Ex vivo culture system and methods of use thereof

Abstract

An ex vivo culture system is provided, comprising a culture medium and precision-cut tissue slices placed on tissue culture inserts, wherein the precision-cut tissue slices are maintained in a high-oxygen atmosphere containing at least 50% oxygen, and the culture is subjected to rotational agitation to facilitate intermittent immersion of the tissue slices in the culture medium. Methods for culturing tissue and using the culture system to select drugs for disease treatment are also provided.

Description

[Technical field]

[0001] The present invention, in some embodiments thereof, relates to ex vivo culture systems and methods of use thereof. [Background technology]

[0002] Traditionally, the selection of anticancer therapeutic regimens is primarily based on the primary cancer tissue, the stage and grade of the tumor, and the overall health of the patient. Although such treatment regimens are effective in some patients, they are toxic and induce a number of adverse effects, some of which may be life-threatening.

[0003] The availability of targeted drug therapy and the identification of patient-specific tumor markers have allowed for personalized treatment options to be directed, but at the same time require extensive use of genetic profiling [see, for example, Dancey et al. (2012) Cell 148, 409-420 and Garraway et al. (2013) J. Clin. Oncol. Off. J. Am. Soc. Clin. Oncol. 31, 1806-1814]. However, extrapolation of genetic data to specific treatment regimens is complex, and prediction of patient response is unreliable. For example, when a patient's tumor is characterized by more than one targetable mutation, it is extremely difficult to predict which of the candidate drugs will be most effective. The same is true when more than one drug is available for a specific mutation. Thus, treatment selection is largely trial and error.

[0004] To directly test the response of a patient's cancer cells to a specific treatment, various techniques are available, such as tumor-derived cell lines generated from the patient's tumor or patient-derived xenograft (PDX) models [see, for example, Crystal et al. (2014) Science 346, 1480-1486, Clevers et al. (2016) Cell 165, 1586-1597, and Hidalgo et al. (2014) Cancer Discov. 4, 998-1013]. However, these models are expensive and time-consuming, and most importantly, they do not retain the original tumor microenvironment, which may have a significant impact on drug sensitivity [Straussman et al. (2012) Nature 487, 500-504].

[0005] Ex-vivo organ culture (EVOC) systems used in cancer biology are the culture of precision-cut slices of a patient's tumor.EVOCs have been used in a variety of applications including the study of drug toxicity, viral uptake, and tumor sensitivity to radiation or specific anticancer drugs [e.g. Vaira et al. (2010) Proc. Natl. Acad. Sci. USA 107, 8352-8356; Vickers et al. (2004) Chem. Biol. Interact. 150, 87-96; de Kanter et al. (2002) Curr. Drug Metab. 3, 39-59; Stoff-Khalili et al. (2005) Breast Cancer Res. BCR 7, R1141-1152; Merz et al. (2013) Neuro-Oncol. 15, 670-681; Gerlach et al. (2014) Br. J. Cancer 110, 479-488; Meijer et al. al.(2013)Br.J.Cancer 109,2685-2695, Grosso et al.(2013)Cell Tissue Res.352,671-684, Vaira et al.(2010)PNAS 107,8352-8356, Roife et al.(2016)Clin.Cancer Res.June 3,1-10, Maund et al.(2014)Lab.Invest.94,208-221, Vickers et al.(2004)Toxicol Sci.82(2):534-44, Zimmermann et al.(2009)Cytotechnology 61(3):145-152), Parajuli et al.(2009)In Vitro Cell.Dev.Biol.-Animal 45:442-450, Koch et al. (2014) Cell Communication and Signaling 12:73, Graaf et al. Nature Protocols (2010) 5:1540-1551, Majumder et al. Nat. Commun. 6,6169 (2015), U.S. Patent Application Publication Nos. US2014 / 0228246, US2010 / 0203575 and US2014 / 0302491, and International Patent Application Publication No. WO2002 / 044344. [Prior art documents]

Patent Documents

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[0008] According to an aspect of some embodiments of the present invention, there is provided a culture system comprising a culture medium and precision-cut tissue slices placed on tissue culture inserts, wherein the precision-cut tissue slices are maintained in an oxygen-enriched atmosphere containing at least 50% oxygen and the culture is rotary agitated to facilitate intermittent immersion of the tissue slices in the culture medium. According to some embodiments of the invention, the culture is in a tissue culture plate.

[0009] According to some embodiments of the invention, the culture system comprises a drug.

[0010] According to an aspect of some embodiments of the present invention, there is provided a method of culturing tissue comprising culturing precision-cut tissue slices on tissue culture inserts in culture medium under a high oxygen atmosphere containing at least 50% oxygen, and agitating the culture with rotation facilitating intermittent immersion of the tissue slices in the culture medium.

[0011] According to some embodiments of the invention, the method further comprises adding a drug to the culture medium.

[0012] According to some embodiments of the invention there is provided a method for determining the potency of a drug or a batch of a drug, comprising the steps of: (i) culturing a tissue according to the method of the invention; (i) adding a drug or a batch of drug to the culture medium; and (iii) determining the effect of a drug or batch of a drug on a tissue; wherein the sensitivity of the tissue to the drug indicates the efficacy of the drug.

[0013] According to some embodiments of the invention, the tissue is a pathological tissue.

[0014] According to some embodiments of the present invention, there is provided a method of selecting a drug for treating a disease in a subject in need thereof, comprising: (i) culturing a pathological tissue obtained from a subject according to the method of the present invention; (ii) adding a drug to the culture medium; and (iii) determining the effect of drugs on tissues wherein the sensitivity of the tissue to the drug indicates the efficacy of the drug for treating a disease in a subject.

[0015] According to some embodiments of the invention, there is provided a method of treating a disease in a subject in need thereof, comprising: (a) selecting a drug according to the method of the present invention; and (b) administering to the subject a therapeutically effective amount of a drug that is effective for treating the disease in the subject, thereby treating the disease in the subject. A method is provided that includes:

[0016] According to some embodiments of the invention, the drug is a drug combination.

[0017] According to some embodiments of the invention, the tissue or precision-cut tissue sections are freshly isolated.

[0018] According to some embodiments of the invention, the tissue or precision-cut tissue sections are stored at 4°C.

[0019] According to some embodiments of the invention, the tissue or precision-cut tissue sections are cryopreserved.

[0020] According to some embodiments of the invention, the determining is carried out by morphology assessment, viability assessment, proliferation assessment and / or cell death assessment.

[0021] According to some embodiments of the present invention, the determination is performed by morphology evaluation.

[0022] According to some embodiments of the invention, the determination is performed within 3-5 days of culture.

[0023] According to some embodiments of the invention, the disease is cancer.

[0024] According to some embodiments of the invention, the cancer is selected from the group consisting of ovarian cancer, colorectal cancer, lung cancer, pancreatic cancer, gastric cancer, gastroesophageal cancer and breast cancer.

[0025] According to some embodiments of the invention, the pathological tissue is cancerous tissue.

[0026] According to some embodiments of the invention, the tissue is selected from the group consisting of ovarian tissue, colorectal tissue, lung tissue, pancreatic gastric tissue, gastroesophageal tissue and breast tissue.

