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WO2015193702A1 - Procédé de détermination de sensibilité ou de résistance de cellules cancéreuses à un médicament anticancéreux et/ou une molécule thérapeutiquement active - Google Patents

Procédé de détermination de sensibilité ou de résistance de cellules cancéreuses à un médicament anticancéreux et/ou une molécule thérapeutiquement active Download PDF

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WO2015193702A1
WO2015193702A1 PCT/IB2014/062303 IB2014062303W WO2015193702A1 WO 2015193702 A1 WO2015193702 A1 WO 2015193702A1 IB 2014062303 W IB2014062303 W IB 2014062303W WO 2015193702 A1 WO2015193702 A1 WO 2015193702A1
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cells
anticancer drug
therapeutically active
cancer
cell
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Marialuisa Lavitrano
Leda DALPRA'
Donatella CONCONI
Emanuela Grassilli
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Bionsil SRL
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Bionsil SRL
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics

Definitions

  • the present invention relates to methods to determine the sensitivity or resistance of at least one cancer cell to at least one anticancer drug, to at least one molecule with therapeutic activity that is capable of inhibiting growth (proliferation and/or metabolism) of a tumor cell and/or inducing death, and/or capable of restoring in resistant tumor cells the sensitivity to an anticancer drug and to at least one combination of said at least one anticancer drug and said at least one molecule with therapeutic activity.
  • colon cancer is the third most common form of cancer in both men and women and the fourth leading cause of death worldwide (de Gramont, 201 1a), with 608,000 deaths per year worldwide.
  • Surgery is the first and most important therapeutic intervention. Subsequent evaluation takes into account the possibility to perform medical therapy in order to avoid relapses (recurrence). Medical therapy is indicated for treating the patient to avoid recurrence of disease after surgery.
  • stage A disease according to Dukes (B1 according to the Astler- Coller classification) cannot be candidates for surgery, which is indicated for patients with stage C disease.
  • stage B disease stage II according to the TNM classification
  • B2-3 stage II according to the TNM classification
  • oxaliplatin (OxPt) has been added to the standard 5-FU therapy, raising to 66.4% the percentage of disease-free patients at 5 years after completion of chemotherapy (de Gramont, 201 1 a).
  • chemotherapy is not yet effective in a very high percentage of patients, due to the onset of drug resistance.
  • combination chemotherapy protocols have been developed (such as 5-FU in combination with OxPt, irinotecan, capecitabine, tegafur or biologic agents such as bevacizumab and cetuximab) in order to overcome this drawback.
  • Tumor cells may display very high resistance levels.
  • first-line therapy based on 5FU determines an effective response only in 10-15% of patients with advanced stage colon carcinoma.
  • Combination of 5FU with other chemotherapy drugs leads to an increase in response up to 40- 50%, a value not yet completely satisfactory for effective therapeutic action against cancer pathologies (Longley, 2003).
  • a new isoform has been identified for the protein encoded by the BTK gene, with molecular weight comprised between 65-68kDa, that is shorter than the previously known BTK isoform with molecular weight of 77 kDa.
  • the BTK gene has always been known to be expressed only in bone marrow derived cells, such as B cells, mast cells, platelets and erythroid progenitors. Instead the aforementioned patent led to the discovery that the new shorter BTK protein isoform is expressed in several carcinoma cell lines.
  • WO 2011/123103 describes a method to evaluate the ability of a chemotherapeutic drug to induce apoptosis in a cancer cell line.
  • Apoptosis is a well known programmed cell death process normally used by organisms to eliminate cells that are too old or damaged.
  • apoptotic bodies which are phagocytosed by macrophages.
  • the method described in patent WO 201 1/123103 makes use of the iCK (microcu!ture kinetic) assay in order to assess whether cancer cells derived from known tumor cell lines undergo apoptosis in response to a particular drug which is known to be active against one or more types of cancer.
  • Such assay evaluates the morphological changes of the cell membrane that take place in cancer cells after administration of a drug, by means of temporal kinetics. Therefore this method is substantially, if not almost exclusively, addressed to screen for candidate anticancer drugs in known tumor cell lines.
  • the technical task of the present invention is to eliminate the drawbacks of the prior art, in particular to allow identification of anticancer drugs or their combination to which patient's cells are sensitive, and identification of patients in whom chemotherapy is ineffective due to the development of drug resistance.
  • the invention relates to a method to assess sensitivity or resistance of at least one cancer cell to at least one anticancer drug, at least one therapeutically active molecule capable of inhibiting growth (proliferation and/or metabolism) or of inducing death of said tumor cell, and/or capable of restoring in resistant tumor cells the sensitivity to said at least one anticancer drug, and at least one combination of said at least one anticancer drug and said at least one therapeutically active molecule, comprising the steps of:
  • tumor sample solid or liquid tumor biopsy
  • saline solution and/or culture medium supplemented with antibiotics and antifungals
  • step b) in the case of solid tumor biopsy, mechanically disrupt cells of the tumor sample obtained from step a) followed by enzymatic digestion to obtain cells to be seeded in the subsequent step;
  • step b) seed the cells obtained in step b) in cell culture dishes and treat a first portion of said cells with said at least one anticancer drug, a second portion of said cells with said at least one therapeutically active molecule, a third portion of said cells with said at least one combination of said anticancer drugs, a fourth portion of said cells with said at least one combination of said therapeutically active molecules, a fifth portion of said cells with said at least one combination of said at least one anticancer drug and said at least one therapeutically active molecule, a sixth portion of said cells is cultured without addition of drugs (untreated cells, NT);
  • step d) quantify cell growth (proliferation and/or metabolism) and/or viability and/or death of the first, second, third, fourth, fifth and sixth portions of said cells of step c) by exposure to a reagent selective for live cells or a reagent selective for metabolicaily active cells or a dedicated reagent for detection of energy consumption;
  • step e) analyze data obtained from step d) to classify cells according to their response to the treatment with an anticancer drug and/or a therapeutically active molecule.
