CN114908164A - Application of ILT5 in colorectal cancer diagnosis and treatment - Google Patents

Abstract

The invention provides the application of ILT5 in the diagnosis and treatment of colorectal cancer, and belongs to the technical fields of biomedicine and molecular biology. The present invention finds that ILT5 is highly expressed in CRC cells, and as a biomarker of poor prognosis, tumor-derived ILT5 can inhibit the infiltration of T cells, especially the infiltration of CD8 ⁺ T cells, and guide the M2-like polarity of TAMs to create an inhibitory tumor immune microenvironment. Inhibition of tumor-derived ILT5 restores the immunosuppressive tumor microenvironment and inhibits colorectal cancer progression. The research results of this application discovered the expression of ILT5 in solid tumor cells for the first time, and suggested that ILT5 can be used as a potential immune target and prognostic marker for colorectal cancer, so it has good practical application value.

Description

Application of ILT5 in colorectal cancer diagnosis and treatment

Technical Field

The invention belongs to the technical field of biological medicine and molecular biology, and particularly relates to application of ILT5 in colorectal cancer diagnosis and treatment.

Background

The information disclosed in this background of the invention is only for enhancement of understanding of the general background of the invention and is not necessarily to be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person skilled in the art.

Colorectal cancer (CRC) is the third most common, second most lethal cancer worldwide, accounting for 1/10 cases of cancer-related deaths. Since most colorectal cancer patients are diagnosed at an advanced stage, refractory to treatment, systemic therapy including chemotherapy, radiation therapy and targeted therapy is the primary strategy for colorectal cancer treatment. Immunotherapy targeting the PD-1/PD-L1 pathway has altered the therapeutic profile of solid tumor therapy. However, due to the unique inhibitory immune microenvironment of colorectal cancer, little clinical benefit was observed in colorectal cancer patients. Therefore, there is an urgent need to explore how tumor cells regulate immune microenvironment to develop new immunotherapeutic targets.

Immunoglobulin-like transcript (ILT)5, also known as LILRB3/LIR3/CD85a, is an inhibitory member of the activating and inhibitory immunoglobulin-like transcript (ILT) family, regulating activation of immune cells. ILT5 is expressed primarily in myeloid lineage cells, including monocytes, macrophages, Dendritic Cells (DCs), granulocytes, basophils, and eosinophils. The homologous gene of mouse ILT5 is paired immunoglobulin-like receptor B (PIR-B). It has been shown that ILT5 acts as a negative immune regulator in autoimmune diseases, sepsis and HIV infection. However, the expression of ILT5 in tumor cells and its role in anti-tumor immunity is not clear.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provideFor the application of ILT5 in the diagnosis and treatment of colorectal cancer. The research of the invention finds that ILT5 is highly expressed in CRC cells and is used as a poor prognosis biomarker, and the ILT5 of tumor origin can inhibit infiltration of T cells, particularly CD8 ⁺ Infiltration of T cells and directing M2-like polarization of TAMs creates an inhibitory Tumor Immune Microenvironment (TIME). Inhibition of tumor-derived ILT5 restores the immunosuppressive Tumor Microenvironment (TME) and inhibits the progression of colorectal cancer. The research result of the application discovers the expression of ILT5 in solid tumor cells for the first time, and proposes that ILT5 can be used as a potential immune target and a prognostic marker of colorectal cancer. The present invention has been completed based on the above results.

In a first aspect of the present invention, there is provided the use of a substance for detecting the expression level of ILT5 gene and its expression products in the manufacture of a product for diagnosing, detecting, monitoring or predicting the progression of colorectal cancer.

The product can diagnose, detect, monitor or predict colorectal cancer progression by monitoring the expression level of ILT5 gene and/or ILT5 expression product, and the invention finds that ILT5 is highly expressed in colorectal cancer through experimental research, and simultaneously the high expression of ILT5 in colorectal cancer predicts a later disease stage and poorer patient survival, namely, the prognosis (including OS and PFS) of colorectal patients is worse.

Wherein, the ILT5 gene and the expression product thereof can be derived from human; more specifically, the ILT5 is tumor (colorectal cancer) -derived ILT 5.

In a second aspect of the invention, there is provided a product for diagnosing, detecting, monitoring or prognosing colorectal cancer progression comprising a substance for detecting transcription of ILT5 in a sample based on a high throughput sequencing method and/or based on a quantitative PCR method and/or based on a probe hybridization method; or a substance for detecting the expression of ILT5 protein based on an immunodetection method.

Preferably, liquid phase hybridization, a Northern hybridization method, a miRNA expression profile chip, a ribozyme protection analysis technology, a RAKE method and in-situ hybridization are adopted to detect the ILT5 gene expression (transcription) condition;

the immunoassay method preferably comprises Western blot, ELISA, colloidal gold test paper, protein chip and the like;

the sample may be a colorectal-related sample of the subject, such as colorectal cells, tissue, and blood, lymph fluid, and the like.

The product may be a kit.

More preferably, the product further comprises any one or more of the following:

a1) detecting CD3 ⁺ T/CD8 ⁺ (ii) a substance of T expression profile;

a2) substances for detecting the expression of M2-like tumor-associated macrophages.

The invention proves that the high expression of ILT5 in colorectal cancer causes CD3 through research ⁺ And CD8 ⁺ T cell infiltration is reduced, an independent predictor of low patient survival. CD3 ⁺ /CD8 ⁺ The T in combination with ILT5 can better predict the prognosis (including OS and PFS) and the clinical pathological stage of a patient; meanwhile, ILT5 regulates M2-like polarization of Tumor Associated Macrophages (TAMs), and particularly, the high expression of ILT5 regulates the polarization of M2-like TAM, so that the ILT5 is an unfavorable predictor for prognosis of colorectal cancer patients.