[0027] According to some embodiments of the invention, the tissue is selected from the group consisting of ovarian tissue, colorectal tissue, lung tissue, pancreatic gastric tissue, gastroesophageal tissue, breast tissue, liver tissue, cartilage tissue and bone tissue.

[0028] According to some embodiments of the invention, the addition is carried out 12 to 24 hours after the start of the culture.

[0029] According to some embodiments of the invention, the drug is an anti-inflammatory drug or an anti-cancer drug.

[0030] According to some embodiments of the invention, the drug is selected from the group consisting of 5-Flurouricil (5FU), Oxaliplatin, Irinotecan, Cisplatin, 4-Hydroperoxycyclophosphamide, Docetaxel, Doxorubicin, Navalbine, Gemcitabine, Gefitinib, Tamoxifen, Olaparib, Trametinib, Everolimus, and Palbociclib.

[0031] According to some embodiments of the invention, the drug concentration is higher than the IC50 dose of that drug in the cell line.

[0032] According to some embodiments of the invention, drug concentrations are derived from dose ranging experiments in which a dose-dependent response is observed.

[0033] According to some embodiments of the invention, the culturing is carried out for at least 4 days.

[0034] According to some embodiments of the invention, the culturing is carried out for at least 5 days.

[0035] According to some embodiments of the invention, the culture is carried out for up to 7 days.

[0036] According to some embodiments of the invention, the culturing is carried out in tissue culture plates.

[0037] According to some embodiments of the present invention, the precision cut sections are 200-300 μm.

[0038] According to some embodiments of the invention, the slice is placed in the center of the cell culture insert.

[0039] According to some embodiments of the invention, the section is a single section.

[0040] According to some embodiments of the invention, the slice is in direct contact with the tissue culture insert.

[0041] According to some embodiments of the invention, the tissue culture insert is a titanium grid insert.

[0042] According to some embodiments of the invention, the tissue is human tissue.

[0043] According to some embodiments of the present invention, the oxygen-rich atmosphere contains at least 70% oxygen.

[0044] According to some embodiments of the present invention, the oxygen-rich atmosphere contains less than 95% oxygen.

[0045] According to some embodiments of the invention, the method is performed in combination with genetic profiling.

[0046] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains.Although similar or equivalent methods and materials to those described herein may be used in the practice or testing of embodiments of the present invention, exemplary methods and materials are described below.In case of conflict, the present patent specification, including definitions, will control.In addition, the materials, methods and examples are illustrative only and are not necessarily intended to be limiting. Brief description of each figure in the drawing

[0047] Some embodiments of the invention are described herein, by way of example only, with reference to the accompanying drawings, in which: Detailed and specific reference will now be made to the drawings, it being stressed that the particulars shown are by way of example and are intended for the purposes of explanatory discussion of embodiments of the invention, in which respect it will become apparent to those skilled in the art how embodiments of the invention may be practiced, upon reading the description together with the drawings, in which: [Brief description of the drawings]

[0048] [Figure 1]Representative histological micrographs showing the morphology of sections of human ovarian cancer-derived xenografts after 5 days of ex vivo culture. Precision-cut sections of harvested tissues were incubated on titanium inserts in 6-well plates (left column) or directly in 6-well plates (right column) under 21% O2 (bottom row) or 80% O2 (top row) for 5 days and stained with H&E. Scale bar represents 50 μm. For each condition, tissues were tested in triplicate. [Diagram 2] Representative histological micrographs showing the morphology of sections of human breast cancer-derived xenografts after 5 days of ex vivo culture in different media. Precision-cut sections of harvested tissues were incubated on titanium inserts in DMEM / F12 (left panel) or M199 (right panel) medium under 80% O2 for 5 days and stained with H&E. Scale bar represents 50 μm. Tissues were tested in duplicate for each condition. [Diagram 3] FIG. 1 shows that the developed ex vivo culture method can predict the response of human breast cancer xenografts to anticancer drug treatment. Shown is a graph produced by Jackson Laboratories showing the in vivo response of two human breast cancer xenografts to treatment with 4-hydroperoxycyclophosphamide, showing that TM00096 breast cancer is resistant to the treatment, whereas TM00098 breast cancer is sensitive to the treatment. The y-axis shows the tumor size in mm3. Representative histological micrographs show the morphology of sections of TM00096 (left column) and TM00098 (right column) breast cancer after 5 days of ex vivo culture on titanium inserts under 80% O2, treated for 4 days with the indicated concentrations of 4-hydroperoxycyclophosphamide. Scale bar represents 50 μm. Tissues were tested in duplicate for each condition. [Figure 4]FIG. 1 shows that the developed ex vivo culture method can predict the response of human colorectal carcinoma (CRC) derived xenografts to anticancer drug treatment. Shown is a graph produced by Jackson Laboratories showing the in vivo response of three CRC derived xenografts to treatment with oxaliplatin, showing that TM00134CRC is resistant to the treatment, while TM00170 tumors are partially sensitive to the treatment and TM00164CRC is highly sensitive. The y-axis shows the tumor size in mm3. Representative histological micrographs show the morphology of sections of TM00134CRC (left panel), TM00170CRC (middle panel) and TM00164CRC (right panel) after 5 days of ex vivo culture on titanium inserts under 80% O2, treated for 4 days with 0.2 μM oxaliplatin. Treatment with DMSO served as control. Scale bar represents 50 μm. For each condition, tissues were tested in triplicate. [Diagram 5] Figure 1 shows that tumor tissue slices cultured with the developed ex vivo culture method exhibit differential sensitivity to different anticancer drugs. Human colorectal cancer (CRC) tumor tissues were harvested from metastatic lesions in the peritoneum removed during surgery for cytoreduction and intraperitoneal chemotherapy. Shown are representative histological micrographs showing the morphology of tumor slices after 5 days of ex vivo culture on titanium inserts under 80% O2, treated with the indicated anticancer drugs 5-Fluoricil (5-FU), Irinotecan, or Oxaliplatin for 4 days. Treatment with DMSO served as a control. Note that the tissues were sensitive to Oxaliplatin and Irinotecan treatments in a dose-dependent manner, but resistant to 5-Fluoricil. Scale bar represents 50 μm. For each condition, tissues were tested in triplicate. [Figure 6]Figure 1 shows that tumor tissue slices obtained from different individuals and cultured in the developed ex vivo culture method exhibit differential sensitivity to the same anticancer drug. Human colorectal cancer (CRC) tumor tissues were harvested from metastatic foci in the peritoneum removed during surgery for cytoreduction and intraperitoneal chemotherapy. Shown are representative histological micrographs showing the morphology of tumor slices after 5 days of ex vivo culture on titanium inserts under 80% O2, treated for 4 days with the indicated anticancer drugs 5-Fluorouricil (5-FU) or oxaliplatin. Treatment with DMSO served as control. PR indicates partial response and NR indicates no response. Note that the tumor from patient #8 had a strong partial response to oxaliplatin and no response to 5-Fluorouricil, whereas another patient, patient #6, responded partially to both drugs. Scale bar represents 50 μm. For each condition, tissues were tested in duplicate. [Figure 7] Figure 1 shows that the developed ex vivo culture method is able to predict sensitivity to targeted therapy as predicted by molecular profiling. Shown are representative histological micrographs showing the morphology of sections of triple-negative breast cancer tumors from two patients after 5 days of ex vivo culture on titanium inserts under 80% O2, treated for 4 days with AZD4547 (FGFR inhibitor). Treatment with DMSO served as control. Note that patient #1, whose tumor harbors an amplification of FGFR1, was sensitive to AZD4547 (FGFR inhibitor), whereas patient #2, who does not harbor this mutation, was resistant to the treatment. Scale bar represents 50 μm. For each condition, tissues were tested in duplicate. [Figure 8]Figure 1 shows that Ki67 staining for proliferation can be used to assess the sensitivity of tissues to different treatments. Shown are representative histological micrographs showing morphology (by H&E staining) and proliferation (by Ki67 staining) from one patient after 5 days of ex vivo culture on titanium inserts under 80% O2, treated for 4 days with the indicated drugs. The graph on the right was produced by Jackson Laboratories and shows the in vivo response of this CRC-derived xenograft to treatment with oxaliplatin and 5-fluorescein. The y-axis shows tumor size in mm3. This patient was partially sensitive to 5-fluorescein but highly sensitive to oxaliplatin both in vivo and ex vivo. DMSO treatment served as control. Scale bar represents 50 μm. Tissues were tested in duplicate for each condition. [Figure 9] FIG. 1 shows the processing of core biopsies for EVOC. In this example, three core biopsies are placed side-by-side in agarose liquid in a metal tray. Agarose is added and the tray is cooled. The biopsies are excised from the agarose, the block fixed onto a plunger with contact adhesive, embedded in more agarose (using the same method as for tissue sections) and then cut on a microtome. Biopsy sections are handled and processed in a similar manner as tissue sections. Scale bar represents 100 μm. [Figure 10] Histological micrographs showing that sectioning the tissue and storing it in media for up to 48 hours before starting the experiment has no detrimental effect on the tissue or change in outcome. Shown are colon cancer tissues treated with control DMSO or 5-fluorouricil (5-FU) for 4 days either immediately after sectioning or after 48 hours of refrigeration. 18 hours before the end of the experiment, BrdU was added and tissues stained with anti-BrdU antibody to identify dividing cells. Note that in both conditions, the 5-FU treated tissues are still somewhat viable, but begin to respond to the treatment with increased cell death and decreased BrdU. Scale bar represents 50 μM. [Figure 11]Representative histological images showing the indicated tissues after 5 days of ex vivo culture and a table summarizing the tissues examined, all of which maintained their tissue architecture and viability for at least 5 days in culture. Precision-cut sections of collected tissues were incubated on titanium inserts in DMEM / F12 under 80% O2 for 5 days and stained with H&E. All experiments were performed in duplicate for two different cases of the indicated cancers. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0049] The present invention, in some embodiments thereof, relates to ex vivo culture systems and methods of use thereof.

