast tumors and cell lines have demonstrated that cancer stem cell-like cells or cancer starting cells can also be isolated and propagated as "tumor balls" in a similar assay (26). All mammosphere initiating cells in normal human mammary glands are contained within the ALDEFLUOR positive population (17). To characterize the alfeluor positive population from BCL, alfeluor positive and negative populations from 184a1, SUM149, and SUM159 were compared for their ability to form tumor spheres. In each cell line, the aldeluor positive population showed increased tumor sphere forming ability compared to aldeluor negative cells.

ALDEFLUOR-positive BCL cells have cancer stem cell characteristics in vivo. To determine the hierarchical organization of BCL, the ALDELUOR positive and negative populations of MDA-MB-453, SUM159 and BrCa-MZ-01 cell lines were analyzed for stem cell characteristics. The ALDELUOR positive population of these three BCLs constituted between 3.54 + -1.73% and 5.49 + -3.36% of the total cell population (FIGS. 1A-B, G-H; FIGS. 2A-B). As shown in fig. 1F, L, the size and latency of tumor formation correlated with the number of alfelulor positive cells injected. Significantly, 500 ALDEFLUROR-positive cells from MDA-MB-453 and 1,000 ALDEFLUROR-positive cells from SUM159 were able to form tumors. The tumorigenic capacity was maintained during serial passages, indicating the self-renewal capacity of these cells. In contrast, ALDEFLUOR-negative cells were unable to produce tumors, although limited growth occurred upon injection of 50,000 ALDEFLUOR-negative MDA-MB-453 cells. H & E staining of fat pad sections confirmed that tumors formed by ALDEFLUOR positive cells contained malignant cells, while only residual matrigel, apoptotic cells and mouse tissue were seen at the ALDEFLUOR negative cell injection sites (fig. 1E, K). Consistent with the alfeluor positive population having characteristics of cancer stem cells, tumors formed from this population recapitulate the phenotypic heterogeneity of the original tumor at similar alfeluor positive and negative cell rates (fig. 1C, I). This indicates that the ALDEFLUOR-positive cells are capable of self-renewal, producing ALDEFLUOR-positive cells, and are capable of differentiation, producing ALDEFLUOR-negative cells.

When BrCa-MZ-01 cells were split into ALDELUOR positive and negative fractions, both were able to generate tumors. Tumors generated from the ALDEFLUOR positive population consist of ALDEFLUOR positive and negative cells that recapitulate the phenotypic heterogeneity of the original tumor. In contrast, tumors produced by aldeluor-negative cells cause slowly growing tumors, which contain only aldeluor-negative cells. Serial passage of aldeluor-negative tumors resulted in reduced tumor growth, in contrast to the capacity of aldeluor-positive cells to be serially transplanted, with no growth after three passages. This suggests that the ALDEFLUOR-positive component of BrCa-MZ-01 cells contains cells with stem cell characteristics, whereas the ALDEFLUOR-negative cells contain progenitor cells that are capable of undergoing limited growth but are not capable of self-renewal.

Gene expression profiles of ALDELFUOR positive and negative cell populations. To determine whether ALDEFLUOR-positive cells isolated from different BCLs express a common set of "cancer stem cell" genes, ALDEFLUOR-positive and negative cell populations isolated from 8 BCLs (184A1, BrCa-MZ-01, HCC1954, MDA-MB-231, MDA-MB-453, SK-BR-7, SUM49, and SUM159) were analyzed using Affymetrix whole genome oligonucleotide microarrays. Unsupervised hierarchical clustering (which was applied to 16 samples and 13,550 filtered genes/ESTs) did not separate the ALDEFLUOR positive and negative populations. Instead, ALDEFLUOR positive and negative populations are clustered together with the parental cell line. This suggests that differences in mRNA transcripts between clonal cell lines replace differences between aldefluoror-positive and aldefluoror-negative cells. This further suggests that only a limited number of genes are differentially expressed between the putative cancer stem cell and its progeny.

To determine which genes discriminate between ALDEFLUOR positive and negative populations, the Mann and Whitney U tests were applied to all genes except those with low and poorly measured expression, i.e., 25,285 probe sets. This test identifies and grades 413 genes/ESTs that distinguish ALDEFULUOR positive and negative cell populations after FDR correction. The 28 overexpressed genes corresponding to the unique genes are shown in table 1, while the most frequently underexpressed genes are shown in table 2.

TABLE 1 Up-regulated genes

TABLE 2 Down-regulated genes

The classification weight of this discriminative signature was demonstrated by classifying 16 ALDELUOR positive and negative samples with 413 differentially expressed genes/ESTs. Hierarchical clustering ranked 15 of the 16 samples (fig. 2A).

Many genes known to play a role in stem cell biology are upregulated in the aldefluoror positive population (table 1), including NFYA, NOTCH2, PCNX, RBM15, ST3GAL3, and TPRXL. Other genes encode proteins with putative or uncharacterized effects on stem cell function, such as ARID1B, RAD51L1, and chemokine receptor CXCR1/IL8RA (27). Genes under-expressed in the ALDEFLUOR positive population are involved in cell differentiation, apoptosis, RNA splicing, and mitochondrial metabolism.

To increase the stringency of the analysis, the threshold of the Mann and Whitney analyses was increased to risk 0.5 and a list of 49 genes/ESTs (genes with asterisks in tables 1-2) that distinguished the positive and negative populations of ALDELFLUOR was obtained. From this list, all ALDEFLUOR positive cells (except SK-BR-7) were pooled together. Of these 49 genes/ESTS, 45 correspond to the unique genes identified; only 3 of these 45 were overexpressed in the ALDEFLUOR positive group, while 42 were underexpressed. The characterized overexpressed genes encoded the F-box proteins FBXO21 and CXCR1/IL8 RA. Underexpressed genes include those encoding mitochondrial proteins (MRPL41, MRPL42, MRPL47, MRPL54, MRPS23, IMMP1L) and differentiation (NACA) and pre-mRNA splicing factors (LSM3, pre-mRNA processing factors PRPF39 and PRPF 4B). Leave-one-out cross-validation (LOOCV), with a risk of 0.5%, assesses the predictive accuracy of the identifier molecular signature and predicts 88% of samples in the correct species with this "cancer stem cell signature", confirming the supervised analysis.

Quantitative RT-PCR assessment confirmed a significant increase in CXCR1 and FBXO21 in ALDEFLUOR positive cells. Quantitative RT-PCR analysis was performed on 5 discriminatory genes (CXCR1/IL8RA, FBXO21, NFYA, NOTCH2, and RAD51L1) that were overexpressed in the ALDEFLUOR positive population. The 3 cell lines used in the profiling (BrCa-MZ-01, MDA-MB-453, SUM159) and two additional luminal cell lines (MCF7, S68) were sorted by ALDELUOR assay and ALDELUOR positive and negative populations were treated separately for quantitative RT-PCR analysis. Quantitative RT-PCR expression levels of CXCR1 and FBXO21 are presented in fig. 2B and C. The results obtained using the DNA microarray were confirmed by quantitative RT-PCR determined gene expression levels, where CXCR1 and FBXO21mRNA levels were elevated in the ALDEFLUOR positive population compared to the ALDEFLUOR negative population (p < 0.05).

IL8 promotes cancer stem cell self-renewal. Profiling studies suggest that the IL8 receptor CXCR1/IL8RA is consistently expressed in ALDELUOR positive cell populations. To confirm this association, protein expression of CXCR1/IL8RA was measured by flow cytometry in aldeluor positive and negative populations. ALDEFLUOR positive and negative populations from 4 different cell lines were isolated by FACS, fixed and stained with phycoerythrin-labeled CXCR1 monoclonal antibody. As shown in fig. 3A, aldeluor-positive cells were highly enriched in CXCR 1-positive cells compared to the aldeluor-negative population.

To determine whether IL8 signaling is important in stem cell function, 4 BCLs were treated with human recombinant IL8 to determine their effect on cancer stem cell populations as measured by tumor sphere formation and by ALDH enzyme activity. As shown in fig. 3B, the addition of IL8 increased the formation of primary and secondary tumor spheres in a dose-dependent manner. In addition, IL8 increased the aldeluor positive population in a dose-dependent manner in each of the 4 BCL analyzed (fig. 3C). This demonstrates the ability of the "CSC tag" to identify pathways that can play a role in stem cell function.