In a third aspect of the present invention, there is provided a use of a substance inhibiting ILT5 gene and expression product and/or activity reduction thereof for at least one of the following b1) -b 3):

b1) reversing an immunosuppressive T cell/TAM microenvironment, or preparing a product for reversing an immunosuppressive T cell/TAM microenvironment;

b2) inhibiting tumor growth, or preparing a product for inhibiting tumor growth;

b3) colorectal cancer treatment or preparation of a product for colorectal cancer treatment.

Wherein the T cell is effector T cell, specifically including but not limited to CD3 ⁺ T cells and CD8 ⁺ T cells. The study proves that the knock-down of ILT5 induces CD3 ⁺ And CD8 ⁺ T cell infiltration, inhibition of CD163 ⁺ Monocytes infiltrate and limit tumor growth.

The tumor is colorectal cancer.

Substances that inhibit the ILT5 gene and its expression products and/or reduced activity include, but are not limited to, RNA interfering molecules or antisense oligonucleotides directed against ILT5, small molecule inhibitors, shRNA, siRNA, substances that effect lentiviral infection or gene knock-out, and specific antibodies directed against ILT5 itself or molecules upstream and downstream thereof, such as anti-ILT 5 antibody; further, the antibody is a human antibody or a murine antibody.

In a fourth aspect of the present invention, there is provided a composition comprising a substance that inhibits reduction of ILT5 gene and its expression product and/or activity.

Substances that inhibit the reduction of ILT5 gene and its expression products and/or activity, including but not limited to RNA interference molecules or antisense oligonucleotides against ILT5, small molecule inhibitors, shRNA, siRNA, substances that effect lentiviral infection or gene knock-out, and specific antibodies against ILT5 itself or molecules upstream and downstream thereof, such as anti-ILT 5 antibody; further, the antibody is a human antibody or a murine antibody.

In a fifth aspect of the invention, there is provided the use of a composition as described above in any one or more of:

b1) reversing an immunosuppressive T cell/TAM microenvironment, or preparing a product for reversing an immunosuppressive T cell/TAM microenvironment;

b2) inhibiting tumor growth, or preparing a product for inhibiting tumor growth;

b3) colorectal cancer treatment or preparation of products for colorectal cancer treatment.

The product may be a pharmaceutical or a test agent, which may be used for basic research.

According to the invention, when the product is a medicament, the medicament further comprises at least one pharmaceutically inactive ingredient.

The pharmaceutically inactive ingredients may be carriers, excipients, diluents and the like which are generally used in pharmacy. The composition can be prepared into oral preparations such as powder, granule, tablet, capsule, suspension, emulsion, syrup, and spray, external preparations, suppositories, and sterile injectable solutions according to a conventional method.

Such pharmaceutically inactive ingredients, which may include carriers, excipients and diluents, are well known in the art and can be determined by one of ordinary skill in the art to meet clinical criteria.

Preferably, the carriers, excipients and diluents include, but are not limited to, lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, and the like.

Preferably, the medicament of the present invention can be administered into the body by known means. For example, by intravenous systemic delivery or local injection into the tissue of interest. Optionally via intravenous, transdermal, intranasal, mucosal or other delivery methods. Such administration may be via a single dose or multiple doses. It will be understood by those skilled in the art that the actual dosage to be administered in the present invention may vary greatly depending on a variety of factors, such as the target cell, the type of organism or tissue thereof, the general condition of the subject to be treated, the route of administration, the mode of administration, and the like.

Preferably, the subject to which the medicament is administered may be a human or non-human mammal, such as a mouse, rat, guinea pig, rabbit, dog, monkey, chimpanzee, or the like.

At the same time, the above-described protocol is equally effective for the cognate molecule of ILT5, paired immunoglobulin-like receptor B (PIR-B), in mice.

The beneficial technical effects of the technical scheme are as follows:

the above technical scheme proves that ILT5 is highly expressed in CRC cells, and as a poor prognosis biomarker, ILT5 derived from tumor can inhibit infiltration of T cells, especially CD8 ⁺ Infiltration of T cells and directing M2-like polarization of TAMs creates an inhibitory Tumor Immune Microenvironment (TIME). Inhibition of tumor-derived ILT5 restores immunosuppressive TME and inhibits the progression of colorectal cancer.

The research result of the application discovers the expression of ILT5 in solid tumor cells for the first time, and provides that ILT5 can be used as a potential immune target and a prognostic marker of colorectal cancer, so that the application has good value in practical application.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.

FIG. 1 high expression of ILT5 in colorectal cancer.

(A-C) ILT5 is enriched in colorectal cancer tissue relative to adjacent normal tissue. (A) IHC analysis showed a typical image of ILT5 expression in colorectal cancer tissue. Brown particles are defined as positive staining. ILT5 is mainly expressed in the cell membrane and cytoplasm of tumor cells. A scale: 50 μm. (B) Statistical results for ILT5 expression in 129 colorectal cancer tissues. (C) The GEO dataset was used to compare the expression of ILT5 in colorectal cancer and corresponding normal tissues, including 90 primary colon tumors and normal colon tissue (GSE33113), 203 primary rectal tumors and 160 matched mucosal controls (GSE 87211). (D-F) most colorectal cancer cell lines show higher expression of ILT5 at the mRNA and protein levels compared to the human colonic epithelial cell line NCM 460. (D) Analysis by real-time quantitative pcr (qpcr) showed gene expression of ILT5 in different human colorectal cancer cell lines. mRNA levels in each cell line were normalized to relative values for GAPDH expression, and then ILT5 levels in NCM460 cells were adjusted to 1. The results shown in the histogram are the mean ± standard deviation of three independent experiments. (E) The protein level of ILT5 was measured in different cell lines by western blot. The statistical results of ILT5 expression from three independent western blot analyses are shown in (F). P <0.05 × p <0.01 × p <0.001 × p <0.0001.