[0050] Before describing at least one embodiment of the present invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or illustrated by the examples, as the invention is capable of other embodiments and of being practiced or carried out in various ways.

[0051] Personalized treatment options for diseases such as cancer typically involve genetic profiling and testing the response of a patient's cells to a specific treatment using expensive and time-consuming models such as tumor-derived cell lines or patient-derived xenograft (PDX) models generated from the patient's tumor. Recently, ex vivo organ culture (EVOC) systems have been proposed for use in a variety of applications, including studying drug toxicity, viral uptake, and tumor sensitivity to radiation or specific anticancer drugs.

[0052] In practicing the present invention, the inventors have now developed an EVOC system that can preserve the structure and viability of precision-cut tissue sections for up to seven days in culture, and thus predict the response of pathological tissues to various drugs.

[0053] As illustrated below and in the Examples section below, the newly developed EVOC system is based on culturing single tissue slices (in this case in the center of a reusable grid made of titanium) directly on a culture insert that allows intermittent immersion of the slices in the medium, thereby facilitating the diffusion of nutrients and gases throughout the slice, in a high oxygen atmosphere containing oxygen (80%), using standard culture medium supplemented only with fetal calf serum (FCS) and antimicrobial agents (Example 1, Table 1, Figures 1-2 and 9). EVOC systems of various cancerous tissues, including ovarian, colorectal, lung, pancreatic and breast tissues cultured according to this method exhibited high viability after 4-6 days of culture (Example 1, Table 1, Figure 11).

[0054] Next, we show that the developed EVOC system can be used to predict tumor response to anti-cancer drugs (Example 2). Specifically, we show that the sensitivity of ex vivo cancerous tissue slices obtained from various patient-derived xenograft (PDX) models to anti-cancer treatments is consistent with the in vivo response of PDX models to treatments (Example 2, Table 2, Figures 3-4), and that the sensitivity of ex vivo triple-negative breast cancer tissue slices obtained from patients to targeted therapy is consistent with molecular profiling data (Example 2, Figure 7). Furthermore, we show that EVOC systems of human tumors from various patients exhibit differential sensitivity to different anti-cancer treatments (Example 2, Tables 3-4, Figures 5-6 and 8). Importantly, the tissues can be stored in culture medium for at least 48 hours before starting the experiment without any detrimental effects on the tissues or drug sensitivity (Example 2, Figure 10).

[0055] Taken together, the newly developed EVOC system preserves the tissue microenvironment, architecture, viability and genetic heterogeneity. This system allows the response of human tissues (e.g. cancer tissues) to be studied rapidly, reliably and cost-effectively. As a result, the present teachings further propose the use of the developed EVOC system for preclinical and basic research, and for qualifying the efficacy of drugs for disease treatment in general and for individual personalized therapy.

[0056] Thus, according to a first aspect of the present invention there is provided a culture system comprising a culture medium and precision-cut tissue slices placed on tissue culture inserts, wherein the precision-cut tissue slices are maintained in an oxygen-enriched atmosphere containing at least 50% oxygen and the culture is rotary agitated to facilitate intermittent immersion of the tissue slices in the culture medium.

[0057] According to another aspect of the invention, there is provided a method of culturing tissue comprising culturing precision-cut tissue slices on tissue culture inserts in a culture medium under a high oxygen atmosphere containing at least 50% oxygen, and agitating the culture with rotation facilitating intermittent immersion of the tissue slices in the culture medium.

[0058] As used herein, the term "culture system" refers to at least precision-cut tissue slices, inserts and culture medium in an ex vivo environment.

[0059] According to specific embodiments, the culture system maintains the structure and viability of the precision-cut tissue slices for at least 2-10 days, 2-7 days, 2-5 days, 4-7 days, 5-7 days, or 4-5 days in culture. According to a specific embodiment, the precision-cut tissue slices maintain viability for at least 5 days, 6 days, 7 days, or even 10 days. According to a specific embodiment, the precision-cut tissue slices maintain viability for at least 5 days.

[0060] According to specific embodiments, at least 60%, at least 70%, at least 80% of the cells in the precision-cut tissue remain viable after 4-5 days in culture, e.g., as determined by morphological analysis of the optimal area of ​​viability.

[0061] As used herein, the phrase "optimal viability zone" refers to a microscopic field of tissue (e.g., at 20x magnification) that, as assessed by a pathologist, has the greatest number of viable cells per unit area compared to a pre-EVOC sample of the same species.