The IL8/CXCR1 axis is involved in cancer stem cell invasion. The IL8/CXCR1 axis has been reported to play a role in cancer stem cell invasion (28, 29). The invasion capacity of ALDELUOR positive and negative cell populations from three different cell lines (HCC1954, MDA-MB-453, SUM159) was examined using matrigel invasion assay using serum as a decoy. As shown in fig. 4A, it was shown that aldeluor-positive cell invasion was 6 to 20 times higher (p < 0.01) by matrigel than aldeluor-negative population. When used as a chemoattractant, IL8(100ng/ml) increased the invasion of ALDEFLUOR-positive cells (p < 0.05) (FIG. 4A). In contrast to its effect on ALDEFLUOR-positive cells, IL8 had no effect on the invasive capacity of ALDEFLUOR-negative cells. These results indicate that cancer stem cells exhibit invasive behavior and that IL8 promotes this process.

ALDEFLUOR-positive cells have increased metastatic potential. CSCs have been proposed to play a crucial role in cancer metastasis (30, 31). The above experiments demonstrate that ALDELUOR-positive cells have an increased invasive capacity compared to ALDELUOR-negative cells. To determine the relationship between ALDEFLUOR positivity and metastatic capacity, HCC1954, MDA-MB-453 and SUM159 were infected with the luciferase lentivirus reporter system. Luciferase-infected cells were sorted using the ALDEFUOR assay and introduced into NOD/SCID mice by intracardiac injection. A suspension of 100,000 cells from each population was injected and metastasis was assessed by bioluminescent imaging. Mice inoculated with ALDEFLUOR positive cells developed metastases at different sites, and mice inoculated with ALDEFLUOR positive cells exhibited higher photon flux emissions than mice inoculated with non-detached cells (no more than one metastasis per mouse) or mice inoculated with ALDEFLUOR negative cells (which only developed sporadic metastases limited to lymph nodes) (FIGS. 4B-J). Histological sections confirmed the presence of metastases at these sites (FIG. 4K-M). Thus, the metastatic capacity of BCL is mainly mediated by CSCs contained in the ALDEFLUOR positive population.

The hypothesis that tumors are organized in CSC-driven cell divisions has fundamental implications for cancer biology and clinical implications for early detection, prevention and treatment of cancer. Evidence for CSC depends largely on the initial and early passage xenograft models (32-34). However, the success rate of establishing breast tumor xenografts is still low, especially for certain molecular subtypes. In contrast to primary tumors, cell lines are available in unlimited amounts and provide only a neoplastic population of cancer for molecular analysis, without normal tissue and stroma. In breast cancer, a large number of immortalized cell lines have been generated, representing different molecular subtypes found in primary human breast cancer (2, 20). However, the fundamental question of how closely these cell lines can replicate the biology of human breast cancer remains.

In vivo evidence of stem cells in cell lines. Recent studies have suggested that although cell lines can be derived by cloning, they contain cell fractions representing different stages of cell differentiation. Several studies have used markers (such as CD44+/CD24-) to identify CSCs within breast cancer cell lines. However, its utility is limited by the observation that a large percentage of cells within a cell line often express these putative stem cell markers. For example, greater than 90% of cells in the basal breast cancer cell line exhibit the CD44+/CD 24-phenotype. Indeed, the CD44+/CD 24-phenotype did not isolate a tumorigenic population of these Cell lines (Ginestier et al Cell stem Cell 1: 555-567, the contents of which are incorporated by reference in their entirety). An alternative approach has been to use SPs from cell lines. However, functional studies using Hoechst staining are limited by the toxicity of this agent (35). There was also evidence of lack of functional stem cell activity within the SP (36). ALDH activity assessed by the ALDEFLUOR assay cells with stem cell characteristics were isolated from various cancers (14, 37). In this example, 23 of 33 BCL (primarily basal cell lines) were demonstrated to contain aldelulor positive populations. The absence of the aldeluor positive population in some luminal BCLs may indicate that these luminal BCLs originate from aldeluor negative progenitor cells.

This example uses an in vivo assay in NOD/SCID mice to demonstrate the stem cell characteristics of the alfelulor positive population. Self-renewal was demonstrated by serial passage in NOD/SCID mice, while differentiation was demonstrated by the ability of aldeluor-positive, but not aldeluor-negative, cells to regenerate cellular heterogeneity of the original tumor.

Breast cancer stem cell signature. Using 8 breast cell lines, this example identified 413 genes whose expression distinguished ALDEFLUOR positive and negative cells. This tag contains a number of genes known to play a role in stem cell biology. Genes overexpressed in the ALDEFLUOR positive population include Notch homolog 2(Notch2), which regulates self-renewal and differentiation of mammary stem cells (18, 38), NFYA, which is known to regulate self-renewal and differentiation of stem cells (39, 40), pecanex homolog PCNX, RBM15/OTT, which exerts pleiotropic effects in hematopoietic stem cells (41) and affects myeloid differentiation via Notch signal transduction (42), homeobox-like factors TPRXL, ST3GAL3, which encode stage-specific embryonic antigen-4 synthases involved in embryonic development and renal and gastric carcinogenesis (43). Notably, the stage-specific embryonic antigen-4 protein (SSEA-4) is expressed in stem cell populations such as CXCR4+/CD133+/CD34 +/lin-stem cells and quiescent breast stem cells in human cord blood (44).

Genes under-expressed in the ALDEFLUOR positive population are involved in cell differentiation, apoptosis and mitochondrial oxidation. They include genes encoding the following proteins: the complex alpha subunits NACA, apoptosis proteins PDCD5 and PDCD10, mitochondrial ribosomal protein L41(MRPL41) which induces apoptosis via BCL2 and caspase via P53-dependent and independent modes, and proteins involved in mitochondrial processing such as oxidative phosphorylation in mitochondria (NDUFA2, ATP5J2, IMMP1L) and protein synthesis (MRPL42, MRPL47, MRPL54, MRPS23) related to nascent polypeptides. Down-regulation of apoptotic genes in CSCs may play a role in the resistance of these cells to radiation and chemotherapy (45, 46). ALDH1a1 was not identified as a differentially expressed gene in the ALDEFLUOR positive signature. However, examination of the gene expression profiles of individual BCLs revealed that while some showed differential expression of ALDH1a 1in the ALDEFLUOR positive population, others showed differential expression of ALDH1A3 (i.e., a different ALDH isoform) in this population. This suggests that expression of different ALDH isoforms may contribute to the ALDEFLUOR positive phenotype.

From chemokines to "stem cell factor (stemokine)". Expression of CXCR1, an IL8 receptor, is elevated in a variety of cancers (47-50). Although IL8 expression is associated with ER negative breast cancer (51), this chemokine has not previously been reported to play a role in stem cell function. Its implications in regulating growth and metastasis are well established in androgen-dependent prostate cancer (52). Furthermore, the expression level of IL8 is associated with tumorigenicity and metastasis via VEGF production and angiogenesis (53, 54). Gene expression data was validated in three ways. First, quantitative RT-PCR analysis confirmed a significant elevation of CXCR1mRNA in the aldeluor positive population from cell lines both included and excluded from profiling. Second, using flow cytometry, it was demonstrated that CXCR 1-containing cells were found only within the ALDEFLUOR positive population. Third, recombinant IL8 increased mammosphere formation and the percentage of alfelulor positive cells in BCL. Thus, the IL8/CXCR1 axis appears to regulate breast stem cell proliferation or self-renewal. Because endothelial and stromal cells secrete IL8, this chemokine appears to play a role in mediating the interaction between tumor stem cells and the tumor microenvironment.

Recent studies have suggested a role for interleukins/chemokines in the regulation of CSCs (55, 56). This includes the role of IL6 in breast CSCs and IL4 in mediating chemoresistance in colon CSCs (56-59). These factors may be involved in the association between inflammation and cancer. This also includes CCL5(RANTES), a role of a chemokine secreted by mesenchymal stem cells, which acts as a paracrine molecule and enhances breast cancer cell motility, invasion and metastasis (55).