FIG. 2 tumor-derived ILT5 predicts advanced disease and poor patient survival.

(A-D) ILT5 expression was positively correlated with tumor infiltration depth, regional lymph node involvement, distant metastasis and TNM staging. The high ILT5 group had deeper tumor infiltration (a), more regional lymph node involvement (B), more distant metastasis (C), and late TNM staging (D) compared to the low ILT5 group. Fractions of IHC > 5 and <5 were defined as high and low expression of ILT5, respectively. (E) In our patient cohort, OS was significantly better in the ILT5 low group patients than in the ILT5 high group patients than in the ILT5 high group patients. The assay contained 129 patients, with cut-off values for high and low expression of ILT5 identical to those in (A-D). (F) In the GSE33113 dataset, low ILT5 expression was associated with PFS elongation. The cut-off values for high and low expression of ILT5 are median. P <0.05 × p <0.01 × p <0.001 × p <0.0001. Abbreviations: HR, hazard ratio; overall survival rate; PFS, progression free survival.

FIG. 3 expression of ILT5 was negatively correlated with T cell infiltration in colorectal cancer.

(A-C) ILT5 expression in TME vs CD3 by IHC analysis ⁺ T cell infiltration is inversely related. (A) ILT5 expression and CD3 ⁺ /CD4 ⁺ /CD8 ⁺ /FOXP3 ⁺ Representative images of T cell infiltration. ILT5 and CD3 were performed on each paraffin-embedded tissue ⁺ /CD4 ⁺ /CD8 ⁺ /FOXP3 ⁺ And (6) dyeing. A scale: 50 μm. (B-C)129 patients CD3 ⁺ Statistics of the number of T cells (B) and the proportion of TILs (C). (D-E) expression of ILT5 with CD3 ⁺ CD4 in T cells ⁺ /CD8 ⁺ /FOXP3 ⁺ The proportion of T cells was not relevant. (D) IHC analysis showed that all T cell subsets (including CD 4) were present in patients with high levels of ILT5 compared to patients with low levels of ILT5 ⁺ 、CD8 ⁺ And FOXP3 ⁺ T cells) were significantly reduced. (E) CD4 in T cells based on differences in expression of ILT5 ⁺ 、CD8 ⁺ And FOXP3 ⁺ The proportion of T cells did not change significantly. (F) Expression of ILT5 was independent of IFN- γ levels in T cells. Based on the difference in expression of ILT5, IFN-. gamma. ⁺ There was no difference in the number and ratio of T cells. P<0.05**p<0.01***p<0.001****p<0.0001。

FIG. 4 high expression of ILT5 and CD3 ⁺ /CD8 ⁺ T cell depletion in combination is a more powerful indicator of poor patient prognosis. (A-B) Low CD3 ⁺ /CD8 ⁺ T cell infiltration predicts low overall survival of patients. And CD3 ⁺ (A)/CD8 ⁺ (B) CD3 in patients with lower T cell infiltration ⁺ /CD8 ⁺ T cell infiltration is relatively highMany patients showed good OS. Infiltrative CD3 ⁺ Or CD8 ⁺ The mean value of the T cell number was defined as the critical value for high and low infiltration. (C) And high CD8 ⁺ Low CD8 compared to patients with T cell infiltration ⁺ Patients with T cell density had deeper tumor infiltration. (D) High ILT5 levels in combination with low CD3 ⁺ T cell frequency is a stronger predictor of poor patient OS. The OS of the ILT5 high CD3 low group patients was significantly shorter than that of the ILT5 low CD3 high group patients, with higher HR values (2.515) compared to any single marker group (ILT 5 group only HR ═ 1.759, CD3 only ⁺ T cell group HR 1.819). (E) Patients in the ILT5 high CD3 low group showed deeper depth of infiltration and later tumor stage compared to patients in the ILT5 low CD3 high group. (F) High ILT5 levels in combination with low CD8 ⁺ T cell infiltration is a stronger predictor of OS in poor patients. OS (3.202) in patients with ILT5 high CD8 low group was significantly shorter than in ILT5 low CD8 high group, even in combination with ILT5 and CD3 ⁺ T cell infiltration (HR 2.515) was also higher. (G) Patients in the ILT5 high CD8 low group showed deeper tumor infiltration and later tumor stage compared to other patients. P<0.05**p<0.01***p<0.001****p<0.0001。

Abbreviations: HR, hazard ratio; OS, overall survival; PFS, progression free survival.

FIG. 5.ILT5 high expression predicts M2-like polarization of TAM.