[0062] Thus, according to specific embodiments, the culture is carried out for 2 to 10 days, 2 to 7 days, 2 to 5 days, 4 to 7 days, 5 to 7 days or 4 to 5 days.

[0063] According to a specific embodiment, the culturing is carried out for at least 4 days.

[0064] According to a specific embodiment, the culturing is carried out for at least 5 days.

[0065] According to one specific embodiment, the culture is carried out for up to 7 days.

[0066] As used herein, the term "tissue" refers to a portion of a solid organ of an organism (i.e., non-blood) that has some degree of vascularity, contains two or more cell types, and maintains, at least to some extent, the macrostructure of the in vivo tissue from which it is excised.

[0067] Examples include, but are not limited to, ovarian tissue, colorectal tissue, lung tissue, pancreatic tissue, breast tissue, brain tissue, retina, skin tissue, bone, heart tissue and kidney tissue. According to a specific embodiment, the tissue is selected from the group consisting of ovarian tissue, colorectal tissue, lung tissue, pancreatic tissue, gastric tissue, gastroesophageal tissue and breast tissue. According to a specific embodiment, the tissue is selected from the group consisting of ovarian tissue, colorectal tissue, lung tissue, pancreatic gastric tissue, gastroesophageal tissue, breast tissue, liver tissue, cartilage tissue and bone tissue. According to a specific embodiment, the tissue is metastatic cancer tissue obtained from sites such as, but not limited to, liver, bone, lung and peritoneum.

[0068] According to a specific embodiment, the tissue is not liver tissue.

[0069] According to a specific embodiment, the tissue is not prostate tissue.

[0070] According to a specific embodiment, the tissue is a mammalian tissue.

[0071] According to a specific embodiment, the tissue is a human tissue.

[0072] According to another specific embodiment, the tissue is mouse tissue or rat tissue.

[0073] According to a specific embodiment, the tissue is healthy tissue.

[0074] According to another specific embodiment, the tissue is a pathological tissue. The method may use multiple selected precision-cut tissue sections (e.g., each on a separate insert), each of which may be from a pathological tissue, a healthy tissue, or a combination thereof (e.g., where a healthy tissue is a control and the healthy tissue is taken from the same tissue source as the pathological tissue).

[0075] According to a specific embodiment, the tissue is a pathological tissue.

[0076] The term "pathological tissue" as used herein refers to tissue that is the cause of a disease. Elimination of such tissue is therefore expected to lead to a treatment as further defined below. Specific examples of diseases that are amenable to treatment according to some aspects of the present invention are detailed below. According to a specific embodiment, the pathological tissue is inflammatory tissue, fibrotic tissue or cancerous tissue. According to a specific embodiment, the pathological tissue is cancerous tissue.

[0077] According to specific embodiments, the tissue is obtained surgically, obtained by biopsy, laparoscopy, endoscopy, or obtained as a xenograft, or any combination thereof.

[0078] The tissue may be dissected and cultured immediately after tissue harvest (i.e., primary tissue) or may be dissected and cultured following implantation into an animal model (i.e., patient-derived xenografts (PDXs)); each option represents a separate embodiment of the present invention.

[0079] Tissues or tissue sections according to some embodiments of the invention can be freshly isolated, stored, for example at 4° C., or cryopreserved (ie, frozen), for example in liquid nitrogen.

[0080] According to a specific embodiment, the tissue or tissue section is freshly isolated (i.e., not more than 24 hours after collection from the subject and not subjected to preservation processing), as further disclosed below.

[0081] According to a specific embodiment, the tissue is cryopreserved after tissue harvest and prior to sectioning.

[0082] According to a specific embodiment, the tissue is thawed prior to cutting.

[0083] According to a specific embodiment, the tissue sections are cryopreserved after cutting.

[0084] According to a specific embodiment, the tissue slices are thawed prior to culturing.

[0085] According to a specific embodiment, the tissue is stored after tissue collection, for example in culture medium at 4° C., prior to sectioning.

[0086] According to a specific embodiment, the tissue sections are stored after cutting, for example in culture medium, at 4° C., prior to culturing.

[0087] According to specific embodiments, storage at 4° C. is carried out for up to 120 hours, up to 96 hours, up to 72 hours, or up to 48 hours.

[0088] According to a specific embodiment, storage at 4° C. is carried out for 24 to 48 hours.

[0089] Thus, according to a specific embodiment, the method further comprises obtaining the tissue from the subject or from an animal model comprising the tissue.

[0090] As used herein, the phrase "patient-derived xenograft (PDX)" refers to a tissue created by transplantation of a primary tissue into an animal of a different species from the donor of the primary tissue. According to a specific embodiment, a PDX is a tissue created by transplantation of a human primary tissue (e.g., a cancerous tissue) into an immunodeficient mouse.

[0091] Following tissue removal, the tissue is sectioned into precision cut sections.

[0092] As used herein, the phrase "precision-cut tissue section" refers to a viable section obtained from an isolated solid tissue and having a reproducible, well-defined thickness (e.g., thickness variation between sections is ±5%).

[0093] Typically, tissue sections are mini-models of tissues that contain the cells of the tissue in their natural environment and retain the three-dimensional connectivity, such as cell-cell and cell-matrix interactions, of intact tissues, without selecting specific cell types among the various cell types that make up the tissue or organ. Precision cutting reduces sources of error due to variability in section thickness and damage to the cut surface (both of which contribute to uneven gas and nutrient exchange across the tissue section), improves reproducibility, and allows adjacent sections to be assessed for tissue structure and compared pairwise under different experimental conditions.

[0094] Thin sections can be cut in various directions (e.g., anterior-posterior, dorso-ventral, or naso-temporal) and at various thicknesses. The size / thickness of the tissue section is based on the tissue source and the method used for sectioning. According to specific embodiments, the thickness of the precision cut sections allows for the maintenance of tissue architecture during culture.

[0095] According to specific embodiments, the thickness of the precision-cut sections allows complete access of the inner cell layers to oxygen and nutrients so that the inner cell layers are exposed to sufficient oxygen and nutrient concentrations.

[0096] According to specific embodiments, the thickness of the precision-cut sections allows complete access of the inner cell layer to oxygen and nutrients such that the inner cell layer is exposed to the same oxygen and nutrient concentrations as the outer cell layer.

[0097] According to specific embodiments, the precision cut sections are 50-1200 μm, 100-1000 μm, 100-500 μm, 100-300 μm, or 200-300 μm.

[0098] According to one specific embodiment, the precision cut sections are 200-300 μm.

[0099] Methods for obtaining tissue sections are known in the art and are described, for example, in the Examples section below, as well as in Roife et al. (2016) Clin. Cancer Res. June 3, 1-10, Vickers et al. (2004) Toxicol Sci. 82(2): 534-44, Zimmermann et al. (2009) Cytotechnology 61(3): 145-152), Koch et al. (2014) Cell Communication and Signaling 12: 73, and Graaf et al. Nature Protocols (2010) 5: 1540-1551, the contents of each of which are incorporated herein by reference in their entirety. Such methods include, but are not limited to, vibratome sectioning, agarose embedding followed by microtome sectioning, or matrix sectioning.