  • Another aspect of the invention relates to a method to determine sensitivity or resistance of at least one tumor cell to a combination of at least one anticancer drug and at least one therapeutically active molecule able to inhibit growth (proliferation and/or metabolism) or induce death of said tumor cell and/or able to restore in resistant tumor cells the sensitivity to said at least one anticancer drug, comprising the steps of:
  • step b) in the case of solid tumor biopsy, mechanically disrupt cells of the tumor sample obtained from step a) followed by enzymatic digestion to obtain cells to be seeded in the subsequent step;
  • step b) seed cells obtained from step b) in cell culture dishes and treat said cells with said at least one combination of said anticancer drugs, with said at least one combination of said therapeutically active molecules, with said at least one combination of said at least one anticancer drug and said at least one therapeutically active molecule;
  • step d) quantify growth (proliferation and/or metabolism) and/or viability and/or death of said cells of step c) by exposure to a selective reagent for live cells, or to a selective reagent for metabolically active cells, or to a dedicated reagent for detection of energy consumption;
  • step e) analyze data obtained from step d) to classify cells according to their response to the treatment with an anticancer drug and/or a therapeutically active molecule.
  • the methods surprisingly allow the development of a personalized therapy for the patient. Cancer patients very often require a personalized and targeted therapy due to tumor type, genetics and heterogeneity. Therefore the possibility to obtain, through the methods according to the present invention, a rapid and reliable response about treatment success, makes possible considerable cost and time saving, in addition to being an advantage for the patient.
  • the methods according to the present invention make possible, by a surprisingly efficient and reliable single test lasting few hours, to .determine whether cancer cells of the cancer patient are sensitive or resistant to an anticancer drug, to a therapeutically active drug or to combinations thereof, with obvious saving of time and costs.
  • tumor cell it is meant to include neoplastic cells of any tumor stage or classification, derived from epithelial tumors, mesenchymal tumors, ematological tumors or tumors of the nervous tissue, in particular cells from colon carcinoma, breast cancer, ovarian cancer, stomach cancer, lung carcinoma, renal carcinoma, thyroid carcinoma or pancreatic carcinoma, but also circulating cancer cells, sarcomas and melanomas;
  • anti-cancer drug it is meant to comprise classical chemotherapeutic drugs such as 5-fluorouracil (5-FU), irinotecan or capecitabine, oxaliplatin and other platinum coordination compounds; and
  • therapeutically active molecule refers to a molecule capable of inhibiting growth (proliferation and/or metabolism) or inducing death of said tumor cell and/or capable of restoring in resistant cancer cells the sensitivity to said at least one anticancer drug.
  • biological drugs such as monoclonal antibodies, preferably Cetuximab, Panitumumab, Bevacizumab and kinase inhibitors such as gefitinib, erlotinib, vermurafenib, sorafenib, ibrutinib and dasatinib;
  • first portion refers to six equal amounts of a pool of cells to be defined depending on cell type, anticancer drug and therapeutically active molecule involved.
  • Figure 1 shows the results obtained with the method according to the present invention from colon carcinoma-derived cell lines HCT1 16 and HCT1 16p53KO known to be chemo-sensitive and chemo-resistant, respectively.
  • Cell lines were treated with 5-fluorouracil (5-FU) and the effect of the drug was detected by exposure to a reagent which is excluded selectively from live cells (Trypan blue). Evaluation of the sensibility/resistance to 5FU by using the Trypan Blue test.
  • 5-fluorouracil 5-fluorouracil
  • Sensitive cells HCT1 16
  • HCT116p53KO e resistant cells
  • Figure 2 shows the results obtained with the method according to the present invention from colon carcinoma-derived cell lines, chemo-sensitive HCT1 16 and chemo-resistant HCT116p53KO.
  • Cell lines were treated with 5-FU and the effect of the drug was detected upon exposure to a dedicated reagent for detection of energy consumption (ATP).
  • ATP energy consumption
  • Sensitive cells HCT116
  • HCT116p53KO e resistant cells
  • Figure 3 shows the results obtained with the method according to the present invention from colon carcinoma-derived cell lines, chemo-sensitive HCT116 and chemo-resistant HCT1 16p53KO.
  • Cell lines were treated with 5-FU and the effect of the drug was detected by a selective reagent for metabolically active cells (MTT).
  • MTT metabolically active cells
  • Figure 4 shows the results obtained with the method according to the present invention from the chemoresistant colon carcinoma-derived cell lines HCT1 16p53KO, DLD-1 and SW480.
  • Cell lines were treated with 5-FU and the effect of the drug was detected by exposure to a reagent which is excluded selectively from live cells (Trypan blue).
  • HCT1 16p53KO, DLD-1 , SW480 resistent cells are treated with 200 uM 5FU for 72hs and then cells are counted after Trypan Blue colouring.
  • Figure 5 shows the results obtained with the method according to the present invention from the chemoresistant colon carcinoma-derived cell lines HCT1 16p53KO, DLD-1 and SW480. Cell lines were treated with Oxaliplatin (OxPt) and the effect of the drug was detected by exposure to a reagent which is excluded selectively from live cells (Trypan blue).
  • Oxaliplatin Oxaliplatin
  • HCTl 6p53KO, DLD-1 , SW480 resistent cells are treated with 50 uM OxPt for 72hs and then cells are counted after Trypan Blue colouring.
  • Figure 6 shows the results obtained with the method according to the present invention from the chemoresistant colon carcinoma-derived cell lines HCT 16p53KO, DLD-1 and SW480.