High expression of (A-C) ILT5 revealed M2-like (CD 163) ⁺ ) Macrophages rather than total (CD 68) ⁺ ) The TAM wetting increased. (A) Representative images of ILT5 expression, M2-like and total TAM infiltration in colorectal cancer are shown. A scale: 50 μm. (B) shows statistics of the total TAM density expressed by 129 patients based on ILT 5. (C) Expression of ILT5 was positively correlated with the number and proportion of M2-like TAMs in 129 patients. (D-E) high levels of ILT5 in the public database positively correlated with m 2-like TAM infiltrates. The correlation (%) of ILT5 with the level of infiltration of m 2-like TAMs was analyzed using the online tools TIMER2.0(D) and GEPIA2 (E). P<0.05**p<0.01***p<0.001****p<0.0001。

FIG. 6.ILT 5-regulated TAMs M2-like polarized patients had poor prognosis. (A) M2-like (CD 163) ⁺ ) Infiltration of TAMs is poorly correlated with patient OS. By infiltrating CD163 ⁺ Average of TAMsThe values are used as the boundary values of the high and low m 2-like TAMs. (B) Patients with high M2-like TAM infiltration had deeper tumor infiltration depth, more frequent lymph node involvement and advanced tumor progression compared to patients with low M2-like TAM infiltration. (C) High ILT5 combined with high M2-like TAMs predicted patient OS to be significantly shorter compared to low ILT5 and low M2 TAM infiltrates. Notably, the HR value for OS is 2.277, which is much higher than the values for ILT5(HR ═ 1.759) or M2-like TAM (HR ═ 1.773) alone. (D) Patients with high ILT5 and M2-like TAM infiltrates had further depth of infiltration, node involvement, and TNM staging than the ILT 5-low M2-low group.

*p<0.05；**p<0.01；***p<0.001；****p<0.0001。

Abbreviations: HR, hazard ratio; OS, overall survival; PFS, progression free survival.

FIG. 7 PIR-B gene knock-down restores immunosuppressive T cell/TAM environment and tumor growth. PIR-B gene knockout significantly inhibited the growth of C57BL/6 mouse transplantable tumors. A mouse colon cancer cell line MC38 (1X 10) infected with PIR-B knockout lentivirus or control lentivirus ⁶ Cells/mouse) were subcutaneously inoculated into 6-8 week old female C57BL/6 mice. Tumor size was measured every 3 days and expressed as mean ± SD (n-5 mice/group). PIR-B gene knock-out in (B-C) MC38 cells significantly reduced the final size and weight of the transplanted tumor. At the end of the experiment, the tumor size (B) and weight (C) of each group were expressed as mean ± SD. Scale bar: 1 cm. (D-F) PIR-B inhibition significantly increased CD3 ⁺ And CD8 ⁺ Infiltration of T cells, but decreased infiltration of M2-like TAMs. Serial colorectal cancer sections were stained for PIR-B, CD3, CD8, F4/80 and CD 163. Representative images of IHC staining of the above molecules are shown in (D) and statistical results in (E and F). A scale: 50 μm. (G and H) PIR-B Gene knockout significantly increased CD3 in peripheral blood of tumor-bearing mice as analyzed by flow cytometry ⁺ And CD8 ⁺ Percentage of T cells (G), but reduced CD163 ⁺ Percentage of monocytes. Total white blood cell count as CD45 ⁺ And (4) calculating cells. F4/80 staining labeled monocytes.

*p<0.05；**p<0.01；***p<0.001；****p<0.0001.

Abbreviations: LV shNC, MC38 cells transfected with control lentivirus; LV-shPIR-B, transfected PIR-B lentivirus knockout MC38 cells.

FIG. 8.CD3 ⁺ /CD8 ⁺ The relationship between T cell expression and patient prognosis. (A-C) Low levels of CD3 were associated with deeper infiltration depth (A), increased lymph node involvement (B) and advanced tumor stage (C). Infiltrative CD3 ⁺ The mean number of T cells was defined as the cutoff value for the high CD3 and low CD3 groups. (D-E) Low levels of CD8 were associated with increased lymph node involvement (D) and advanced tumor stage (E). Infiltrative CD8 ⁺ The mean number of T cells was defined as the cutoff value for the high CD8 and low CD8 groups.

FIG. 9 is a graph of PIR-B knock-out efficiency determined by real-time PCR and Western blot analysis. (A-C) the expression of PIR-B in MC38 cells transfected with shRNA vectors was detected by real-time PCR (A) and western blot (B), the statistical results of which are shown in (C). P < 0.05; p < 0.01; p < 0.001; p <0.0001.

Abbreviations: LV shNC, MC38 cells transfected with control lentivirus; LV-shPIR-B, transfected PIR-B lentivirus knockout MC38 cells.

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

The present invention will now be further described with reference to specific examples, which are provided for the purpose of illustration only and are not intended to be limiting. If the experimental conditions not specified in the examples are specified, the conditions are generally as usual or as recommended by the reagents company; reagents, consumables and the like used in the following examples are commercially available unless otherwise specified.

The invention is further illustrated by the following examples, which are not to be construed as limiting the invention thereto. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The following examples are test methods in which specific conditions are indicated, and are generally carried out under conventional conditions.

Examples

Materials and methods

Patient and tissue samples

Under approval by the review board and ethics committee, we collected paraffin-embedded tumor and normal tissues of 129 consecutive colorectal cancer patients during the 1 month of 2013 to 12 months of 2015. All patients received a first stage of surgery without chemotherapy, radiation therapy or targeted therapy.