[0100] As a non-limiting example, tissue is isolated and immediately placed into physiological dissection medium (eg, ice-cold PBS), optionally supplemented with antibiotics.

[0101] According to specific embodiments, the warm ischemic time is less than 2 hours, less than 1.5 hours, or less than 1 hour.

[0102] According to specific embodiments, the cold ischemic time is less than 96 hours, less than 72 hours, less than 48 hours, less than 24 hours, less than 12 hours, less than 5 hours, or less than 2 hours.

[0103] Prior to sectioning, the tissue is attached to a tissue sectioning device, for example using contact adhesive, and then embedded in, for example, low melting point agarose gel. The tissue is then cut into precision-cut sections. Many suitable tissue sectioning devices are commercially available, for example, but not limited to, Compresstome™ VF-300 (Precisionary Instruments Inc., North Carolina, USA), Brendel-Vitron tissue sectioning device (Tucson, Arizona), Krumdieck precision tissue sectioning device (model number MD4000-01, Alabama R&D), and Leica VT1200S vibrating blade microtome (Leica, Wetzlar, Germany). According to a specific embodiment, the sectioning device is filled with ice-cold medium, such as Williams medium E or Krebs-Henseleit buffer (KHB). Those skilled in the art will know the media and conditions for each tissue type for sectioning and for storage of tissue and tissue sections before culturing.

[0104] The tissue slice is then placed on a tissue culture insert in a tissue culture vessel filled with culture medium. One slice or multiple slices can be placed on a single tissue culture insert. According to a specific embodiment, one slice is placed on a single tissue culture insert.

[0105] According to a specific embodiment, the culture vessel is filled with culture medium up to the bottom of the tissue slices (eg, 4 ml of medium for a 6-well plate containing an insert).

[0106] The cultures can be in glass, plastic or metal vessels that can provide a sterile environment for tissue culture. According to specific embodiments, the culture vessels include dishes, plates, flasks, bottles and vials. Culture vessels such as COSTAR®, NUNC® and FALCON® are commercially available from various manufacturers.

[0107] According to specific embodiments, the culture vessel is a tissue culture plate, such as a 6-well plate, a 24-well plate, a 48-well plate and a 96-well plate.

[0108] According to one specific embodiment, the culture vessel is a 6-well tissue culture plate.

[0109] According to specific embodiments, the culture vessel is not pre-coated with proteins extracted from the corresponding tissue (e.g., if the tissue is cancerous, the culture vessel is not pre-coated with stage- and grade-corresponding tumor-extracted proteins). Non-limiting examples of such proteins include ECM proteins, such as collagen, fibronectin, laminin, vibronectin, cadherin, filamin A, vimentin, osteopontin, decorin, tenascin-X, basement membrane proteins, cytoskeletal and matrix proteins, and growth factors.

[0110] The culture medium used according to the present invention can be an aqueous medium containing a combination of salts, nutrients, minerals, vitamins, amino acids, nucleic acids and / or proteins, such as cytokines, growth factors and hormones, all of which are necessary for cell growth and can maintain tissue structure and viability. For example, the culture medium can be a synthetic tissue culture medium, such as DMEM / F12 (e.g., available from Biological Industries), M199 (e.g., available from Biological Industries), RPMI (e.g., available from Gibco-Invitrogen Corporation products), M199 (e.g., available from Sigma-Aldrich), Ko-DMEM (e.g., available from Gibco-Invitrogen Corporation products), supplemented with necessary additives, as further described below. Preferably, all components contained in the culture medium of the present invention are tissue culture grade and substantially pure.

[0111] Those skilled in the art will know how to select a culture medium for each tissue type envisioned.

[0112] According to a specific embodiment of the invention, the culture medium comprises serum, for example fetal calf serum (FCS, available, for example, from Gibco-Invitrogen Corporation products).

[0113] According to a specific embodiment, the culture medium contains less than 10% serum.

[0114] According to a specific embodiment, the culture medium contains less than 2% serum obtained from the same species as the tissue being cultured.

[0115] According to a specific embodiment, the culture medium lacks serum obtained from the same species as the tissue being cultured.

[0116] According to a specific embodiment, the culture medium contains less than 2% serum that is autologous (i.e., from the same subject) to the tissue being cultured.

[0117] According to a specific embodiment, the culture medium lacks serum autologous (i.e., from the same subject) to the tissue being cultured.

[0118] According to a specific embodiment, the culture medium contains less than 2% human serum.

[0119] According to some embodiments of the invention, the culture medium lacks human serum.

[0120] According to some embodiments of the invention, the culture medium is devoid of any animal contaminants, i.e., animal cells, body fluids, or pathogens (e.g., viruses that infect animal cells), i.e., xeno-free.

[0121] According to some embodiments of the invention, the culture medium may further comprise an antibiotic (e.g., penicillin, streptomycin, gentamicin), an antimycotic (e.g., amphotericin B), L-glutamine or a non-essential amino acid (NEAA).

[0122] According to a specific embodiment, the medium comprises serum and antibiotics.

[0123] According to one specific embodiment, the medium comprises DMEM / F12, 5% FCS, glutamine, penicillin, streptomycin, gentamicin and amphotericin B.

[0124] It should be noted that the culture medium may be periodically refreshed to maintain adequate levels of supplements and to remove metabolic waste products that may damage the tissue. According to specific embodiments, the culture medium is refreshed every 12-72 hours, every 24-72 hours, every 24-48 hours, or every 12-48 hours.

[0125] According to a specific embodiment, the culture medium is renewed every 12 to 48 hours.

[0126] According to one specific embodiment, the culture medium is refreshed once after 12-24 hours, and then approximately every 48 hours.

[0127] The phrase "tissue culture insert" as used herein refers to a porous membrane suspended in a container for tissue culture, which is compatible with the subsequent ex vivo culture of tissue slices. The pore size can support the tissue slice and at the same time allows the culture medium to pass through, thereby allowing the passage of nutrients and metabolic waste to and from the slice, respectively. According to a specific embodiment, the tissue slice is placed on the tissue culture insert, which allows the culture medium to access both the apical and basal surfaces of the tissue slice.

[0128] According to specific embodiments, the pore size is 0.1 μm to 20 μm, 0.1 μm to 15 μm, 0.1 μm to 10 μm, 0.1 μm to 5 μm, 0.4 μm to 20 μm, 0.4 μm to 10 μm, or 0.4 μm to 5 μm.

[0129] According to specific embodiments, the hole size is 0.4 mm to 4 mm, 0.4 mm to 1 mm, 1 mm to 4 mm, 1 mm to 3 mm, or 1 mm to 2 mm.

[0130] According to a specific embodiment, the tissue culture insert is sterile.

[0131] According to a specific embodiment, the tissue culture inset is disposable.

[0132] According to specific embodiments, the cell culture inserts are reusable and autoclavable.