The root of the transfer. CSCs may be responsible for mediating tumor metastasis. The link between CSCs and metastases was first suggested by identifying the stem cell genes among the 11 gene signatures generated using a transgenic mouse model of prostate cancer and a comparative profile of metastatic and primary tumors in cancer patients (60). This signature is also a powerful predictor of disease recurrence, post-treatment death, and distant metastasis in a variety of cancer types. This example has demonstrated that ALDEFLUOR positive cells are more metastatic than ALDEFLUOR negative cells, and that IL8 (previously reported to play a role in tumor metastasis) promotes the invasion and chemotaxis of cancer stem cells that preferentially express the IL8 receptor CXCR 1. The ability to isolate metastatic cancer stem cells from cell lines should facilitate the study of the molecular mechanisms by which cancer stem cells mediate tumor metastasis.

Example 2

CXCR1 inhibition and combination therapy

This example describes various methods for testing the effect of CXCR1 inhibition on tumor cells, as well as CXCR1 inhibition in combination with an anti-mitotic agent (docetaxel).

CXCR1 inhibited the effect of aldeluor positive population on cell growth and on SUM159 cell line.

SUM159 cell lines were cultured in adherent conditions and cells were treated with either CXCR1/CXCR2 inhibitor Repertaxin or two blocking antibodies specific for CXCR1 or CXCR 2. After 4 days of treatment, the MTT assay was used to analyze the effect on cell growth (fig. 5A) and the aldeluor assay was used to analyze the effect on cancer stem cell populations (fig. 5B). More than 95% inhibition of cell growth was observed in cells treated with either Repertaxin or CXCR1 blocking antibodies, while no effect was observed on cells treated with CXCR2 blocking antibodies (fig. 5A). Interestingly, a similar effect was observed on the ALDEFLUOR positive population, where the ALDEFLUOR positive population was reduced by 80% and 50% in cells treated with reportaxin and CXCR1 blocking antibodies, respectively (fig. 5B).

Reertaxin treatment induces bystander effects mediated by FAS/FAS ligand signaling

SUM159 cell line cells were cultured in adherent conditions and then treated with Resertaxin alone or in combination with a FAS antagonist. Interestingly, the cell growth inhibition induced by the reportaxin treatment was partially rescued by the addition of FAS antagonist (anti/FAS ligand from BD pharmingen (catalog No. 556371)). Furthermore, cells treated with FAS agonist showed similar inhibition of cell growth as cells treated with repotaxin. These results indicate that Repertaxin treatment induces bystander effects mediated by FAS/FAS ligand signaling.

Effect of Repertaxin treatment on FAK, AKT and FOXOA3 activation.

To evaluate the effect of Repertaxin treatment on CXCR1 downstream signaling, SUM159 cells were cultured in adherent conditions in the absence or presence of 100nM Repertaxin over a2 day period and stained by immunofluorescence with antibodies against p-FAK, p-AKT, and FOXOA 3. In untreated cells (FIG. 7A), 30% of the cells expressing p-FAK and 10% of the cells expressing p-AKT showed inactivation, while the cells treated with Repetaxin showed complete inactivation of p-FAK and p-AKT (FIG. 7B). Untreated SUM159 cells showed FOXOA3 positive in the cytoplasm of 80% of the cells. Interestingly, SUM159 cells treated with reportaxin showed positive FOXOA3 in the nucleus of 80% of the cells. Changes in FOXOA3 cellular localization from cytosolic to nuclear indicate activation of FOXOA3 protein.

Tumor growth curves after treatment with Resertaxin, docetaxel, or combination

The effect of Reertaxin, docetaxel or a combination thereof was evaluated using one breast cancer cell line (8A, SUM159) and three human breast cancer xenografts generated from different patients (8B, MC 1; 8C, UM 2; and 8D, UM 3). For each sample, 50,000 cells were injected into the mammary fat pad of NOD-SCID mice of monitored tumor size. The injection was started when the tumor size was about 4 mm. Twice a day (15mg/Kg) injection of Repertaxin for 28 days or once a week, i.p. injection of docetaxel (10mg/Kg), or with a combination (Repertaxin/docetaxel). Fig. 8 shows the tumor size before and during each of the prescribed treatment procedures (arrows, start treatment). Similar results were observed for each sample (SUM159, MC1, UM2, UM3), where the tumor size was statistically significantly reduced (p < 0.01) when treated with docetaxel alone or combined Repertaxin/docetaxel compared to the control, while no significant difference was observed between the growth of the control tumor and the tumor treated with Reperataxin.

Assessing the effect of Reertaxin, docetaxel or combination treatment on cancer stem cell populations by ALDEFLUOR assay

ALDH activity was assessed by ALDEFLUOR assay for analysis of cancer stem cell population size in each tumor (9a. sum159, 9b.mc1, 9c.um2, 9d.um3) treated with reportaxin, docetaxel or a combination. Similar results were observed for each sample. Docetaxel treated tumor xenografts showed similar or increased percentages of alfeluor-positive cells compared to controls, whereas treatment with Repertaxin alone or in combination with docetaxel resulted in statistically significant reductions in alfeluor-positive cells and 65% to 85% fewer cancer stem cells (p < 0.01) compared to controls.

The effect of reportaxin, docetaxel or combination treatment on cancer stem cell populations was assessed by implanting secondary mice.

Serial dilutions of cells (10a. sum159, 10b. mc1, 10c. um2, 10d. um3) obtained from untreated (control) and primary tumors treated with reportaxin, docetaxel, or a combination were implanted into the mammary fat pads of secondary NOD-SCID mice. All diluted primary tumors treated with control and docetaxel formed secondary tumors, while only higher concentrations of primary tumors treated with repeatxin or in combination with docetaxel were able to form delayed secondary tumors that were significantly smaller in size (p < 0.01) than the control and docetaxel treated tumors. In addition, 1000 and 100 cells initially treated with the combination failed to form secondary tumors for 3 of 4 samples (SUM159, UM2, UM 3).

Repetaxin treatment reduces the metastatic potential of SUM159 cell lines

SUM159 cell line was infected with luciferase-expressing lentivirus and 250,000 luciferase-infected cells were seeded into the heart of NOD/SCID mice. Mice were divided into two groups. 12 hours after the intracardiac injection, two groups of mice were treated by s.c. injection of saline solution or s.c. injection of Repertaxin (15mg/kg) twice daily during 28 days. Bioluminescence imaging was used to monitor metastasis formation (11B: mice treated with saline solution; 11C: mice treated with Repetaxin). Quantification of the normalized photon flux measured at weekly time intervals after inoculation revealed that the formation of metastases in the group of mice treated with saline solution was statistically significantly increased compared to the group of mice treated with reportaxin (11A).

Example 3

Treatment of cancer stem cells by CXCR1 blockade

This example demonstrates the effect of CXCR1 inhibition on tumor cells via in vitro assays and mouse models.

Dissociation of mammary tissue. 100-200g of normal breast tissue from mammoplasty was minced with a scalpel, enzymatically dissociated, and single cells were cultured in suspension to generate mammospheres or in adherent conditions on collagen substrates to induce cell differentiation (Dontu et al Genes Dev.17: 1253-1270, the contents of which are incorporated herein by reference in their entirety).

And (5) culturing the cells. The breast Cancer cell line was cultured using the recommended culture conditions (Charafe-Jauffret et al Cancer Res.69: 1302-1313, the contents of which are incorporated herein by reference in their entirety). Breast cancer cell lines were treated with Repertaxin (Sigma-Aldrich), anti-human CXCR1 mouse monoclonal antibody (clone 42705, R & D systems), anti-human CXCR2 mouse monoclonal antibody (clone 48311, R & D systems), anti-human CD95 mouse monoclonal antibody (clone DX2, BD Pharmingen) as FAS signaling agonist, anti-human FAS ligand mouse monoclonal antibody (clone NOK-1, BD Pharmingen) as FAS signaling antagonist, or docetaxel (Taxotere, Sanofi-Aventis) in adhesion conditions.