Immunohistochemical analysis

Immunohistochemical staining was used to detect expression of ILT5, infiltration of T cell and macrophage subsets in paraffin-embedded human and mouse colorectal cancer tissues. The following primary antibodies were used: anti-human ILT5(1:100, Immunoway, Cat # YN1914), anti-human CD3(ready-to-use, Fuzhou Maixin, Cat # Kit-0003), anti-human CD4(ready-to-use, Fuzhou Maixin, Cat # RMA-0620), anti-human CD8(ready-to-use, Fuzhou Maixin, Cat # MAB-0021), anti-human FOXP3(1:200, Abcam, Cat # 215206), anti-human IFN- γ (1:400, Abcam, Cat # 036), anti-human CD68(ready-to-use, Fuzhou Xinxin, Cat # Kit- γ), anti-human CD 3876 (ready-to-CD 39163, Cat # To-0026), anti-human CD68(ready-to-use, Cat # Tou Mainy # Toxin, Cat # 0026), anti-human CD68 (ready-CD 1, Cat # Tou-US # 4, Cat # Tou-0023, Cat # To # Toxin, Cat # To # 0026), anti-CD 389 # To # 19, anti-CD 2, anti-human CD 2, anti-CD 15, anti-human CD 2, Cat # To # 3, No. 2, No. 3, No. 2, No. 3, No. 2, No. To # To, abcam, Cat # ab183685), anti-mouse CD8(1:500, Abcam, Cat # ab217344), anti-mouse F4/80(1:500, CST, Cat #70076T), anti-mouse CD163(1:500, Abcam, Cat # ab 182422). Sections incubated with mouse or rabbit anti-igG antibody served as negative controls. The primary antibody was detected by a mouse/rabbit streptomyces bifidobacterium detection system (universal SP method).

Evaluation of ILT5 expression and immune cell infiltration

Each slide was randomly sectioned under a 400x magnification microscope for 5 fields of view and evaluated by two independent investigators. The staining intensity of ILT5 was determined according to staining intensity and percentage of positive cells. The staining intensity was defined as: 0, none, 1, weak, 2, medium, and 3, strong. The percentage scores of the positive cells are respectively 1 point (less than or equal to 25 percent), 2 points (26-50 percent), 3 points (51-75 percent) and 4 points (more than or equal to 76 percent). The final score of the slice is determined by the product of the two scores. The cut-off values for low and high expression of ILT5 staining intensity were respectively<5 and 5 or more. Cell infiltration was counted under a uniform 400X microscope. Using CD3 ⁺ T proportion in Total immune cells the infiltration rate of T cells was determined using CD4 ⁺ /CD8 ⁺ /FOXP3 ⁺ /IFN-γ ⁺ T cells in CD3 ⁺ Ratios among T cells the proportion of different T cell subsets was calculated. The proportion of M2 macrophages was calculated from the ratio of cell counts of CD163 in CD 68. The mean value of T cell counts and the mean percentage of M2-like TAMs infiltration were chosen as the cut-off value for the intensity of immune cell infiltration.

Bioinformatics analysis

ILT5 expression in colorectal cancer and corresponding normal tissues was analyzed using the data series GSE33113 and GSE87211 from the GEO dataset, including 90 primary colon cancers and 6 normal colon tissues, 203 primary rectal tumors and 160 matched mucosal controls. Patient clinical data from GSE33113 was used for analysis of Progression Free Survival (PFS) based on ILT5 expression. Tumor immune evaluation resources (TIMER2.0) on-line tool and GEPIA2 on-line tool were used to analyze the correlation of ILT5 with the level of M2-like macrophage infiltration.

Tumor cell line experiments and PIR-B Gene knockout

Human CRC cell lines (DLD-1, SW620, SW480, HT29, HCT116, LOVO) and mouse CRC cell line MC38 were purchased from the cell resource center of Chinese academy of sciences (Beijing, China). The human colonic epithelial cell line NCM460 was purchased from American Type Culture Collection (Manassas, VA, USA). DLD-1, SW620, SW480, LOVO and MC38 cells were cultured in RPMI-1640 medium (Gibco, Thermo Fisher Scientific, USA) and 10% fetal bovine blood was addedQing (FBS; Gibco, Thermo Fisher Scientific, USA). NCM460, HCT116 and HT29 cells were cultured in DMEM medium (Gibco, Thermo Fisher Scientific, USA) supplemented with 10% fetal bovine serum. Lentiviruses used for PIR-B knockdown were purchased from Genecopoeia. MC38 at 1 × 10 ⁵ Was seeded in six well plates. After 24 hours, 1ml of fresh medium containing PIR-B knockdown or control lentivirus (MOI: 5-10) was added to each well. PIR-B knockdown MC38 cells were purified 48 hours after transfection by adding 2. mu.g/ml puromycin for 5 days.

RNA extraction and real-time fluorescent quantitative PCR

Total RNA was extracted from human and mouse CRC cell lines using the RNAfast200 RNA extraction kit (Fastagen, Shanghai, China) and reverse transcribed using the Evo M-MLV RT premix (Accurate Biology). q-PCR was performed using 2 × AccurateTaq Master Mix (Accurate Biology). The relative expression levels of ILT5 and PIR-B mRNA were analyzed according to the comparative Ct method and normalized to GAPDH. Primer sequence (5 '-3'):

human ILT5 sense strand: ATGCTGCTGTGAAGGACACA

Human ILT5 antisense strand: AGATGCAGCAGCCTCAGTG

Murine PIR-B sense strand: ACCCAGGAAGAAAGCCTATATG

Murine PIR-B antisense strand: GTTCAGTTGTTCCCTTGACATG are provided.

Western blot analysis

After lysis with Ripa containing protease inhibitors, protein lysates of human CRC cell line (SW480/SW620/DLD-1/HCT1LOVO/HT29) and colonic epithelial cell line (NCM460) were collected. The protein concentration of the lysates was determined using the BCA kit (p0010s, Beyotime, China). The gel was electrophoresed with 10% SDS polyacrylamide gel, and then transferred onto a PVDF membrane in a membrane transfer solution. After the electrotransformation was completed, the PVDF membrane was blocked with 5% milk at room temperature for 1 hour, and then incubated with an anti-ILT 5 antibody (1:1000, immunoway, Cat # YN1914) or an anti-GAPDH antibody (1: 10000; Wuhan Sanying Biotechnology; Cat #10494-1-ap) overnight at 4 ℃. The goat anti-rabbit secondary antibody was then conjugated to horseradish peroxidase (1: 10000; Wuhan Sanying Biotechnology; Cat # sa00001-2) with a primary antibody. Protein bands were observed using Enhanced Chemiluminescence (ECL) detection reagent (CWBIO, Beijing, China).