[0133] The cell culture insert may be synthetic or natural, inorganic or polymeric, such as titanium, alumina, polytetrafluoroethylene (PTFE), Teflon, stainless steel, polycarbonate, nitrocellulose, and cellulose esters. According to a specific embodiment, the cell culture insert is a titanium insert. Cell culture inserts that can be used in specific embodiments of the present invention are commercially available, such as from Alabama R&D, Millipore Corporation, Costar, Corning Incorporated, Nunc, Vitron Inc., and SEFAR, including, but not limited to, MA0036-well plate inserts, BIOCOAT™, Transwell®, Millicell®, Falcon®-Cyclopore, Nunc® Anapore, titanium screens, and Teflon screens.

[0134] According to a specific embodiment, the tissue culture insert is a titanium grid insert, such as, but not limited to, a titanium MA003 6 well plate insert (Alabama R&D).

[0135] According to a specific embodiment, the tissue culture insert is not coated with an organic material such as collagen, fibronectin or polyethylene glycol (PEG).

[0136] According to specific embodiments, the tissue culture inserts are not coated with proteins extracted from the corresponding tissue (e.g., if the tissue is cancerous tissue, the tissue culture inserts are not pre-coated with stage- and grade-corresponding tumor-extracted proteins).

[0137] According to a specific embodiment, the tissue slice is in direct contact with the tissue culture insert.

[0138] According to specific embodiments, the tissue slices are not separated from the culture medium by a foreign organic material, such as collagen, fibronectin or a synthetic polymer (that is not part of the culture vessel), e.g. polyethylene glycol (PEG).

[0139] According to a specific embodiment, the tissue slice is in direct contact with the culture medium.

[0140] According to a specific embodiment, the tissue slice is placed in the center of the cell culture insert, thus for example when applying a titanium grid such as a titanium MA0036 well plate insert, the tissue slice is placed in a depression located in the center of the insert.

[0141] The tissue sections are then cultured (or maintained) at a physiological temperature, eg, 37° C., in a highly oxygenated, humidified atmosphere containing at least 50% oxygen and, eg, 5% CO 2 .

[0142] According to specific embodiments, the oxygen-rich atmosphere contains at least 60%, at least 70% or at least 80% oxygen.

[0143] According to a specific embodiment, the oxygen-rich atmosphere contains at least 70% oxygen.

[0144] According to another specific embodiment, the oxygen-rich atmosphere contains less than 95% oxygen.

[0145] According to one particular embodiment, the oxygen-rich atmosphere contains about 80% oxygen.

[0146] According to one specific embodiment, during the culturing process the culture is agitated with rotation facilitating intermittent immersion of the tissue slices in the culture medium.

[0147] As used herein, the phrase "rotationally agitated to facilitate intermittent immersion of the tissue slice in the culture medium" or "rotationally agitated to facilitate intermittent immersion of the tissue slice in the culture medium" refers to agitation that allows for periodic immersion of the tissue slice in the medium in a manner that facilitates diffusion of nutrients and gases throughout the medium and throughout the tissue slice.

[0148] According to a specific embodiment, the stirring is orbital stirring.

[0149] According to a specific embodiment, the agitation is an angled agitation, for example an angle of 30° to 45°.

[0150] According to a specific embodiment, agitation is performed by a tilt rotator (eg, an MD2500 incubation unit available from Alabama Research and Development).

[0151] According to a specific embodiment, the stirring speed is 50 to 200 rpm, 50 to 150 rpm, or 50 to 100 rpm.

[0152] According to a specific embodiment, the stirring speed is about 70 rpm.

[0153] The tissue cultures and methods of the present invention can be adapted for many applications, including but not limited to the following: 1. Preclinical and basic research, e.g. -Research into mechanisms involved in health and disease; - screening of pathological tissue (e.g. tumor tissue) for the presence of specific markers; - Screening and / or development of new drugs or new drug combinations, such as anti-cancer drugs; - Determining the potency of a batch of a drug, - Studying mechanisms of sensitivity and resistance in human tissues. Thus, for example, treatment of the same tumor with multiple drugs and drug combinations can be utilized for detailed mechanistic studies that cannot be easily performed in human patients. -The study of the tumor microbiome, i.e. the bacteria, viruses or fungi that may be present in cancer tumors. 2. Study of the effect of novel drugs or drug combinations on tumor sections to prioritize drugs or drug combinations for further drug development and mechanistic studies, preclinical in vivo testing and clinical trials. 3. Predicting a patient's response to drugs (e.g., anti-cancer drugs) and drug combinations, and then using the predictions to tailor a specific treatment regimen for that patient (i.e., personalized medicine). 4. Because the tissue slices maintain their structure and exhibit high viability after 5 days of culture, this system allows modeling of acute as well as chronic toxicity studies, including the metabolic activity of the tissue. 5. Cultures exhibiting a positive ex vivo response can be used as a criterion for patient inclusion in clinical trials. This process can therefore preselect patients who are likely to respond, thereby improving the chances of clinical trial success. In such cases, the ex vivo tissue cultures may later become part of the criteria for drug eligibility.

[0154] Thus, according to a specific embodiment, the culture system comprises a drug.

[0155] According to another specific embodiment, the method of the invention described above comprises the addition of a drug or drug combination, as further described below.

[0156] The phrase "drug" as used herein refers to an agent having anti-pathological effect, including small molecules and biological drugs (e.g., nucleic acid agents, polypeptides, antibodies, aptamers, etc.). Typically, the drug is exogenous to the precision-cut tissue section or in the human body or human tissue from which the precision-cut tissue section is derived. The drug may be a drug approved by a regulatory agency for the treatment of the pathology, or a drug under development. According to a specific embodiment, the drug is a batch of drugs.

[0157] According to a specific embodiment, the drug is an anti-inflammatory drug.

[0158] Non-limiting examples of anti-inflammatory drugs that can be used in specific embodiments of the present invention include the following drugs: alclofenac; alclometasone dipropionate; algestone acetonide; alpha amylase; amcinafal; amcinafide; amfenac sodium; amiprilose hydrochloride; anakinra; anilolac; anitrazafen; apazone; balsalazide disodium; bendazac; benoxaprofen; benzydamine hydrochloride; bromelain; broperamol; budesonide; carprofen; cycloprofen; synthazone; cliprof cyclohexyl;clobetasol propionate;clobetasone butyrate;clopirac;cloticasone propionate;cormethasone acetate;cortodoxone;deflazacort;desonide;desoximetasone;dexamethasone dipropionate;diclofenac potassium;diclofenac sodium;diflorasone diacetate;diflumidone sodium;diflunisal;difluprednate;diphthalone;dimethyl sulfoxide;drocinonide;endrysone;enlimomab;enolicam sodium;epirizole;etodolac;etofenamate;felbinac;phenam ol;fenbufen;fenclofenac;fenclorac;fendosal;fenpiparone;fentiazac;flazaron;fluazacort;flufenamic acid;flumizole;flunisolide acetate;flunixin;flunixin meglumine;fluocortin butyl;fluorometholone acetate;fluquazone;flurbiprofen;fluretofen;fluticasone propionate;flaprofen;flobufen;halcinonide;halobetasol propionate;halopredone acetate;ibufenac;ibuprofen;ibuprofen aluminium;ibuprofen Lofenpiconol;Ilonidap;Indomethacin;Indomethacin sodium;Indoprofen;Indoxol;Intrazole;Isoflupredone acetate;Isoxepac;Isoxicam;Ketoprofen;Lofemizole hydrochloride;Lomoxicam;Loteprednol etabonate;Meclofenamate sodium;Meclofenamic acid;Meclorisone dibutyrate;Mefenamic acid;Mesalamine;Meseclazone;Methylprednisolone suleptanate;Momifurumate;Nabumetone;Naproxen;Naproxen sodium;Naproxol;Nimazone;Olsalazine sodium;Orgotein;Orpanoxin;Oxaprozin;Oxyphenbutazone;Paraniline hydrochloride;Pentosan sodium polysulfate;Phenbutazone sodium glycerate;Pirfenidone;Piroxicam;Piroxicam cinnamate;Piroxicam olamine;Pirprofen;Prednazate;Priferon;Prodolic acid;Proquazone;Proxazole;Proxazole citrate;Rimexolone; Lomazarit;Sarcolex;Sarnasedin;Salsalate;Sanguinarium chloride;Seclazone;Selmetacin;Sudoxicam;Sulindac;Suprofen;Talmetacin;Talniflumate;Talosalate;Tebufelone;Tenidap;Tenidap sodium;Tenoxicam;Tesicam;Tesimide;Tetridamine;Tiopinac;Tixocortol pivalate;Tolmetin;Tolmetin sodium;Triclonide;Triflumidate;Zidometacin;Zomepirac sodium;