Cell viability. For the MTT assay, cells were dispensed in 96-well plates at 5,000 cells per well in adherent conditions. After 1 day, treatment with Repertaxin was started. The effect of Repertaxin treatment on cell viability was assessed at different time points by adding 20 μ l of MTT solution (5mg/mL in PBS) to each well. Then, cells were incubated at 37 ℃ for 1 hour, followed by the addition of 50 μ L DMSO to each well. The absorbance was measured at 560nm in a fluorescence analyzer (Spectrafluor, Tecan). For TUNEL assay, cells were dispensed in 6-well plates at 50,000 cells per well in adherent conditions. After 1 day, treatment with Repertaxin was started. The number of apoptotic cells was assessed after 4 days of treatment. Cells were fixed in 3.7% formaldehyde and stained using a TACS TdT kit (R & D systems). Nuclei were counterstained with DAPI/antidade (Invitrogen). Sections were examined with a fluorescence microscope (Leica, Bannockborn, IL, USA) in which apoptotic cells were detected in green.

ALDELUOR assay. Clusters with high ALDH enzyme activity were isolated using FACStarPLUS (Becton Dickinson), using the ALDELUOR kit (StemCell technologies), as previously described (Ginestier et al Cell Stem Cell 1: 555-. To clear mouse-derived cells from xenografted tumors, cell populations were stained with anti-H2 Kd antibody (BD biosciences, 1/200, 20 min on ice) followed by Phycoerythrin (PE) -labeled secondary antibody (Jackson labs, 1/250, 20 min on ice).

And (4) performing ELISA measurement. To measure the level of secreted soluble FAS ligand in the culture medium of cells treated with or without Repertaxin, human sFAS ligand elisa (bender medsystems) was used. The absorbance was read on a spectrophotometer using 450nm as the dominant wavelength.

Western blotting. Cells were lysed in Laemmli buffer and loaded onto SDS-polyacrylamide gels. The blots were incubated with the respective primary antibody diluted in TBST (containing 0.1% Tween20 and 2% BSA) overnight at 4 ℃ or 2 hours at room temperature. The blot was washed and incubated with an appropriate secondary antibody (GE Healthcare, UK) and detected using SuperSignal West Pico chemiluminescent substrate (Pierce).

And (4) immunostaining. For immunofluorescent staining, sorted CXCR1 positive cells were fixed with 95% methanol at-20 ℃ for 10 minutes. The cells were rehydrated in PBS and incubated with the respective antibodies for 1 hour at room temperature. The primary antibodies used were P-FAK (1: 50, Cell Signaling Technology), P-AKT (1: 300, Cell Signaling Technology), and FOXO3a (1: 250, Cell Signaling Technology). Slides were then washed and incubated with PE-conjugated secondary antibodies (Jackson labs) for 30 minutes. Nuclei were counterstained with DAPI/antistade (Invitrogen) and coverslipped. Sections were examined with a fluorescence microscope (Leica, Bannockborn, IL, USA). Immunohistochemistry was performed on paraffin sections to detect ALDH1 (1: 100, BD biosciences), P-FAK, P-AKT, FOXO3a expression (Ginestier et al, am. J Pathol. 161: 1223-1233, the contents of which are incorporated herein by reference in their entirety). Staining was performed using Histostainplus kit (Zymed laboratories). Diaminobenzidine (DAB) or 3-amino-9-ethylcarbazole (AEC) was used as a chromogen, and sections were counterstained with hematoxylin.

An animal model. The tumorigenicity of ALDELFUOR-positive/CXCR 1-positive and ALDELUOR-positive/CXCR 1-negative SUM159 cells was evaluated in NOD/SCID mice (Ginestier et al Cell stem Cell 1: 555-567, the contents of which are incorporated herein by reference in their entirety). The efficacy of Resertaxin treatment on tumor growth was determined using the SUM159 Cell line and 3 primary human breast cancer xenografts (MC1, UM2, UM3) generated from three different patients (Ginestier et al Cell StemCell 1: 555-. Cells from these tumors were orthotopically transplanted into humanized cleared fat pads (cleared fat-pads) of NOD/SCID mice without in vitro culture. Fat pads were prepared as previously described (Ginestier et al Cell Stem Cell 1: 555-567, the contents of which are incorporated herein by reference in their entirety). 50,000 cells from each xenograft were injected into the humanized fat pad of NOD/SCID mice and tumor growth was monitored. At a tumor size of about 4mm, repteraxin alone (s.c., 15mg/Kg twice a day during 28 days), docetaxel alone (i.p., 10mg/Kg once a week during 4 weeks), combination (reptaxin/docetaxel) treatment or saline injection control (i.p., once a week and twice s.c. a day during 28 days) was started. Animals were euthanized at a maximum tumor diameter of about 1.5cm to avoid tumor necrosis, and according to vertebrate study use regulations. A portion of each fat pad injected was fixed in formalin and embedded in paraffin for histological analysis. The remaining portion of tumor cells was re-transplanted into secondary NOD/SCID mice. Serial dilutions of cells were used for re-transplantation, with 10,000, 1,000 and 100 cells injected for each treated tumor.

Anchorage independent culture. BCL treated with Repertaxin (100nM), anti-CXCR 1 antibody (10 μ g/ml), or anti-CXCR 2(10 μ g/ml) in adherent conditions were dissociated and plated as single cells at low density (5000 viable cells/ml) in ultra-low attachment plates (Corning, Acton, MA). Cells were cultured as previously described (Charafe-Jauffret et al Cancer Res.69: 1302-1313, the contents of which are incorporated herein by reference in their entirety). Subsequent cultures after dissociation of the primary tumor spheres were plated at a density of 5000 viable cells/ml on ultra-low attachment plates. The ability of cells to form tumor spheres was quantified after the first (primary tumor spheres) and second (secondary tumor spheres) passages.

RNA extraction and qRT-PCR. After treating SUM159 cells, total RNA was isolated using Rneasy Mini kit (QIAGEN) and used in ABI7900 the real-time quantitative RT-PCR (qRT-PCR) assay was performed in the HT sequence detection system. The primers and probes used in the Taqman system were selected from the group consisting of Applied Biosystems wet site (www dockelibiosystems. com) (FAS ligand assay ID: Hs-00899442-mi; IL8 assay ID: Hs-00174103-mi; TBP assay ID: Hs-00427620-mi). The relative expressed mRNA levels of FAS ligand and IL8 were calculated relative to an internal standard TBP gene to normalize for changes in RNA quality and input cDNA amount, as previously described (Ginestier et al clin. cancer Res.12: 4533-4544., the contents of which are incorporated herein by reference in their entirety).

Flow cytometry analysis. CD44/CD24/Lin staining was performed (Ginestier et al Cell Stem Cell 1: 555-567, the contents of which are incorporated herein by reference in their entirety). CD95/FAS staining was performed using anti-CD 95 labeled APC (1: 20, BDbiosciences). For CXCR1 and CXCR2 staining, primary anti-CXCR 1 (1: 100, clone 42705, R & D systems) and anti-CXCR 2 (1: 100, clone 48311, R & D systems) were followed by PE-labeled secondary anti-mouse (dilution 1: 250, Jackson Labs). Fresh cells were stained with 1. mu.g/ml PI (Sigma) for 5 min to examine viability.

And (4) viral infection. Two different lentiviral constructs were generated for expressing the luciferase gene (Lenti-LUC-VSVG) (Charafe-Jauffret et al Cancer Res.69: 1302-1313, the contents of which are incorporated herein by reference in their entirety) and for inhibiting PTEN expression (Lenti-PTEN-SiRNA-DsRed) (Korkaya et al PLoS biol.7: e1000121, the contents of which are incorporated herein by reference in their entirety), respectively. All lentiviral constructs were prepared from University of michigan Vector (University of michigan Vector). Also utilized is an adenovirus construct (Ad-FAK-GFP) for over-expression of FAK (Luo et al Cancer Res.69: 466-474, the contents of which are incorporated herein by reference in their entirety). Infection of cells with different vectors was performed as previously described (Charafe-Jauffret et al Cancer Res.69: 1302-1313, the contents of which are incorporated herein by reference in their entirety). Infection efficiency was verified by measuring DsRed or GFP expressing cells.

Intracardiac vaccination. 6-week-old NOD/SCID mice were anesthetized with a 2% isoflurane/air mixture and injected with 100 μ L of Ca-deficient mice in the left ventricle of the heart²⁺And Mg²⁺250,000 cells in sterile dubber PBS. For each of the three cell lines (HCC1954, MDA-MB-453 and SUM159) and for each treatment (saline or Repetaxin), 6 animals were injected. 12 hours after the intracardiac injection, mice were injected with repteraxin or control saline twice daily.