  • Cell lines were treated with a combination of 5FU and OxPt and the effect of the drug was detected by exposure to a reagent which is excluded selectively from live cells (Trypan blue).
  • HCT1 16p53KO, DLD-1 , SW480 resistent cells are treated with the combination of 200 uM 5FU + 50 uM OxPt for 72hs and then cells are counted after Trypan Blue colouring.
  • Figure 7 shows the results obtained with the method according to the present invention from the chemo-resistant colon carcinoma-derived cell line HCT 6p53KO after treatment with a combination of 5FU and Dasatinib.
  • the effect of the drug was detected by exposure to a reagent which is excluded selectively from live cells (Trypan blue).
  • HCT 6p53KO resistent cells with 200 uM 5FU, 50 nM Dasatinib or with the combination of both for 72hs and cell count after Trypan Blue colouring.
  • Figure 8 shows the results obtained with the method according to the present invention from the chemo-resistant colon carcinoma-derived cell line HCT1 16p53KO after treatment with "biological" drugs antagonist of Epidermal Growth Factor (Cetuximab and Panitumumab) and Vascular Endothelial Growth Factor (bevacizumab).
  • the effect of the drug was detected upon exposure to a selective reagent for metabolically active cells (MTT). Evaluation of the sensibility/resistance to biological drugs by using the test which evaluates the energy consumption (Cell Titer Glo Luminescent Cell Viability Assay).
  • HCT1 16p53KO chemioresistent cells have been treated with Cetuximab 10 pg/ml , Panitumumab 75 pg/ml, Bevacizumab 25 pg/m for 72hs before undergoing the test. Cetuximab vs NT p>0.05; Panitumumab vs NT p>0.05; .
  • Figure 9 shows the results obtained with the method according to the present invention from the chemo-resistant colon carcinoma-derived cell line HCT 6p53KO after treatment with "biological" drugs antagonist of Epidermal Growth Factor (Cetuximab and Panitumumab) and Vascular Endothelial Growth Factor (bevacizumab).
  • the effect of the drug was detected by exposure to a selective reagent for live cells (Calcein assay).
  • HCT1 16p53KO chemioresistent cells have been treated with Cetuximab 10 pg/ml , Panitumumab 75 pg/ml,
  • Figure 10 shows the results obtained with the method according to the present invention from the chemo-resistant ovarian carcinoma-derived cell line SKOV3 after treatment with classical chemotherapeutic agents (carboplatin, paclitaxel) and "biological" drugs antagonist of Vascular Endothelial Growth Factor (bevacizumab).
  • classical chemotherapeutic agents carboplatin, paclitaxel
  • bevacizumab "biological" drugs antagonist of Vascular Endothelial Growth Factor
  • Figure 1 shows the distribution of cell samples from colon tumors from patients tested with the method according to the present invention, who were treated only with 5-FU. The effect of the drug was detected by exposure to a reagent selective for metabolically active cells (MTT).
  • MTT metabolically active cells
  • D Distribution of delta (D) values obtained as the difference between the average of triplicate samples for the untreated and the average of triplicate samples for treatment in 135 colon samples. Each symbol represents one patient. On the left are the delta (D) of samples found to be resistant with Student's T test, on the right are the delta of samples found to be sensitive according to the same test .
  • Figure 12 shows the distribution of delta values obtained as difference between the average of triplicate samples for the untreated condition and the average of triplicate samples for the treated condition in 135 colon samples treated with 5FU, Ibrutinib or with a combination of the two drugs. Drug effect was detected by exposure to a reagent selective for metabolically active cells ( TT).
  • TT metabolically active cells
  • Distribution of delta (D) values obtained as the difference between the average of triplicate samples for the untreated and the average of triplicate samples for treatment in colon samples.
  • Figure 13 shows the distribution of delta values obtained as difference between the average of triplicate samples for the untreated condition and the average of triplicate samples for the treated condition in 135 colon samples treated with 5FU, Dasatinib or with a combination of the two drugs. Drug effect was detected by exposure to a reagent selective for metabolically active cells (MTT).
  • MTT metabolically active cells
  • Distribution of delta (D) values obtained as the difference between the average of triplicate samples for the untreated and the average of triplicate samples for treatment in 25 colon samples.
  • Figure 14 shows the distribution of delta values obtained as difference between the average of triplicate samples for the untreated condition and the average of triplicate samples for the treated condition in 29 samples from the ovary. Drug effect was detected by exposure to a reagent selective for metabolically active cells (MTT). Distribution of delta (D) values obtained as the difference between the average of triplicate samples for the untreated and the average of triplicate samples for treatment in 29 ovary samples
  • MTT metabolically active cells
  • the invention therefore relates to a method for determination of the sensitivity or resistance of at least one cancer cell to at least one anti-tumor drug, at least one therapeutically active molecule capable of inhibiting growth (proliferation and/or metabolism) or of inducing death of said tumor cell and/or capable of restoring in resistant tumor cells the sensitivity to said at least one anticancer drug, and at least to one combination of said at least one anticancer drug and said at least one therapeutically active molecule, comprising the steps of:
  • tumor sample solid or liquid tumor biopsy
  • saline solution and/or culture medium supplemented with antibiotics and antifungals
  • step b) in the case of solid tumor biopsy, mechanically disrupt cells of the tumor sample obtained from step a) followed by enzymatic digestion to obtain cells to be seeded in the subsequent step;
  • step b) seed the cells obtained in step b) in cell culture dishes and treat a first portion of said cells with said at least one anticancer drug, a second portion of said cells with said at least one therapeutically active molecule, a third portion of said cells with said at least one combination of said anticancer drugs, a fourth portion of said cells with said at least one combination of said therapeutically active molecules, a fifth portion of said cells with said at least one combination of said at least one anticancer drug and said at least one therapeutically active molecule, a sixth portion of said cells is cultured without addition of drugs (untreated cells, NT);
  • step d) quantify cell viability and/or death of the first, second, third, fourth, fifth, sixth portion of said cells from step c) by exposure to a reagent selective for live cells or to a reagent selective for metabolically active cells or to a reagent dedicated to detection of energy consumption; e) analyze data obtained from step d) to classify cells according to their response to the treatment with an anticancer drug and/or a therapeutically active molecule.