[0159] According to another specific embodiment, the drug is an anti-cancer drug.

[0160] As used herein, the phrase "anti-cancer drug" refers to agents that have anti-tumor effects, including chemotherapeutic agents, small molecules, biological drugs, hormonal therapeutic agents, antibodies and targeted therapeutic agents.

[0161] Anti-cancer drugs that can be used in specific embodiments of the present invention include, but are not limited to, the following drugs: acivicin; aclarubicin; acodazole hydrochloride; acronine; adriamycin; adzelesin; aldesleukin; altretamine; ambomycin; amethanthrone acetate; aminoglutethimide; amsacrine; anastrozole; anthramycin; asparaginase; asperlin; azacitidine; azetepa; azotomycin; batimastat; benzodepa; bicalutamide; bisantrene hydrochloride; bis mesylate Nafide;Bizelesin;Bleomycin sulfate;Brequinar sodium;Bropirimine;Busulfan;Cactinomycin;Calsterone;Caracemide;Carbetimer;Carboplatin;Carmustine;Carbicin hydrochloride;Carzelesin;Cedefingol;Chlorambucil;Ciloremycin;Cisplatin;Cladribine;Crisnatol mesylate;Cyclophosphamide;Cytarabine;Dacarbazine;Dactinomycin;Daunorubicin hydrochloride;Decitabine;Dexormaplatin;Desaguanine;Desaguanine mesylate;Diazicon;Docetaxel;Doxil Sorubicin;Doxorubicin hydrochloride;Droloxifene;Droloxifene citrate;Dromostanolone propionate;Duazomycin;Edatrexate;Eflornithine hydrochloride;Elsamitrucin;Enloplatin;Empromate;Epipropizine;Epirubicin hydrochloride;Elburozole;Esorubicin hydrochloride;Estramustine;Estramustine sodium phosphate;Etanidazole;Etoposide;Etoposide phosphate;Etoprine;Fadrozole hydrochloride;Fazarabine;Fenretinide;Floxuridine;Fludarabine phosphate;Fluorouracil Sil;Flurocitabine;Fosquidone;Fostriecin sodium;Gemcitabine;Gemcitabine hydrochloride;Hydroxyurea;Idarubicin hydrochloride;Ifosfamide;Irmofosine;Interferon alpha-2a;Interferon alpha-2b;Interferon alpha-n1;Interferon alpha-n3;Interferon beta-Ia;Interferon gamma-Ib;Iproplatin;Irinotecan hydrochloride;Lanreotide acetate;Letrozole;Leuprolide acetate;Liarozole hydrochloride;Lometrexol sodium;Lomustine;Rosoxantrone hydrochloride;Masoprocol;Maytansine;Mechlorethamine hydrochloride;Megestrol acetate;Melengestrol acetate;Melphalan;Menogaril;Mercaptopurine;Methotrexate;Methotrexate sodium;Metoprine;Meturedepa;Mitindomide;Mitocalcine;Mitochromine;Mitogillin;Mitomarcin;Mitomycin;Mitosper;Mitotane;Mitoxantrone hydrochloride;Mycophenolic acid;Nocodazole;Nogalamycin;Ormaplatin;Oxisuran ;Paclitaxel;Pegaspargase;Periomycin;Pentamustine;Peplomycin sulfate;Perfosfamide;Pipobroman;Piposulfan;Piroxantrone hydrochloride;Plicamycin;Promestane;Porfimer sodium;Porfiromycin;Prednimustine;Procarbazine hydrochloride;Puromycin;Puromycin hydrochloride;Pyrazofurin;Riboprin;Rogletimide;Safingol;Safingol hydrochloride;Semustine;Simtrazene;Sparfose tosodium;sparsomycin;spirogermanium hydrochloride;spiromustine;spiroplatin;streptonigrin;streptozocin;sulofenur;tallysomycin;taxol;tecogalan sodium;tegafur;teroxantrone hydrochloride;temoporfin;teniposide;teroxylon;testolactone;thiamiprine;thioguanine;thiotepa;tiazofuirin;tirapazamine;topotecan hydrochloride;toremifene citrate;trestron aceta ate;Triciribine phosphate;Trimetrexate;Trimetrexate glucuronate;Triptorelin;Tubrozole hydrochloride;Uracil mustard;Uredep;Vapreotide;Verteporfin;Vinblastine sulfate;Vincristine sulfate;Vindesine;Vindesine sulfate;Binepidine sulfate;Vinglisinate sulfate;Vinleurosine sulfate;Vinorelbine tartrate;Vinrocidine sulfate;Vinzolidine sulfate;Vorozole;Zeniplatin;Zinostatin;Zorubicin hydrochloride. Additional antineoplastic agents include those disclosed in Goodman and Gilman's "The Pharmacological Basis of Therapeutics", 8th Edition, 1990, McGraw-Hill, Inc. (Health Professions Division), pages 1202-1263, Chapter 52, Antineoplastic Agents (Paul Calabresi and Bruce A. Chabner) and its introduction.;