And (4) detecting bioluminescence. Initial bioluminescence was assessed before and weekly after inoculation. The bioluminescence detection procedure was performed as previously described (Charafe-Jauffret et al Cancer Res.69: 1302-1313, the contents of which are incorporated herein by reference in their entirety). The normalized photon flux represents the ratio of the photon flux detected weekly after inoculation to the photon flux detected before inoculation.

CXCR1 expression subdivides the cancer stem cell population. The identification of signal transduction pathways that modulate Cancer Stem Cells (CSCs) provides potential therapeutic targets in cell populations. Gene expression profiling-based breast CSC tags have been identified that contain several genes that may be involved in breast CSC regulatory pathways (Charafe-Jauffret et al Cancer Res.69: 1302-1313, the contents of which are incorporated herein by reference in their entirety). Among the genes overexpressed in the breast CSC population, CXCR1, a receptor that binds the proinflammatory chemokine IL-8/CXCL8, appears to be a promising candidate because recombinant IL-8 stimulates self-renewal of breast CSCs (Charafe-Jauffret et al Cancer Res.69: 1302-1313, the contents of which are incorporated herein by reference in their entirety). CXCR1 protein expression was measured in the breast CSC population using flow cytometry as assessed by the ALDELFUOR assay of the human breast cancer cell lines HCC1954, MDA-MB-453 and SUM 159. Cells with functional stem cell characteristics in NOD/SCID mouse xenografts are contained within ALDELUOR positive cell populations (Charafe-Jauffret et al cancer Res.69: 1302-1313, the contents of which are incorporated herein by reference in their entirety). The CXCR1 positive population (which represents less than 2% of the total population) was almost exclusively contained within the ALDEFLUOR positive population (see fig. 12A and table 4).

Table 4.

CXCR2 expression was also assessed. CXCR2 is a receptor that also binds IL-8/CXL8, albeit with reduced affinity compared to CXCR 1. In contrast to CXCR1 positive cells, CXCR2 positive cells were evenly distributed among the aldeluor positive and aldeluor negative populations (see fig. 12A). To determine the hierarchical structure of cancer stem cell populations based on CXCR1 expression, populations of aldeluor positive/CXCR 1 positive and aldeluor positive/CXCR 1 negative cells were sorted and injected into NOD/SCID mice (see fig. 13). Both cell populations give rise to tumors. Tumor growth kinetics are related to the latency and size of tumor formation and the number of injected cells. Tumors generated from the aldeluor positive/CXCR 1 positive population reconstitute the phenotypic heterogeneity of the original tumor after serial passage, while the aldeluor positive/CXCR 1 negative population generates tumors containing only aldeluor positive/CXCR 1 negative cells. These results suggest that CSC cell grading is organized according to CXCR1 expression, however, both cell populations exhibit similar tumorigenic capacity.

CXCR1 blockade reduces the breast cancer stem cell population in vitro. Three different cell lines were treated with Repertaxin (100nM), a CXCR1/2 inhibitor, to assess the effect of CXCR1 blockade on the mammary CSC population (Bertini et al proc. natl. acad. sci. u.s a 101: 11791-11796, the contents of which are incorporated herein by reference in their entirety). For SUM159, a 5-fold decrease in the proportion of ALDEFLUOR positive cells was observed three days after treatment (see fig. 12B). Similar effects were observed after treatment of SUM159 cells with anti-CXCR 1 blocking antibodies. In contrast, no effect was observed after treatment with anti-CXCR 2 blocking antibody, indicating that the effect of Repertaxin on alfeluor positive population is mediated by CXCR 1.

Data from breast tumors and cell lines indicate that Cancer stem-like cells or Cancer initiating cells can also be isolated and propagated as "tumor balls" in suspension culture (Ponti et al Cancer Res.65: 5506-5511, the contents of which are incorporated herein by reference in their entirety). After three days of treatment with Repertaxin or with anti-CXCR 1 blocking antibody, an 8-fold reduction in primary and secondary tumor sphere formation was observed when the cells were separated and cultured in suspension compared to the control. In contrast, anti-CXCR 2 blocking antibodies had no effect on tumor sphere formation (see fig. 14).

Unexpectedly, after 5 days of treatment with reportaxin, we observed a large reduction in viability of the entire cell population, with only 3% of the cells still viable, as assessed by the MTT assay (see fig. 12C). Similar results were observed with anti-CXCR 1 blocking antibodies instead of anti-CXCR 2 blocking antibodies, thus indicating that this effect is dependent on CXCR1 blocking. This effect of reportaxin was delayed, with loss of cell viability beginning three days after treatment (see fig. 15A). Reertaxin treatment induced a similar effect on the HCC1954 breast Cancer cell line, whereas no effect was observed on MDA-MB-453 cells containing PTEN mutations (Holleselle et al Cancer Res.5: 195-201, the contents of which are incorporated herein by reference in their entirety) (see FIG. 14, FIGS. 15B-C and FIG. 16).

SUM159 cells were stained 4 days after treatment with Reertaxin using the TUNEL assay, and a large decrease in cell viability due to apoptosis induction was observed, with 36% of apoptotic cells detected after Reertaxin treatment (see FIG. 12D). The results indicate that blockade of CXCR1 results in a reduction in the breast CSC population, followed by induction of massive apoptosis in the remaining large tumor population.

CXCR1 blocks the induction of cell death in CXCR1 negative cells via a bystander effect. The observation that either Repertaxin or anti-CXCR 1 block antibodies induced massive cell death, although the CXCR1 positive population accounted for less than 2% of the total cell population, suggests that CXCR 1in XCR1 positive cells blocks induction of CXCR1 negative cell death via bystander effect. Sorted CXCR1 positive and CXCR1 negative populations were treated with Repertaxin (see fig. 12E). Reportaxin reduced cell viability in the CXCR1 positive population within 3 days, while no effect was observed in the CXCR1 negative population. Reportaxin induces massive cell death in unseparated cells. The effect of Repertaxin on cell viability of the unseparated and CXCR1 positive populations was dose-dependent (see fig. 12E). The results are consistent with a Repertaxin treatment that targets the CXCR1 positive population, followed by induction of CXCR1 negative cell death via bystander effect.

To determine whether this effect is mediated by soluble factors induced by Repertaxin, conditioned media was collected from the CXCR1 positive population three days after Repertaxin treatment and this media was dialyzed using a membrane with a 3.5KDa exclusion to remove the Repertaxin from the media, while retaining molecules greater than 3.5 KDa. The dialyzed conditioned medium induced a substantial decrease in cell viability in both CXCR1 negative and unseparated populations but not in the CXCR1 positive population (see fig. 12F). These results demonstrate that CXCR 1in the CXCR1 positive population blocks the induction of cell death in the CXCR1 negative population via soluble, non-dialyzable factors. Although the CXCR1 positive population is sensitive to reportaxin, it is resistant to transducible death factors.

The bystander effect induced by CXCR1 blockade is mediated by FAS ligand/FAS signaling. The FAS ligand/FAS interaction is activated in different physiological states (such as mammary gland degeneration) or in conditions of tissue injury (including induction by chemotherapy) (Chhipa et al J Cell biochem.101: 68-79, Song et al J Clin. invest 106: 1209-1220, the contents of which are incorporated herein by reference in their entirety). ELISA assays were used to determine the level of soluble FAS ligand in the culture media of Repertaxin-treated SUM159 cells to assess the role of the FAS ligand/FAS interaction in mediating the apoptotic bystander effect induced by CXCR1 blockade. More than a 5-fold increase in soluble FAS ligand was observed in the culture medium of four-day cells treated with reportaxin compared to untreated cells (see fig. 17A). The transcriptional regulation of FAS ligand by Reertaxin treatment obtained by measuring FAS ligand mRNA levels was confirmed by RT-PCR (see FIG. 17B). A 4-fold increase in FAS ligand mRNA levels was observed in Repertaxin-treated cells compared to untreated cells. Similar results were observed after treatment with FAS agonists that activate FAS signaling, indicating that the FAS ligand is the target for FAS signaling that produces a positive feedback loop. As determined by flow cytometry, 100% of SUM159 cells expressed FAS protein. Treatment of SUM159 cells with FAS agonist reproduced the killing effect observed with the repotaxin treatment with a large reduction in cell viability (see fig. 17C). The anti-FAS ligand-blocking antibody partially reversed the effect of Repertaxin treatment on cell viability, with 44% of the cells remaining viable after treatment with Repertaxin and anti-FAS ligand antibody, compared to only 3% of the cell viability in the case of Repertaxin alone (see fig. 17C). The results indicate that the massive cell death induced by reportaxin is due to a FAS ligand/FAS pathway mediated bystander effect.