Flow cytometry analysis

After surface staining with different fluorescently labeled specific antibodies, mouse CD163 was determined by flow cytometry ⁺ Markers for monocytes and T cells. The following mouse antibodies were used: APC-anti-mouse CD163(Cat #155305, PE-anti CD80), FITC anti-mouse F4/80(Cat #123108), FITC-anti-CD3(Cat #100204), Percp5.5-anti-CD4(Cat #100434), APC-anti-CD8(Cat #100712), PE-anti-CD45(Cat # 147712). All antibodies were purchased from Biolegend. Stained cells were analyzed on a FACS Calibur flow cytometer (BD Bioscience) and data were analyzed using FlowJo10 software (Tree Star, Inc; Ashland; OR).

In vivo experiments

Approved by the animal welfare ethics committee at the central hospital in south China, female C57BL/6 mice (6-8 weeks old) were purchased from Beijing Viewsolidd Biotechnology, Inc. (Beijing, China) and raised under specific SPF conditions. Mice were randomly divided into two groups (n-5/group). MC38 cells infected with control lentivirus or PIR-B knock-out lentivirus were injected subcutaneously into the flank of mice (1X 10) ⁶ Cell/mouse). Tumor size was measured every 3 days and tumor volume was calculated to be 0.5X length X width ² 。

When the tumor grew to the size limit (20 mm), the mice were euthanized and the tumors were isolated and weighed. Peripheral blood was isolated and the frequency of immune cell infiltration was analyzed by flow cytometry. Immunohistochemistry detects the infiltration of immune cells in tumor tissues.

Survival follow-up and statistical analysis

All patients were followed up on the phone to obtain time-to-live data. The last follow-up date was 20/8/2021. Statistical analysis was performed using GraphPad Prism 8.0 Software (GraphPad Software inc., USA). The correlation of ILT5 expression with clinical pathology was analyzed using Fisher's exact test, and differences between groups were assessed using unpaired Student's t test. Survival curves were plotted using Kaplan-Meier and log rank test. Statistical significance was set at P < 0.05.

Results

High expression of ILT5 in colorectal cancer

We collected 129 paraffin-embedded tumor tissues of colorectal cancer patients and 70 corresponding paracancerous normal tissues and assessed expression of ILT5 by IHC staining. We found that ILT5 is mainly expressed in the cytoplasm of colorectal cancer cells (fig. 1A), and that ILT5 is significantly higher in colorectal cancer tissues than in adjacent normal tissues (fig. 1A, 1B). We also determined the expression of ILT5 in the colon and rectal cancer datasets of the GEO database. As shown in fig. 1C, ILT5 expression was significantly improved in both colon and rectal cancer tissues compared to the corresponding normal tissues. To further validate the expression of tumor-specific ILT5, we tested the expression of ILT5 at the mRNA and protein levels in six colorectal cancer cell lines and the colorectal epithelial cell line NCM 460. We found that mRNA expression of ILT5 was much higher in most CRC cell lines than NCM460 (fig. 1D). Meanwhile, almost all tumor cell lines showed significantly higher expression of ILT5 protein compared to NCM460 by western blot analysis (fig. 1E, 1F). In conclusion, ILT5 is highly expressed in colorectal cancer cells.

2. Tumor-derived ILT5 is predictive of later disease stages and poor patient survival

To determine the significance of ILT5 expression, we analyzed the clinicopathological features and survival rate of 129 colorectal cancer patients based on ILT5 levels (table 1). We found that the ILT5 high expressing group patients showed deeper tumor infiltration (fig. 2A), more regional lymph node involvement (fig. 2B), more frequent distant metastasis (fig. 2C) and later TNM staging (fig. 2D) compared to the ILT5 low expressing group patients. Survival analysis showed that OS was longer in patients with ILT5 low expression compared to the ILT5 high expression group (fig. 2E). We also analyzed the predictive value of ILT5 for PFS using the GEO database. As expected, low ILT5 expression correlated with PFS elongation (fig. 2F). Taken together, high expression of ILT5 in colorectal cancer is predictive of a later stage of disease and poorer patient survival.

Expression of ILT5 negatively correlated with T cell infiltration

Since T cells are the most important regulators and effectors of tumor immune clearance, we studied ILT5 by IHCCorrelation of expression with T cell density and subpopulation distribution. We found that in colorectal cancer tissues, high ILT5 expression was associated with CD3 in TME ⁺ Decreased T cell infiltration was associated (FIGS. 3A-C). Then we passed the test for CD4 ⁺ 、CD8 ⁺ And FOXP3 ⁺ T cell populations were explored to explore how ILT5 regulates T cell subset distribution. As shown in FIG. 3D, enrichment of ILT5 and CD4 in TME ⁺ T cell, CD8 ⁺ T cells and FOXP3 ⁺ The number of T cells is inversely related. However, the proportion of these T cell subsets in total T cells did not differ significantly (fig. 3E), suggesting that tumor-derived ILT5 may reduce T cell recruitment rather than regulate its differentiation. To investigate whether ILT5 affects T cell killing ability, we analyzed IFN- γ levels in T cells based on ILT5 levels. Unfortunately, we did not observe differences in T cell-produced IFN- γ between the two groups (fig. 3F), indicating that ILT5 did not significantly alter T cell function. These results indicate that ILT5 can inhibit T cell infiltration in colorectal cancer TME, but does not affect T cell subpopulation distribution and killing ability.