  • Another aspect of the invention relates to a method to determine sensitivity or resistance of at least one tumor cell to a combination of at least one anticancer drug and at least one therapeutically active molecule able to inhibit growth (proliferation and/or metabolism) or induce death of said tumor cell and/or able to restore in resistant tumor cells the sensitivity to said at least one anticancer drug, comprising the steps of:
  • step b) in the case of solid tumor biopsy, mechanically disrupt cells of the tumor sample obtained from step a) followed by enzymatic digestion to obtain cells to be seeded in the subsequent step;
  • step b) seed cells obtained from step b) in cell culture dishes and treat said cells with said at least one combination of said anticancer drugs, with said at least one combination of said therapeutically active molecules, with said at least one combination of said at least one anticancer drug and said at least one therapeutically active molecule;
  • step d) quantify growth (proliferation and/or metabolism) and/or viability and/or death of said cells of step c) by exposure to a selective reagent for live cells, or to a selective reagent for metabolically active cells, or to a dedicated reagent for detection of energy consumption;
  • step e) analyze data obtained from step d) to classify cells according to their response to the treatment with an anticancer drug and/or a therapeutically active molecule.
  • a further aim of the invention was to develop a method to determine tumor sensitivity/ esistance, thus allowing not only development of personalized therapy but also an evaluation of the evolution of tumor resistance to drugs.
  • the invention advantageously provides a simple and effective method applicable to any type of tumor and cell line, allowing for determination of the most suitable therapy for certain tumor processes starting from the biological material taken from the patient, and selection of the most appropriate treatment for the case under analysis.
  • the step d) for quantification of cell growth (proliferation and/or metabolism) and/or viability and/or death of the method according to the present invention is advantageously performed by exposure to a reagent selective for live cells or to a reagent selective for metabolically active cells or a dedicated reagent for detection of energy consumption.
  • the step of quantitative analysis of metabolism to verify metabolically active cells and quantify total metabolism is realized by MTT assay (3-(4,5-Dimethylthiazol-2-YI)-2,5- Diphenyltetrazolium Bromide) comprising a colorimetric reagent and a step of measurement of spectrophotometric absorbance at a wavelength of 570 nm.
  • MTT assay 3-(4,5-Dimethylthiazol-2-YI)-2,5- Diphenyltetrazolium Bromide
  • the phase of quantitative analysis of cell energy consumption (or energy metabolism), a quantitative analysis correlated with cell viability, is realized by the Cell Titer Glo Luminescent Cell Viability Assay.
  • the step of quantitative analysis of cell viability is performed by the Calcein-AM assay (acetoxy methyl Calcein) comprising a pro-fluorescent compound, and the phase of fluorimetric measurement of the emission is carried out at a wavelength of 535nm.
  • Calcein-AM assay acetoxy methyl Calcein
  • step e) for data analysis of the method according to the present invention is carried out by statistical analysis of the data obtained from step d) for categorization of cells into classes of response to treatment selected from the group consisting of :
  • the methods according to the present invention allow for determination of sensitivity or resistance of a tumor cell also in function of spectrophotometric measurement evaluating the absorbance (OD) (average value of a triplicate): i) of samples of tumor cells treated (T) with an anticancer drug and/or a therapeutically active molecule and/or a combination thereof, ii) of samples of tumor cells not treated (NT) with a drug or a combination of drugs, followed by exposure to one of said dedicated detection reagents (in particular MTT or calcein).
  • a Student's T test value ⁇ 0.05 from three identical replicas (triplicate) of the NT sample and of the T sample of tumor cells from the same patient identifies cancer cells sensitive to a specific treatment.
  • a Student's T test value ⁇ 0,05 from three identical replicas (triplicate) of the NT sample and of the T sample of tumor cells from the same patient identifies cancer cells resistant to a specific treatment.
  • the delta values ( ⁇ ) obtained as the difference between the mean absorbance (OD, average of triplicate) for not treated (NT) cancer cells and treated (T) cancer cells from sensitive samples are preferably ⁇ 0.04, more preferably from +0.04 to +0.4.
  • the delta values ( ⁇ ) obtained as the difference between the mean absorbance (OD, average of triplicate) of not treated (NT) cancer cells and treated (T) cancer cells for resistant samples are preferably ⁇ +0.02, more preferably from +0.02 to -0.2.
  • the method also identifies tumors containing a mixed population of sensitive and resistant cells wherein the delta values ( ⁇ ) obtained as the difference between the mean absorbance (OD, average of triplicate) of not treated cancer cells (NT) and treated cancer cells (T) are preferably comprised between +0.04 and +0.02.
  • measurement of absorbance is carried out by spectrophotometric or fluorimetric measurement, preferably at a wavelength from 400 to 700 nm, preferably at 570 nm (MTT assay) or 535nm (calcein assay).
  • said at least one tumor cell is a cell derived from epithelial, mesenchymal tumors, ematological tumors or tumors of the nervous tissue.
  • said cell is a carcinoma, in particular from colon cancer, breast cancer, ovarian cancer, stomach cancer, lung cancer, kidney cancer, thyroid cancer or carcinoma of the pancreas, but also circulating cancer cells, sarcomas and melanoma of any stage or tumor class.
  • said at least one tumor cell is a colon or ovarian carcinoma cell.