Treatment of SUM159 cells with FAS agonist resulted in a 10-fold and 3-fold increase in the percentage of CXCR1 positive and ALDEFLUOR positive cells, respectively (see fig. 17D/E and fig. 18). The effect of anti-FAS ligand on both populations was not rescued (see fig. 17D/E), suggesting that the alfeluor positive population containing the CXCR1 positive population, although directly sensitive to CXCR1 blockade, which in turn induces FAS ligand production by these cells, is resistant to FAS ligand/FAS pro-apoptotic signal transduction. In contrast, a large population of cells that are ALDEFLUOR negative do not express CXCR1, but are sensitive to FAS ligand-mediated cell death.

FAS ligand/FAS signaling plays an important role in the process of mammary gland degeneration (Song et al J Clin. invest106: 1209-1220, the contents of which are incorporated herein by reference in their entirety). The effect of CXCR1 blockade on normal human mammary epithelial cells obtained from breast reconstructive plasty was examined. As observed in breast cancer cell lines, CXCR 1-positive normal breast cells were almost exclusively contained within the ALDEFLUOR-positive population (see fig. 19A). To determine whether IL-8 signaling is important in normal breast stem/progenitor cell function, normal mammary epithelial cells cultured in suspension were treated with human recombinant IL-8 and their effect on CSC populations was determined, as measured by mammosphere formation (Dontu et al Genes Dev.17: 1253-. The addition of IL-8 increased primary and secondary mammosphere formation in a dose-dependent manner (see fig. 19B), suggesting that the IL-8/CXCR1 axis may be involved in regulating the proliferation or self-renewal of normal breast stem/progenitor cells. Treatment with Repertaxin or a FAS agonist had no effect on the viability of normal mammary epithelial cells cultured in adherent conditions, even when high concentrations of Repertaxin (500nM) were used (see fig. 16A). However, as observed for breast cancer cell lines, an increase in soluble FAS ligand was detected in the culture media of normal breast epithelial cells treated with reportaxin (see fig. 20B). This observation can be explained by the absence of FAS expression in normal epithelial cells cultured under these conditions (see fig. 20C). This is consistent with studies demonstrating that FAS expression in the mammary gland occurs only during the degenerative process following lactation (Song et al J clin. invest 106: 1209-1220, the contents of which are incorporated herein by reference in their entirety). In contrast to its lack of effect on a large population of normal mammary epithelial cells, reportaxin significantly reduced the mammosphere formation of these cells (see fig. 20C).

These results indicate that the IL-8/CXCR1 axis plays an important role in the regulation and survival of normal and malignant mammary epithelial stem/progenitor cell populations. The ability to affect large cell populations via a bystander effect mediated by the FAS ligand may be correlated with the level of FAS expression in these cells.

The effects of CXCR1 blockade on cancer stem cells are mediated by the FAK/AKT/FOXO3A pathway. CXCR1 functions via a signal transduction pathway involved in Focal Adhesion Kinase (FAK) phosphorylation leading to activation of AKT (waggh et al, cancer res.14: 6735-6741, the contents of which are incorporated herein by reference in their entirety). To assess the effect of CXCR1 blockade on FAK and AKT activation, the levels of FAK and AKT phosphorylated proteins were measured by Western blotting performed on three different cell lines. For SUM159 and HCC1954, with untreated finesIn contrast to cells, we detected FAK Tyr in cells treated with Repetaxin³⁹⁷And AKT Ser⁴⁷³A decrease in phosphorylation, suggesting that the epertaxin effect may be mediated by the FAK/AKT pathway (see fig. 21A and 22). The observation that MDA-MB453 is resistant to Repettaxin treatment can be explained by the presence of a PTEN mutation (919G > A) that activates the PI3K/AKT pathway (Holleselle et al mol. cancer Res.5: 195. sub.201, the contents of which are incorporated herein by reference in their entirety). No FAK Tyr was detected following Reptaxin treatment in the MDAMB453 cell line³⁹⁷And AKT Ser⁴⁷³Modification of phosphorylation (see FIG. 22). To confirm the functional role of the FAK/AKT pathway in mediating the effects of CXCR1 blockade, two viral constructs were used, one knocking out PTEN expression via PTEN shRNA and the other resulting in FAK overexpression. PTEN antagonizes PI3-K/AKT signaling via its lipid phosphatase (Vivanco et al nat. Rev. cancer 2: 489-501, the contents of which are incorporated herein by reference in their entirety). PTEN knock-out results in AKT activation, e.g. by AKT Ser⁴⁷³Increased phosphorylation (see fig. 21A and 22). PTEN knockdown blocks the effect of Repetaxin treatment on FAK and AKT activity, FAK overexpression also blocks the effect of Repetaxin, and induces the activation of FAK and AKT by FAK Tyr ³⁹⁷And AKT Ser⁴⁷³Increase in expression of phosphorylation. These results indicate that the CXCR1 blocking effect is mediated by FAK/AKT signaling.

Using immunofluorescent staining of CXCR1 positive cells, it was confirmed that reportaxin treatment resulted in a significant reduction in phospho-FAK and phospho-AKT expression compared to untreated cells (see fig. 21B). AKT modulates the activity of the forkhead transcription factor FOXO3A via a phosphorylation event that results in sequestration of cytosolic FOXO3A (Brunet et al mol. cell biol. 21: 952-. In contrast, the unphosphorylated form of FOXO3A is transported to the nucleus where it acts as a transcription factor that regulates FAS ligand synthesis (Jonsson et al nat. Med. 11: 666-671), the contents of which are incorporated herein by reference in their entirety. Reportaxin induces cell death via FAS ligand-mediated bystander effects; the effect of Repertaxin on this signal transduction pathway was examined by immunofluorescence staining. FOXO3A was present in untreated cells in cytoplasmic localization, but shuttled to the nucleus after reportaxin treatment (see fig. 21B). This indicates that CxCR1 blocks the induction of FOXO3A activity via inhibition of the FAK/AKT pathway. Cells with PTEN deletion or FAK overexpression showed high levels of phospho-FAK and phospho-Akt expression in both Repertaxin-treated and untreated cells, as detected by immunofluorescence. As shown by the cytoplasmic location of FOXO3A, Repertaxin treatment did not induce FOXO3A activation in cells with PTEN deletion or FAK overexpression (see fig. 21B).

Cells with PTEN deletion or FAK overexpression exhibit resistance to reportaxin treatment due to constitutive activation of the FAK/AKT pathway. Cells with PTEN deletion or FAK overexpression did not show any reduction in cell viability in the case of reportaxin treatment. AKT signaling has been proposed to play a critical role in the biology of CSC (see FIGS. 21B and 22) (Dubrovska et al Proc. Natl. Acad. Sci. U.S. A106: 268-273., Korkaya et al PLoS biol. 7: e1000121., Yilmaz et al Nature 441: 475-482, the contents of which are incorporated herein by reference in their entirety). Activation of the FAK/AKT pathway blocks the effect of reportaxin on CSC populations as shown by the maintenance of aldelfusor positive populations following treatment with inhibitors (see fig. 21B). All results indicate that blockade of CXCR1 directly affected the FAK/AKT/FOXO3A pathway. Reportaxin treatment inhibits AKT signaling, which is critical for CSC activity, and subsequently induces FAS ligand-mediated bystander effects on a large number of tumor cells generated by CSCs.