High expression of ILT5 and CD3 ⁺ /CD8 ⁺ T cell depletion in combination is a more powerful indicator of poor patient prognosis

To elucidate the clinical significance of ILT5 in regulating T cell infiltration, we investigated the OS and clinical pathological features of patients based on the amount of infiltration by different T cell subsets. We found that CD3 compared to patients with low CD3 infiltration ⁺ Patients with high T cell infiltration (high CD3 group) showed beneficial OS (fig. 4A). We also observed deeper tumor infiltration, more lymph node metastasis, and later TNM staging, but no statistical significance, for the low CD3 expression group compared to the high CD3 expression group (fig. 8A-C). Low CD8 in TME ⁺ T cell infiltration was likely associated with poor patient OS (fig. 4B) and deeper tumor infiltration (fig. 4C), more lymph node metastasis and worse TNM staging (fig. 8D-E). However, based on CD4 ⁺ T cells and FOXP3 ⁺ In the results of the number of T cell infiltrates, we did not observe significant survival differences, suggesting that their clinical value may be negligible.

Next, we analyzed the expression based on ILT5 and CD3 ⁺ T/CD8 ⁺ Survival of patients with T cell infiltration. Expression level according to ILT5 and CD3 ⁺ Degree of T cell infiltration, dividing patients into 4 subgroups: ILT5 high CD3 high (n-25), ILT5 high CD3 low (n-43), ILT5 low CD3 high (n-32) and ILT5 low CD3 low (n-29). The results showed that the OS was significantly longer in the ILT5 low CD3 high group than in the ILT5 high CD3 low group, compared to ILT5 expression alone or CD3 ⁺ The risk rate of OS (HR) was higher in the T cell density group compared to the one indicating that the combination biomarker is a stronger predictor of patient survival (fig. 4D). In addition, the combination of high ILT5 and low CD3 predicted more advanced infiltration depth and TNM staging (fig. 4E). Similarly, according to ILT5 expression and CD8 ⁺ T cell infiltration also produced four subgroups: ILT5 high CD8 high (n-24), ILT5 high CD8 low (n-44), ILT5 low CD8 high (n-26) and ILT5 low CD8 low (n-35). Based on ILT5 expression and CD8 ⁺ Survival of patients with T cell infiltration groups was shown to be comparable to ILT5 and CD3 ⁺ Similar trend in T cell association group (fig. 4F). The ILT5 high CD8 low group also showed more advanced infiltration depth and TNM staging (fig. 4G). In conclusion, high expression of ILT5 in colorectal cancer leads to CD3 ⁺ And CD8 ⁺ T cell infiltration is reduced, an independent predictor of low patient survival. CD3 ⁺ /CD8 ⁺ The T in combination with ILT5 can better predict the prognosis and clinical pathological stage of patients.

High expression of ILT5 predicts M2-like polarization of TAMs

TAMs are usually polarized in TME to an M2-like phenotype and are the most common and critical tumorigenic immune cells in colorectal cancer. To investigate the ilm 5-induced TAM infiltration and subpopulation distribution, we performed IHC staining of CD68 and CD163 in 129 colorectal cancer patients. Although we did not find expression of ILT5 and CD68 ⁺ There was a clear correlation between macrophage densities (FIGS. 5A, 5B), but we observed M2-like TAM (CD 163) in TME in patients with high levels of ILT5 (see FIG. 5A, 5B) ⁺ ) Is significantly higher than that of ILT5 low level patients (FIGS. 5A, 5C). These results indicate that ILT5 may polarize rather than recruit TAMs. We have shown similar results in TIMER2.0 and GEPIA2 databases, i.e., ILT5 expression was positively correlated with infiltration of M2-like TAM (FIGS. 5D, 5E). In conclusion, our results show that ILT5 expression and density of M2-like TAMIs in positive correlation.

ILT 5-regulated TAMs M2-like polarization is predictive of poorer prognosis in patients

To further explore the significance of ILT5 in modulating TAM polarization, we analyzed the pathoclinical parameters and OS of colorectal cancer patients based on varying degrees of infiltration by M2-like macrophages. The results showed that patients in the high M2 group had poor OS, deeper infiltration depth, more regional lymph node metastasis and poorer TNM staging (fig. 6A, 6B), indicating that M2-like macrophages are predictors of poor prognosis. Furthermore, we compared the difference in clinical characteristics and patient survival based on ILT5 expression and M2-like TAMs density. As expected, patients with high ILT5 expression and M2-like TAM enrichment showed poorer OS and more advanced depth of infiltration, regional lymph node metastasis and TNM staging compared to those with low M2 expression of ILT5 (fig. 6D). These results indicate that high expression of ILT5 modulates the polarization of M2-like TAMs, an unfavorable predictor of patient prognosis.

PIR-B gene knock-down reversible immunosuppressive T cell/TAM microenvironment and inhibition of tumor growth

To verify how ILT5 modulates the tumor immune microenvironment in vivo, we established a CRC transplantation model in C57BL/6 mice using MC38 cells. The MC38 cells transfected with PIR-B knockdown or control lentivirus were plated at 1X 10 ⁶ The/number was injected subcutaneously into the right flank of the C57BL/6 mice. The efficiency of PIR-B knockdown was determined by real-time PCR and Western blot analysis (FIGS. 9A-C). When the tumor grew to the size limit (20 mm in length), the mice were euthanized, and then the tumor and peripheral blood were isolated for subsequent experiments. We found that PIR-B gene knockdown slowed tumor growth compared to the control group (fig. 7A, 7B). The final tumor weights of each group showed similar results (fig. 7C). The mice tumors were then encapsulated with wax blocks and sections were immunohistochemically stained. As shown in FIGS. 7D-F, PIR-B Gene knockdown in tumor tissue showed CD3 ⁺ And CD8 ⁺ T cell infiltration increased significantly, but M2-like TAMs decreased in density. We also determined immune cell subpopulations in peripheral blood by flow cytometry analysis. As shown in FIG. 7G, PIR-B gene knockdown increased CD3 in peripheral blood TIL ⁺ And CD8 ⁺ Ratio of T cellsExample, the reduction of CD163 in TIL ⁺ Frequency of monocytes (fig. 7H). In conclusion, our results show that knock-down of ILT5 induces CD3 ⁺ And CD8 ⁺ T cell infiltration, inhibition of CD163 ⁺ Monocytes infiltrate and limit tumor growth.