  • said at least one anticancer drug advantageously includes classical chemotherapeutic drugs (alkylating drugs, base analogues, intercalators, microtubule inhibitors, nitrogen mustards, etc.) or biological drugs or kinase inhibitors.
  • said classical chemotherapy drugs comprise but are not limited to 5- fluorouracil (5-FU), irinotecan or capecitabine, oxaliplatin and other platinum coordination compounds
  • said biological drugs comprise but are not limited to monoclonal antibodies, preferably Cetuximab, Panitumumab, Bevacizumab and said kinase inhibitors comprise gefitinib, erlotinib, vermurafenib, sorafenib, ibrutinib and dasatinib.
  • said therapeutically active molecule is Dasatinib or ibrutinib.
  • Said cancer cell is advantageously a colon carcinoma cell and said at least one anticancer drug is 5FU, said at least one therapeutically active molecule is dasatinib or ibrutinib and said combination of said at least one anticancer drug and said at least one therapeutically active molecule is selected from 5-fluorouracil and dasatinib or 5-fluorouracil and ibrutinib.
  • Said cancer cell is advantageously an ovarian cancer cell and said at least one anticancer drug is Paclixitel or Carboplatin, and said at least one therapeutically active molecule is bevacizumab and said combination of anticancer drugs is Paclitaxel + Carboplatin and said combination of said at least one anticancer drug and said at least one therapeutically active molecule is selected from Paclixitel, and Carboplatin, and bevacizumab and combinations of two of Paclixitel, Carboplatin or bevacizumab or Paclixitel, Carboplatin and bevacizumab.
  • the methods for determination of sensitivity or resistance of human tumor cells may include one or a combination of chemotherapeutic drugs and may be used to test new drugs and/or combinations of drugs.
  • the study of new cancer drugs is in fact updated and expanded daily with new activities of new molecules to be tested on each individual patient.
  • the methods according to the present invention have the advantage that they make possible the rapid testing of each new molecule alone or in combination, thereby allowing an effective targeted therapy for the patient.
  • said tumor sample of step a) is pre-stored at a temperature comprised between 0- 8 °C for a storage time above 5 hours and a maximum duration of 7 days, and at a temperature comprised between 0-24 °C for a storage time up to a maximum of 5 hours.
  • said physiological saline solution and/or said culture medium of step a) are supplemented with antibiotics and antifungals in a range comprised between 1-5% of the final volume of penicillin, streptomycin, gentamicin, metronidazole and amphotericin.
  • said physiological saline solution and/or said culture medium of step a) are supplemented with penicillin from 100 to 500 U/ml, streptomycin and gentamycin from 100 to 500 micrograms/ml, metronidazole from 1 to 5 micrograms/ml and amphotericin from 5 to 25 micrograms/ml.
  • cells are grown on a support for cell trofism comprising collagen or type I collagen or type IV collagen or laminin or vitronectin or fibronectin.
  • the invention relates to a method for determining sensitivity or resistance of at least one cancer cell to at least one anti-cancer drug, comprising the steps of: - ex vivo collection of a tumor sample to be tested from a patient; - Storage of the tumor sample in saline solution and/or culture medium supplemented with antibiotics and antifungals to prevent contamination until the next phase of disruption; - in the case of solid tumors, mechanical disruption followed by enzymatic digestion of the tumor sample in order to obtain cells for the subsequent seeding step and preparation of a support substrate for cell trofism in multi-well plates for culturing the so obtained cells; - seeding cells in multi-well plates so coated in culture medium supplemented with antibiotics/antifungals, support factors and growth factors; - treatment of plated cells with said at least one anticancer drug; - execution on treated and untreated cells of a test to quantify cell growth (proliferation and/or metabolism), viability and/or death by exposure to a reagent selective for live
  • a method to determine sensitivity or resistance of at least one cancer cell to at least one therapeutically active molecule capable of inhibiting growth (proliferation and/or metabolism) or inducing death of said tumor cell and/or restoring anticancer drug sensitivity in resistant tumor cells comprising the steps of: - ex vivo collection from a patient of a tumor sample to be tested; - storage of the tumor sample in saline solution and/or culture medium supplemented with antibiotics and antifungals to prevent contamination until the subsequent phase of disruption; - in the case of solid tumors, mechanical disruption followed by enzymatic digestion of the tumor sample in order to obtain cells for the subsequent seeding step and preparation of a support substrate for cell trofism in multi-well plates for culturing the so obtained cells; - seeding cells in multi-well plates so coated in culture medium supplemented with antibiotics/antifungals, support factors and growth factors; - treatment of plated cells with said at least one anticancer drug; - execution on
  • the methods particularly those in claims 1 and 2 surprisingly allow development of personalized therapy for the patient.
  • Cancer patients very often require a personalized targeted therapy due to tumor type, genetics and heterogeneity that change from patient to patient. Therefore the possibility to obtain a rapid and reliable response, by the methods according to the present invention, about the best treatment and its potential effectiveness, allows considerable cost and time saving, in addition to being an advantage for the patient.
  • the methods according to the present invention make possible, in a surprisingly efficient and reliable single test, to determine if the cancer patient has cancer cells sensitive or resistant to an anticancer drug, a therapeutically active drug or a combination thereof, with obvious time and cost saving.
  • This method may be applicable to other cancers, not only tumors of epithelial origin and circulating cancer cells.
  • the tissue sample containing tumor cells is taken over by the pathologist, after surgery, removed and transferred to a test tube containing DMEM medium supplemented with antibiotics and antifungals in a range comprised between 1 and 5% of the final volume of penicillin, streptomycin, gentamicin, metronidazole and amphotericin.