Reportaxin treatment reduces the population of breast cancer stem cells in vivo. Recent evidence suggests that breast CSCs are relatively resistant to chemotherapy and radiation, and may contribute to tumor regrowth following treatment (Phillips et al jnatl. cancer inst.98: 1777-. CSC principles suggest that significant improvements in clinical outcome will require effective targeting of the CSC population (Reya et al Nature 414: 105-111, the contents of which are incorporated herein by reference in their entirety). When a large number of tumor cells are targeted by chemotherapy, several factors are synthesized and secreted during the apoptotic process. Among these factors, FAS ligands amplify the effect of chemotherapy by mediating bystander killing effects (Chhipa et al J CellBiochem.101: 68-79, the contents of which are incorporated herein by reference in their entirety). Chemotherapy may also induce IL-8 production in injured cells. The commonly used chemotherapeutic agent docetaxel induces both IL-8 and FAS ligand mRNA in SUM159 cells (see FIG. 10 a/B). We also detected a 4-fold increase in IL-8mRNA levels after FAS agonist treatment (see FIG. 10B). We have shown that IL-8 is able to modulate CSC populations. This suggests that increasing Repertaxin on cytotoxic chemotherapy can block this effect and target cancer stem cell populations.

The efficacy of reportaxin treatment on tumor growth was probed using SUM159 cell line and three primary human breast cancer xenografts (MC1, UM2, UM3) generated from three different patients. Cells from these tumors were orthotopically transplanted into humanized clean fat pads of NOD/SCID mice without in vitro culture. For each of these xenografts, the CSC population was only contained within the ALDELUOR positive population (Ginestier et al Cell Stem Cell 1: 555-. In each tumor, the CXCR1 positive population was almost exclusively contained within this ALDEFLUOR positive population (see table 5), and the PTEN/FAK/AKT pathway was activated (see fig. 25).

TABLE 5

50,000 cells from each xenograft were injected into the humanized fat pad of NOD/SCID mice and tumor growth was monitored. At a tumor size of about 4mm, treatment was started with Repertaxin alone (15mg/Kg twice a day during 28 days), docetaxel alone (10mg/Kg once a week during 4 weeks), or a combination of the two drugs. Tumor growth was compared to saline injected controls. For each xenograft, significant inhibition of tumor growth induced by docetaxel treatment or the Repertaxin/docetaxel combination was observed (see fig. 26A and fig. 27). Treatment with Repertaxin alone had a moderate effect on tumor growth. After 4 weeks of treatment, animals were sacrificed and residual tumors were analyzed using the ALDEFLUOR assay. Residual tumors treated with docetaxel alone contained an unchanged or increased percentage of aldefuor positive cells compared to untreated controls (see fig. 26B and fig. 27). In comparison, treatment with Repertaxin alone or in combination with docetaxel reduced the alfelulor positive population by more than 75% (see fig. 26B and fig. 27). The results of ALDH1 expression in different xenografts were confirmed by immunohistochemistry. A decrease in ALDH1 positive cells was detected in Repertaxin treated tumors compared to untreated tumors, whereas the percentage of ALDH1 positive cells was unchanged or increased in tumors treated with docetaxel alone (see fig. 26D).

The presence of CD44+/CD 24-cells in these tumors was assessed. Expression markers in breast cancer stem cells have been previously shown (Al Hajj et Al Proc. Natl. Acad. Sci. U.S. A100: 3983-. The overlap between the CD44+/CD 24-phenotype and CXCR1 expression was measured. CXCR1 positive cells were present in both the CD44+/CD 24-cell population and the CD24 expressing or CD44 negative cell population (see table 6).

TABLE 6

In the residual tumors treated with docetaxel alone, an unchanged or increased percentage of CD44+/CD 24-cells was observed, whereas treatment with Repertaxin alone or in combination with docetaxel resulted in a reduction of the CD44+/CD 24-cell population (see fig. 28).

A functional in vivo assay comprising re-transplantation of cells from the treated tumor into secondary NOD/SCID mice provides a direct test to assess the tumor initiating and self-renewal capacity of CSCs remaining after treatment. All dilutions of tumor cells derived from control or docetaxel treated animals showed similar tumor regrowth in secondary NOD/SCID mice. In contrast, Repertaxin treatment with or without docetaxel reduced tumor growth in secondary recipients (see fig. 26C). Those cells from Repertaxin treated animals showed a 2-5 fold reduction in tumor growth when equal numbers of cells were injected compared to cells from control or docetaxel treated animals (see fig. 26C). For each xenograft model, 1000 or 100 tumor cells obtained from animals treated with the combination of Repertaxin and docetaxel failed to form any secondary tumors in NOD/SCID mice (see fig. 26C, fig. 27, and table 7). These studies demonstrate that reportaxin therapy specifically targets and reduces CSC populations.

TABLE 7

Reportaxin treatment inhibits FAKMKT signaling and activates FOXO3A in vivo. The expression of phospho-FAK and phospho-AKT was examined by immunohistochemistry of each xenograft after treatment. Membranous phospho-FAK expression was detected in 50% of cells from control and docetaxel-treated tumors, whereas phospho-FAK expression was abolished in tumors treated with reportaxin alone or in combination with docetaxel (see fig. 26D). Similar results were observed for phospho-AKT expression, where 70% of cells expressed phospho-AKT in untreated tumors, 20% of phospho-AKT positive cells in docetaxel treated tumors, and completely inhibited phospho-AKT expression in tumors treated with reportaxin alone or in combination with docetaxel (see fig. 26D). Nuclear FOXO3A was detected in cells from tumors treated with docetaxel alone, reperaxin alone, and a combination of reperaxin/docetaxel. These in vivo data are consistent with in vitro data and confirm that Repertaxin treatment inhibits FAK/AKT signaling and activates FOXO 3A.

Reertaxin treatment reduces the formation of systemic metastases. To determine whether Reertaxin reduces systemic metastasis, we infected HCC1954, MDA-MB-453, and SUM159 breast cancer cell lines with a luciferase lentivirus reporter system and introduced the cells into NOD/SCID mice by intracardiac injection. A suspension of 250,000 cells of each cell line was injected and metastasis formation was monitored weekly by bioluminescence imaging. Mice were treated twice daily by either injection of Repertaxin or saline injection of controls 12 hours after intracardiac injection. Repertaxin treatment of mice injected with HCC1954 and SUM159 cells significantly reduced metastasis formation, with lower photon flux emission in treated mice compared to untreated mice (see fig. 29A/B). Histological sections confirmed that metastasis was present at several sites in untreated animals (see fig. 29D). Reertaxin treatment had no effect on metastasis formation in mice injected with MDA-MB-453 cells (see FIG. 29C). The photon flux emission and the number of animals forming metastases were similar in both the Repertaxin treated and untreated groups. This result is consistent with data describing MDA-MB-453 as a cell line resistant to Repettaxin due to the presence of a PTEN mutation. These results indicate that blockade of CXCR1 with agents such as Repertaxin can reduce metastasis mediated by CSC populations (Charafe-Jauffret et al Cancer res 69: 1302-1313, the contents of which are incorporated herein by reference in their entirety).

Experiments conducted during the development of embodiments of the present invention have shown that a cellular sub-fraction with stem cell properties drives tumor growth and metastasis (Visvader et al Nat. Rev. cancer 8: 755-768, the contents of which are incorporated herein by reference in their entirety). These cells may contribute to treatment resistance and relapse by virtue of their relative resistance to current forms of treatment (Reya et al Nature 414: 105-111, the contents of which are incorporated herein by reference in their entirety). The present invention provides a method based on blocking CXCR1 cytokine receptors, which are expressed on breast cancer stem cells, to efficiently target cancer stem cell populations and improve treatment outcomes. Experiments conducted in a number of systems during the development of embodiments of the present invention have demonstrated that cytokine networks play an important role in tumorigenesis. There is evidence that several of these cytokines can modulate stem cell behavior. IL-4 is capable of regulating the self-renewal of pancreatic cancer Stem cells, while IL-6 is capable of regulating cancer Stem cells in colon and breast cancers (Todaro et al Cell 1: 389-400402, Sansone et al J Clin. invest 117: 3988-4002, the contents of which are incorporated herein by reference in their entirety). The role of IL-8 in mediating tumor invasion and metastasis has been previously demonstrated (Waugh and Wilson. cancer Res.14: 6735-. In addition, IL-8 enhances the self-renewal of neural stem cells during wound healing in the brain (Beech et al J neuroimunol.184: 198-208, the contents of which are incorporated herein by reference in their entirety). Lung cancer stem cells are described as expressing the chemokine receptor CXCR1(Levina et al plos. one.3: e3077, the contents of which are incorporated herein by reference in their entirety). Experiments conducted during the development of embodiments of the present invention demonstrated that CXCR1 positive populations were almost exclusively contained within the aldeluor positive populations in breast cancer cell lines and primary xenografts and normal breast cells. Chemokine receptors are overexpressed in ALDEFLUROR-positive breast Cancer cell populations (Charafe-Jauffret et al Cancer Res.69: 1302-1313, the contents of which are incorporated herein by reference in their entirety). In breast cancer, IL-8 is produced in the tumor microenvironment by a variety of cell types including inflammatory cells, vascular endothelial cells, tumor-associated fibroblasts, and mesenchymal stem cells (Waugh et al Clin. cancer Res.14: 6735-6741, the contents of which are incorporated herein by reference in their entirety). The cytokine network mediates interactions between these cell types, and thus cancer stem cells can be targeted via blockade of the IL-8 receptor CXCR 1.