T cells and tumor-associated macrophages (TAMs) are the most common and important immune cell components that make up the anti-tumor immune response in the Tumor Microenvironment (TME). T cells are the major contributors and effectors of anti-tumor immune responses. They modulate tumor immunity by a variety of means, either direct or indirect, including direct killing of target cells, assistance or inhibition of B cell production of antibodies, response to specific antigens and mitogens, and production of cytokines. These antigen-specific effector mechanisms are essential for tumor immune monitoring. Among the myriad receptors expressed by T cells, CD3 is a unique molecule capable of converting a specific antigen into an intracellular signal, thereby triggering an immune response to a tumor. CD8 ⁺ T cells, as effector T cells, recognize antigenic molecules and subsequently eliminate tumors by inducing cell death mainly through the perforin-granzyme-and Fas/Fas ligand pathways. In addition to T cells, TAMs are another major immune cell component of TME. They often display the M2 phenotype, which was found to affect almost every step of tumor cell metastasis, including invasion, vascularization, endosmosis, extravasation, establishment of a pre-metastatic niche and maintenance of circulating tumor cell survival. Therefore, the infiltration and phenotype of T cells and macrophages regulated by ILT5 are of great interest in this application.

Taken together, ILT5 is highly expressed in CRC cells and can be a biomarker for poor prognosis. ILT5 inhibits CD3 ⁺ And CD8 ⁺ Infiltration of T cells induces M2-like polarization of TAMs, producing immunosuppressive TME that promotes CRC progression. ILT5 is a potential novel immune target for CRC immunotherapy.

The invention is not the best known technology.

The above embodiments are merely illustrative of the technical ideas and features of the present invention, and the purpose thereof is to enable those skilled in the art to understand the contents of the present invention and implement the present invention, and not to limit the protection scope of the present invention. All equivalent changes and modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention.

Claims (10)

1. Application of substances for detecting expression levels of ILT5 gene and expression products thereof in preparing products for diagnosing, detecting, monitoring or predicting colorectal cancer progression.

2. The use according to claim 1, wherein the ILT5 gene and its expression products are of human origin; more specifically, the ILT5 is a tumor-derived ILT 5.

3. A product for diagnosing, detecting, monitoring or predicting colorectal cancer progression, characterized in that it comprises a substance for detecting transcription of ILT5 in a sample based on a high throughput sequencing method and/or based on a quantitative PCR method and/or based on a probe hybridization method; or a substance for detecting the expression of ILT5 protein based on an immunodetection method.

4. A product according to claim 3, wherein the sample is a colorectal-related sample from a subject, including colorectal cells, tissues, blood, lymph.

5. A product according to claim 3, wherein the product is a kit.

6. A product according to claim 3, further comprising any one or more of the following:

a1) detecting CD3 ⁺ T/CD8 ⁺ (ii) a substance of T expression profile;

a2) substances for detecting the expression of M2-like tumor-associated macrophages.

7. Use of a substance inhibiting the ILT5 gene and the expression product and/or activity reduction thereof in at least one of b1) -b3) as follows:

b1) reversing an immunosuppressive T cell/TAM microenvironment, or preparing a product for reversing an immunosuppressive T cell/TAM microenvironment;

b2) inhibiting tumor growth, or preparing a product for inhibiting tumor growth;

b3) colorectal cancer treatment or preparation of a product for colorectal cancer treatment.

8. The use of claim 7, wherein said T cells are effector T cells comprising CD3 ⁺ T cells and CD8 ⁺ A T cell;

the tumor is colorectal cancer;

preferably, the substance inhibiting the ILT5 gene and its expression product and/or activity reduction includes RNA interference molecule or antisense oligonucleotide against ILT5, small molecule inhibitor, shRNA, siRNA, substance performing lentiviral infection or gene knock-out, and specific antibody against ILT5 itself or its upstream and downstream molecules, including anti-ILT 5 antibody; further preferably, the antibody is a human antibody or a murine antibody.

9. A composition comprising a substance that inhibits reduction of ILT5 gene and its expression product and/or activity;

preferably, the substance for inhibiting ILT5 gene and expression products and/or activity reduction thereof includes RNA interference molecule or antisense oligonucleotide against ILT5, small molecule inhibitor, shRNA, siRNA, substance for performing lentiviral infection or gene knock-out, and specific antibody against ILT5 itself or its upstream and downstream molecules, including anti-ILT 5 antibody; further preferably, the antibody is a human antibody or a murine antibody.

10. Use of a composition according to claim 9 in any one or more of:

b1) reversing an immunosuppressive T cell/TAM microenvironment or preparing a product for reversing an immunosuppressive T cell/TAM microenvironment;

b2) inhibiting tumor growth, or preparing a product for inhibiting tumor growth;

b3) colorectal cancer treatment or preparation of a product for colorectal cancer treatment;

preferably, the product is a medicament or a test agent for use in basic research.

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