  • penicillin can be added in a range from 100 to 500 U/ml
  • streptomycin and gentamicin in a range from 100 to 500 micrograrns/ml
  • metronidazole in a range from 1 to 5 micrograms/ml
  • amphotericin in a range from 5 to 25 micrograms/ml.
  • HBSS Hank's balanced salts solution
  • DMEM medium with antibiotics comprised in a range between 1 and 5%.
  • Storage conditions of the tumor sample vary depending on the time required for the sample to travel from where it is taken to the laboratory. In case of storage above 5 hours, the sample is maintained at a temperature between 0-8 for a maximum of 7 days. Instead in case of storage for a maximum of 5 hours, the sample is maintained at a temperature comprised between 0-24 °C.
  • Antibiotics and antifungals are required in the medium to eradicate the bacterial charge possibly present in the sample. This crucial step made possible to reduce the number of contaminated samples. Tumor cells derived from tissue samples from colon and ovary are at high risk for contamination due to the presence of bacteria, and keeping samples in 5% solution of broad-spectrum antibiotics has made possible to minimize the probability of contamination.
  • the percentage of antibiotics and antifungals vary depending on the site of the tumor under analysis, ranging from the 1 % standard dose used in any cell culture up to 7%. Preferably the range is comprised between 1 % and 5%.
  • the sample is rinsed with HBSS and cut with sterile scissors until it is reduced to fragments of less than one millimeter. Fragments are subjected to enzymatic digestion in 10 ml of trypsin/EDTA for a time ranging from a minimum of 1 hour to a maximum of 1 hour and 30 minutes, depending on the consistency of the tissue.
  • the suspension is filtered through 40 micron nylon filters by crushing the cell suspension with a syringe plunger to facilitate disaggregation of pieces and passage through the filter.
  • the filtrate containing the cells is collected in a centrifuge tube with the addition of HBSS and centrifuged for 10 min at 1000 rpm. After centrifugation the supernatant is removed and the pellet containing the cells is resuspended in 2 ml of lysis solution (0.84% ammonium chloride) and left at room temperature (T) for 10 minutes. After the lysis time, 5 ml of DMEM medium are added (to stop the reaction by dilution) and the suspension is centrifuged at 1000 rpm for 0 minutes.
  • substrates tested for cell adhesion were type I collagen, laminin, vitronectin and fibronectin. All tests performed with the above listed substrates showed compatibility with the experiment; in particular, in the response tests to treatment with chemotherapeutic or therapeutic molecules, measuring viability or cell metabolism, the results were mutually comparable and compatible with those observed when using collagen prepared in the laboratory by rat tail extraction and used in previous experiments.
  • Cells are seeded in a 96-well plate at a minimum concentration of 20,000 cells per well in 100 microliters of complete medium, in triplicate for each treatment. Cell concentration may vary up to a maximum of 40.000/60.000 cells per well. After approximately 1 hour from seeding, cells are treated with the drugs of interest.
  • Treatment of colon carcinoma is with 5FU (e.g., treatment with 5FU 200 ⁇ ), ibrutinib (e.g. , treatment with 20 ⁇ ibrutinib) and Dasatinib (e.g., treatment with Dasatinib at different concentrations ranging from 2 to 100 nM).
  • Treatment of cells from ovarian cancer is with Paclitaxel, Carboplatin and Bevacizumab at final concentrations of 500 nM, 0-100 nM, 50 micrograms/ml, respectively.
  • each well receives 100 ⁇ of a solution obtained by dissolving a solution of Thiazolyl Blue Tetrazolium Bromide (MTT) in DMEM medium at a concentration of 1 mg/ml.
  • MTT Thiazolyl Blue Tetrazolium Bromide
  • the plate is transferred back to the incubator for 3 hours, which is the time required for metabolically active cells to convert the tetrazolium ring of the MTT molecule into a purple color insoluble salt (formazan salt) that precipitates inside the cells.
  • the plate is centrifuged for 10 min at 2000 rpm.
  • the tumor cells ex vivo collected from patients' tumor biopsies are treated with a single drug or a combination of anticancer drugs in order to assess sensitivity/ resistance.
  • Colon carcinoma cells were tested for sensitivity/resistance to classical chemotherapeutic agents (e.g., 5-FU, oxaliplatin, irinotecan).
  • Tumor cells derived from ovarian cancer were in turn tested for sensitivity/ resistance to the chemotherapeutic agents Paclitaxel and Carboplatin as well as to Bevacizumab.
  • chemotherapeutic agents e.g., 5-FU, oxaliplatin, irinotecan
  • Tumor cells derived from ovarian cancer were in turn tested for sensitivity/ resistance to the chemotherapeutic agents Paclitaxel and Carboplatin as well as to Bevacizumab.
  • other anticancer drugs such as those belonging to the families i) of the so-called biological drugs (monoclonal antibodies, e.g
  • Cetuximab, Panitumumab, Bevacizumab ii) of kinase inhibitors (e.g. gefitinib, erlotinib, vemurafenib, sorafenib, ibrutinib, dasatinib), iii) of classical chemotherapeutic agents (e.g. 5-FU, oxaliplatin, irinotecan).
  • chemotherapeutic agents e.g. 5-FU, oxaliplatin, irinotecan.
  • other molecules can be tested with therapeutic activity such as molecules capable of inhibiting cell cycle progression or inducing cancer cell death or restoring sensitivity to chemotherapeutic agents in resistant tumor cells.
  • Dasatinib and Ibrutinib are among these molecules without being limited to these.
  • Dasatinib is a multikinase inhibitor clinically used for the treatment of adult chronic myeloid leukemia (CML), positive for the Philadelphia chromosome (Ph+), in chronic phase, accelerated phase or blast phase with resistance or intolerance to prior therapy including Imatinib mesylate, for the treatment in adults affected by acute lymphoblastic leukemia (ALL) and for the treatment of CML with lymphoid blast phase with resistance or intolerance to prior therapy.