Using in vitro assays, blockade of CXCR1, but not CXCR2 (an alternative IL-8 receptor), was demonstrated to reduce the breast cancer stem cell population. Apoptosis was then induced in the entire remaining cell population that lacked CXCR1 expression. In addition to CXCR1 blocking antibodies, experiments conducted during development of the embodiments demonstrated that reportaxin, an inhibitor of CXCR1/2, induces similar effects by targeting the CXCR1 positive population. In contrast to its direct effect on CXCR1 expressing cancer stem cell populations, reportaxin has no direct effect on a large tumor cell population lacking CXCR1 expression. This indicates that CXCR1 blockade in CXCR1 positive cells induces cell death in CXCR1 negative cells via bystander effect. The experiments described herein demonstrate that the FAS ligand/FAS pathway is a mediator of this bystander killing effect. This phenomenon explains the efficacy of Repertaxin treatment in inducing massive apoptosis in the entire cell population, although CXCR1 positive populations represent less than 1% of the cell population. The effect of the FAS ligand is indicated by the effective blocking of bystander killing by anti-FAS ligand antibodies.

Experiments conducted during the development of embodiments of the present invention have shown that similar cytokine interactions can occur in tumors exposed to cytotoxic chemotherapy. Chemotherapy can directly induce apoptosis in differentiated tumor cells and induce the production of FAS ligand by these dying cells, which in turn induces apoptosis of surrounding tumor cells via FAS-mediated bystander effects. Along with the production of FAS ligand, these injured cells also secrete elevated levels of IL-8 in processes like breast degeneration or wound healing. This is often the case in degenerated breasts, and this IL-8 can stimulate breast cancer stem cells and protect them from apoptosis. This may contribute to the relative increase in cancer stem cells observed after chemotherapy in preclinical model (4) and neoadjuvant clinical trial (5). The effect of chemotherapy on the apoptotic and self-renewal pathways of tumors is shown in figure 30.

To determine whether CXCR1 blockade could target breast cancer stem cells in vivo, the effect of Repertaxin and the cytotoxic agent docetaxel on cancer stem cell compartment and tumor growth in NOD/SCID mice was compared. Docetaxel is currently one of the most effective chemotherapeutic agents for treating women with breast cancer. Cancer stem cell populations were assessed by ALDEFUOR assay and serial transplantation in NOD/SCID mice. Using these assays, it was determined that chemotherapy treatment alone resulted in no change or a relative increase in the cancer stem cell population. In comparison, treatment with Repertaxin alone or in combination with chemotherapy significantly reduces the cancer stem cell population. Although tumor initiation population was significantly reduced, the use of reportaxin alone did not result in significant tumor shrinkage. The combination of Repertaxin and chemotherapy results in a significant reduction in tumor size and cancer stem cell population. Combining these agents to target cancer stem cells and large tumor cell populations maximizes the efficacy of these treatments.

To elucidate the mechanism of action of reportaxin, the downstream pathway of CXCR1 was analyzed. Confirming the interaction between CXCR1, FAK and AKT. CXCR1 blocks specific effects via FAK and AKT activation. Experiments conducted during development of embodiments of the present invention have shown that AKT activation regulates normal and malignant mammary stem cell self-renewal via phosphorylation of GSK3 β, which leads to activation of the WNT pathway (Korkaya et al PLoS biolog.7: e1000121, the contents of which are incorporated herein by reference in their entirety). These results indicate why cells with PTEN knockouts are resistant to reportaxin. The other function of AKT is to regulate cell survival via phosphorylation of forkhead transcription factor FOXO 3A. AKT phosphorylation of FOXO3A results in its cytoplasmic segregation. In comparison, it was demonstrated that CXCR1 blockade results in decreased AKT activation, which results in a translocation of FOXO3A in the cell nucleus, where FOXO3A induces a number of genes in the cell nucleus, including FAS ligands (Jonsson et al nat. med. 11: 666-671, the contents of which are incorporated herein by reference in their entirety). Blocking the induced FAS ligand via CXCR1 in turn caused the observed bystander killing effect (see fig. 30).

In addition to its role in CXCR1 signaling, FAK mediates cellular interaction with extracellular matrix components via integrin receptors (Waugh et al, cancer res.14: 6735-6741, the contents of which are incorporated herein by reference in their entirety). FAK signaling plays a role in regulating self-renewal of normal and malignant mouse mammary stem cells in transgenic models (Luo et al Cancer Res.69: 466-474, the contents of which are incorporated herein by reference in their entirety). FAK activation also promotes cell survival by blocking FADD and RIP mediated apoptosis (Kurenova et al mol. CellBiol.24: 4361-4371, Xu et al J biol. chem.275: 30597-30604, the contents of which are incorporated herein by reference in their entirety). This provides an explanation for the resistance of cancer stem cell populations to FAS/FAS ligand-induced apoptosis.

It has been demonstrated that breast Cancer stem cells play a significant role in tumor invasion and metastasis (Croker et al JCell mol. med.2008, Charafe-Jauffret et al Cancer res.69: 1302-. It is shown herein that IL-8 and CXCR1 also play important roles in these processes. The effect of CXCR1 blockade on the formation of experimental metastases was analyzed using reportaxin. CXCR1 blockade was shown to reduce metastasis formation when administered following intracardiac injection of breast cancer cells.

Clinical studies using reportaxin have demonstrated a lack of toxicity. Strategies directed to interfering with cytokine regulation loops (such as IL-8 and CXCR1) represent approaches for targeting breast cancer stem cells.

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All publications and patents mentioned in the above specification are herein incorporated by reference.

Various modifications and variations of the described methods and systems of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. While the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the relevant fields are intended to be within the scope of the invention.

Claims (11)

1. Use of a compound capable of detecting FBXO21 in the manufacture of a medicament for detecting solid tumor stem cells in a tissue sample taken from a breast cancer tumor of a subject.

2. The use of claim 1, wherein the compound comprises an antibody or antibody fragment directed against FBXO 21.

3. The use of claim 1, wherein the compound comprises Repertaxin.

4. The use of claim 2, wherein the antibody or antibody fragment comprises a signal molecule.

5. The use of claim 4, wherein the signal molecule comprises a fluorescent molecule or an enzyme capable of catalyzing a chromogenic reaction in the presence of a colorimetric substrate.

6. The use of claim 1, wherein no other proteins or nucleic acids are assayed to determine the presence or absence of FBXO21+ solid tumor stem cells.

7. The use of any one of claims 1-5, wherein at least one marker selected from the group consisting of CD44, CD24, ESA, ALDH, TPRLL, NOTCH2, RBM15, ST3GAL3, NFYA, PCNX, TAS2R14, CD300LB, GIPC3, RAD51L1, ARID1B, EPPK1, COL11A2, KLK3, EIF2C2, ZFP41, FAM49B, PSORS1C2 is also detected.

8. An isolated population of breast cancer stem cells, said cancer stem cells being:

a) tumorigenic; and is

b) FBXO21+。

9. The isolated population of cancer stem cells of claim 8, wherein the isolated population comprises at least 60% cancer stem cells and less than 40% non-tumorigenic tumor cells.

10. The isolated population of cancer stem cells of claim 8, wherein said cancer stem cells are enriched by at least two-fold compared to unfractionated, non-tumorigenic tumor cells.

11. The isolated population of cancer stem cells of claim 8, wherein said cancer stem cells are enriched at least four-fold compared to unfractionated non-tumorigenic tumor cells.