  • Ibrutinib is a specific BTK inhibitor still undergoing clinical trials for treatment of chronic lymphocytic leukemia.
  • Dasatinib inhibits the Abl/Src fusion protein, SRC and also BTK (Hantschel et al., Proc Natl Acad Sci USA, 2007, vol 104:13283; Rix et al., Blood 2007, 1 10: 4055).
  • Dasatinib and Ibrutinib were tested for treatment of carcinoma-derived cells, in particular colon carcinoma expressing the shorter BTK protein isoform (65-67 kDa).
  • Such therapeutically active molecules have surprisingly shown in colon carcinoma cells both their own anti-tumor activity and an activity that restores sensitivity to an anticancer drug in colon cancer cells resistant to 5FU.
  • MTT assay which is based on a metabolic reaction that correlates with cell viability.
  • the MTT assay was used to assess viability of cancer cells in presence of a chemotherapeutic drug or of other drugs and/or therapeutic molecules, or the combination thereof.
  • Assays other than MTT can be used to measure cell metabolism.
  • the MTT assay is widely used to assess cell viability. It is a simple, accurate and reproducible assay.
  • the detection assay can also consist of a colorimetric assay measuring energy consumption of live cells (ATP consumption).
  • ATP consumption energy consumption of live cells
  • Ce!ITiter-Glo® assay has been used that allows determination of the number of viable cells in culture, based on quantification of the ATP content.
  • the assay makes use of the lucife n/luciferase principle and, in presence of a specific luminescence filter, can be read at 3600 gain and 0.5 seconds.
  • the assay can be read at a wavelength ranging from 500 to 650 nm.
  • the assay can also consist of a colorimetric assay measuring activity of cellular esterases.
  • the assay employed is the Fluorescent cell counting kit (Sigma) based on the ability of cellular esterases to hydrolyze calcein-AM (3',6'-Di(0-acetyl)-2 ⁇ 7'- bis[N,N-bis32(carboxymethyl)aminomethyl]-fluorescein,tetraacetoxymethyl ester) in calcein.
  • the amount of (fluorescent) calcein produced is proportional to the number of live cells. Fluorescent emission is measured by use of a plate reader (fluorimeter) at 535 nm (excitation at 485 nm).
  • the assay can even be any one of the systems for read-out of cellular death, based on the use of vital dyes, detection of release of intracellular enzymes, cellular metabolism, loss of cell adhesion, morphological parameters or on specific systems for detection of apoptosis or necrosis (Kepp, 201 1 ).
  • HCT1 16p53KO A chemosensitive cell line, HCT1 16, and 3 chemoresistant lines, HCT1 16p53KO, DLD-1 and SW480, have been used.
  • different methods were used to evaluate viability/mortality of untreated cells, cells treated with 5-FU or other drugs commonly used for colon cancer treatment (5FU, OxPt, Cetuximab, Panitumumab, Bevacizumab) or drugs currently used for other cancer pathologies (Dasatinib). Drug concentration was selected based on dose- response curves carried out on available colon carcinoma lines.
  • ovarian cancer-derived cell line SKOV-3 is used to set up the assay and the experimental conditions for ovarian cancer cells.
  • SKOV-3 cells are used to test the concentrations of Paclitaxel, Bevacizumab and Carboplatin. The best results in terms of treatment efficacy with chemotherapy are obtained with 500nM Paclitaxel, 50pg/ml Bevacizumab and 100nM Carboplatin.
  • Figure 1 and 2 were analyzed statistically by ANOVA with Tukey's post-hoc test (significance set at 0,05).
  • Figures 1 -3 show results of experiments comparing the response to 5FU of the chemosensitive HCT 16 line and the chemoresistant HCT1 16p53KO line.
  • Cell mortality/viability was quantitatively assessed by exposure to a reagent selective for live cells (trypan blue, Figure 1 ), quantitative analysis of energy consumption (Cell Titer Glo Luminescent Cell Viability Assay, Figure 2), an assay evaluating metabolic activity ( TT assay, Figure 3).
  • the MTT assay was validated on tumor cells freshly prepared from ex vivo samples of patients from the operating rooms. A total number of 135 samples were used. Setup of the best possible cell culture conditions required several initial trials with the use of 6 tumor samples.
  • the biopsy should be kept in saline solution or in culture medium containing high doses of different antibiotics and antifungals;
  • isolation of cells from the biopsy is performed by mechanical disruption followed by enzymatic treatment with trypsin;
  • optimal concentration of cells for seeding was determined to be 2x10 /well;
  • DMEM supplemented with fetal calf serum and growth factors was found to be the optimal culture medium for cell growth in the case of colon carcinoma.
  • the same protocol was used for ovarian cancer, with 28 samples obtained for primary tumor and two samples for metastatic tumor. All samples were treated with paclitaxel, carboplatin, and bevacizumab.
  • Ovarian cancer cells proved to be easily cultured in vitro, with viability beyond 72 hours of culture, as shown by mean absorbance values (0.356 vs 0.214 in untreated samples from colon).
  • the graph in Figure 1 1 shows the distribution of responses of 135 tumor samples treated with 5FU alone (200 ⁇ ). For each sample of tumor cells, 5FU sensitivity was assessed by the MTT assay comparing the absorbance values of triplicates by the Student's T test relative to untreated cells (control) (p ⁇ 0.05).
  • the absorbance (OD) (average value of a triplicate) was assessed for: i) the sample of tumor cells treated (T) with 5FU; ii) the sample of not treated (NT) tumor cells.
  • a Student's T test value ⁇ 0.05 from three identical replicas (triplicate) of the NT sample and of the T sample of tumor cells from the same patient identifies cancer cells sensitive to a specific treatment.
  • a Student's T test value ⁇ 0,05 from three identical replicas (triplicate) of the NT sample and of the T sample of tumor cells from the same patient identifies cancer cells resistant to a specific treatment.
  • Delta values ( ⁇ ) obtained as the difference between the mean absorbance (OD, average of triplicate) of not treated (NT) cancer cells and treated (T) cancer cells in sensitive samples are preferably > 0.04.
  • Delta values ( ⁇ ) obtained as the difference between the mean absorbance (OD, average of triplicate) of not treated (NT) cancer cells and treated cancer cells (T) in resistant samples (p > 0.05) are preferably ⁇ +0:02.
  • the method also identifies tumors containing a mixed population of sensitive and resistant cells wherein delta values ( ⁇ ) obtained as the difference between mean absorbance (OD, average of triplicate) of not treated (NT) cancer cells and treated (T) cancer cells are preferably comprised between +0.04 and +0.02.
  • Example 6B EVALUATION OF RESPONSES TO TREATMENT WITH 5FU ALONE OR IN COMBINATION WITH IBRUTINIB AND DASATI IB.
  • Figure 12 shows the distribution of responses to 5FU alone, Ibrutinib alone, and to the combination Ibrutinib + 5FU in 68 samples analyzed.
  • Figure 13 shows the distribution of responses to 5FU alone, Dasatinib alone, and to the combination Dasatinib + 5FU in 25 samples analyzed.
  • the absorbance (OD) (average value of a triplicate) is assessed for: i) the sample of 5FU treated (T) cancer cells; ii) the sample of not treated (NT) cancer cells.
  • a Student's T test value ⁇ 0.05 from three identical replicas (triplicate) of the NT sample and of the T sample of tumor cells from the same patient identifies cancer cells sensitive to a specific treatment.
  • a Student's T test value > 0,05 from three identical replicas (triplicate) of the NT sample and of the T sample of tumor cells from the same patient identifies cancer cells resistant to a specific treatment.
  • Delta values ( ⁇ ) obtained as the difference between the mean absorbance (OD, average of triplicate) of not treated (NT) cancer cells and treated (T) cancer cells in sensitive samples are preferably ⁇ 0:04.
  • Delta values ( ⁇ ) obtained as the difference between the mean absorbance (OD, average of triplicate) of not treated (NT) cancer cells and treated cancer cells (T) in resistant samples (p ⁇ 0.05) are preferably ⁇ +0:02.
  • the method also identifies tumors containing a mixed population of sensitive and resistant cells wherein delta values ( ⁇ ) obtained as the difference between mean absorbance (OD, average of triplicate) of not treated (NT) cancer cells and treated (T) cancer cells are preferably comprised between +0.04 and +0.02.
  • Figure 10 shows dose-response curves relative to the different drugs tested on the SKOV-3 cell line, in order to select the most appropriate drug concentration. In light of this, concentrations of 500nM Paclitaxel, 50 pg/ml Bevacizumab and 100nM Carboplatin were selected.
  • Figure 14 shows results obtained for the different treatments on 29 ovarian samples.
  • the tumors analyzed can be divided into subtypes based on their sensitivity/resistance.
  • the absorbance (OD) (average value of a triplicate) was measured for: i) the sample of 5FU treated (T) cancer cells; ii) the sample of not treated (NT) cancer cells.
  • a Student's T test value ⁇ 0.05 from three identical replicas (triplicate) of the NT sample and of the T sample of tumor cells from the same patient identifies cancer cells sensitive to a specific treatment.
  • a Student's T test value ⁇ 0,05 from three identical replicas (triplicate) of the NT sample and of the T sample of tumor cells from the same patient identifies cancer cells resistant to a specific treatment.
  • Delta values ( ⁇ ) obtained as the difference between the mean absorbance (OD, average of triplicate) of not treated (NT) cancer cells and treated (T) cancer cells in sensitive samples are preferably > 0:04.
  • Delta values ( ⁇ ) obtained as the difference between the mean absorbance (OD, average of triplicate) of not treated (NT) cancer cells and treated cancer cells (T) in resistant samples (p ⁇ 0.05) are preferably ⁇ +0:02.
  • the method also identifies tumors containing a mixed population of sensitive and resistant cells wherein delta values ( ⁇ ) obtained as the difference between mean absorbance (OD, average of triplicate) of not treated (NT) cancer cells and treated (T) cancer cells are preferably comprised between +0.04 and +0.02.

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Abstract

L'invention concerne des procédés pour la détermination de sensibilité ou résistance d'au moins une cellule tumorale à un médicament anticancéreux, une molécule thérapeutiquement active pouvant inhiber la croissance ou induire la mort de ladite cellule tumorale et/ou pouvant rétablir la sensibilité de cellules cancéreuses résistant à un médicament à un médicament anticancéreux et une combinaison du médicament anticancéreux et de la molécule thérapeutiquement active.
PCT/IB2014/062303 2014-06-17 2014-06-17 Procédé de détermination de sensibilité ou de résistance de cellules cancéreuses à un médicament anticancéreux et/ou une molécule thérapeutiquement active Ceased WO2015193702A1 (fr)

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RU2803730C1 (ru) * 2023-03-03 2023-09-19 Акционерное Общество "Р-Фарм" Способ определения специфической активности препарата цетуксимаб биологическим методом на культуре клеток

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11040027B2 (en) 2017-01-17 2021-06-22 Heparegenix Gmbh Protein kinase inhibitors for promoting liver regeneration or reducing or preventing hepatocyte death
RU2803730C1 (ru) * 2023-03-03 2023-09-19 Акционерное Общество "Р-Фарм" Способ определения специфической активности препарата цетуксимаб биологическим методом на культуре клеток

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