[0162] Non-limiting examples of approved anti-cancer drugs include the following drugs: abarelix, aldesleukin, aldesleukin, alemtuzumab, alitretinoin, allopurinol, altretamine, amifostine, anastrozole, arsenic trioxide, asparaginase, azacitidine, AZD9291, AZD4547, AZD2281, bevacuzimab, bexarotene, bleomycin, bortezomib, busulfan, calsterone, capecitabine, carboplatin, Carmustine, celecoxib, cetuximab, cisplatin, cladribine, clofarabine, cyclophosphamide, cytarabine, dabrafenib, dacarbazine, dactinomycin, actinomycin D, darbepoetin alfa, darbepoetin alfa, daunorubicin liposomal, daunorubicin, decitabine, denileukin diftitox, dexrazoxane, docetaxel, doxorubicin, dromostanolone propionate, Elliot's B solution Solution), epirubicin, epoetin alfa, erlotinib, estramustine, etoposide, exemestane, filgrastim, floxuridine, fludarabine, fluorouracil 5-FU, fulvestrant, gefitinib, gemcitabine, gemtuzumab ozogamicin, goserelin acetate, histrelin acetate, hydroxyurea, ibritumomab tiuxetan, idarubicin, ifosfamide, imatinib mesylate, interferon alfa-2a, interferon alfa-2b, irinotecan Kampo, lenalidomide, letrozole, leucovorin, leuprolide acetate, levamisole, lomustine, CCNU, meclorethamine, nitrogen mustard, megestrol acetate, melphalan, L-PAM, mercaptopurine 6-MP, mesna, methotrexate, mitomycin C, mitotane, mitoxantrone, nandrolone phenpropionate, nelarabine, nofetumomab, oprelvekin, oxaliplatin, paclitaxel, palbociclib, palifermin, pamidronate, pegademase, pegaspargase, pegfilgrastim, pemetrexed disodium, pentostatin, pipobroman, plicamycinMithramycin, porfimer sodium, procarbazine, quinacrine, rasburicase, rituximab, sargramostim, sorafenib, streptozocin, sunitinib maleate, tamoxifen, temozolomide, teniposide VM-26, testolactone, thioguanine 6-TG, thiotepa, thiotepa, topotecan, toremifene, tositumomab, trametinib, trastuzumab, tretinoin ATRA, uracil mustard, valrubicin, vinblastine, vinorelbine, zoledronate and zoledronic acid.

[0163] According to specific embodiments, the anticancer drug is selected from the group consisting of gefitinib, lapatinib, afatinib, BGJ398, CH5183284, linsitinib, PHA665752, crizotinib, sunitinib, pazopanib, imatinib, ruxolitinib, dasatinib, BEZ235, pictilisib, everolimus, MK-2206, trametinib / AZD6244, vemurafinib / dabrafenib, CCT1 96969 / CCT241161, Barasertib, VX-680, Nutlin 3, Palbociclib, BI2536, Bardoxolone, Vorinostat, Navitoclax (ABT263), Bortezomib, Vismodegib, Olaparib (AZD2281), Simvastatin, 5-Fluorouracil, Irinotecan, Epirubicin, Cisplatin and Oxaliplatin.

[0164] According to specific embodiments, the anticancer drug is selected from the group consisting of 4-hydroperoxycyclophosphamide, 5-flurourisyl (5FU), oxaliplatin, irinotecan, docetaxel, cisplatin, trametinib, palbociclib, and AZD4547.

[0165] According to specific embodiments, the anticancer drug is selected from the group consisting of 5-flurourisyl (5FU), oxaliplatin, irinotecan, cisplatin, 4-hydroperoxycyclophosphamide, decetaxel, doxorubicin, navalbine, gemcitanime, gefitinib, tamoxifen, olaparib, trametinib, everolimus, and palbociclib.

[0166] It will be appreciated that the culture systems and methods described herein can be used to test the differential effects of drug combinations.

[0167] Thus, according to a specific embodiment, the drug is a drug combination.

[0168] The specific concentration of the drug in the culture system and method of the present invention is derived by culturing different drug concentrations in several cancer tissues (of the same or different types) and evaluating the range in which a dose-dependent response is achieved. Typically, this dose-dependent range reflects the region in which the cultured cancer tissue is expected to respond or not respond, giving the predicted outcome.

[0169] According to specific embodiments, the drug concentration in the culture systems and methods of the invention is at least the same as the IC50 dose of that drug in a cell line (eg, a cell line of the same tissue type and / or species).

[0170] According to specific embodiments, the drug concentration in the culture systems and methods of the present invention is higher than the IC50 dose of that drug in a cell line (eg, a cell line of the same tissue type and / or species).

[0171] According to specific embodiments, the drug concentration in the culture systems and methods of the invention is at least 2-fold, at least 3-fold, at least 5-fold, at least 10-fold, at least 25-fold, at least 50-fold, at least 100-fold the IC50 dose of that drug in a cell line (e.g., a cell line of the same tissue type and / or species).

[0172] According to specific embodiments, the culture systems and methods of the present invention are at least 10-fold the IC50 dose of the drug in a cell line (eg, a cell line of the same tissue type and / or species).

[0173] Specific concentrations of anti-cancer drugs that can be used in some embodiments of the present invention are shown in Tables 5-6 below.

[0174] [Table 5]

[0175] [Table 6]

[0176] According to a specific embodiment, several tissue slices are prepared from one tissue and cultured in several culture vessels (eg, wells of a plate), allowing the testing of several drugs and drug combinations.

[0177] According to a specific embodiment, the method of the present invention comprises: (i) determining the effect of a plurality (e.g., at least two) drugs according to the methods of the present invention; and (ii) determining the efficacy of drug combinations shown to be effective in step (i); wherein increased sensitivity of the tissue to the drug combination indicates the efficacy of the drug combination.

[0178] The term "increased sensitivity" as used herein refers to an increase in the effect induced by a drug combination compared to the effect induced by each of the drugs in the combination, which may be an additive or synergistic effect. According to a specific embodiment, the effect is a synergistic effect.

[0179] According to specific embodiments, the increase is at least 1.5-fold, at least 2-fold, at least 3-fold, at least 5-fold, at least 10-fold, or at least 20-fold compared to the effect induced by each of the drugs in the combination.

[0180] According to another specific embodiment, the increase is at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or greater than 100% compared to the effect induced by each of the drugs in the combination.

[0181] As mentioned above, according to specific embodiments, tissues or tissue sections can be stored, i.e., stored at 4°C or frozen, and thawed as desired, allowing for sequential serial testing of several drugs and drug combinations.

[0182] Thus, for example, according to a specific embodiment, the method of the invention comprises (i) determining the effect of at least two drugs according to the methods of the present invention; (ii) culturing additional tissue sections obtained from the pathological tissue obtained from the subject (e.g., after cryopreservation and thawing) according to the present invention; (iii) adding a drug combination determined in step (i) to be effective for treating the disease in the subject; and (iv) determining the effect of said drug combinations on tissue sections; wherein increased sensitivity of the tissue section to the drug combination indicates the efficacy of the drug combination for treating a disease in the subject.

[0183] The drug or drug combination can be added to the culture at various times: According to specific embodiments, the drug is added to the culture 2-48 hours, 2-36 hours, 2-24 hours, 12-48 hours, 12-36 hours or 12-24 hours after the start of the culture.

[0184] According to one specific embodiment, the drug or drug combination is added to the culture 12 to 24 hours after the initiation of the culture.

[0185] According to one specific embodiment, the drug or drug combination is added to the culture 12 to 36 hours after the initiation of the culture.

[0186] According to a specific embodiment, the drug or drug combination is added to the culture 12 to 48 hours after the initiation of the culture.

[0187] Culturing in the presence of the drug or drug combination can be performed throughout the entire cultured organ from the first drug addition, or can be time-limited. Alternatively, or additionally, the drug or drug combination can be added to the culture multiple times, for example when the culture medium is renewed.

[0188] Selection of drug concentrations and incubation times with drugs or drug combinations is well within the capabilities of one of ordinary skill in the art.

[0189] According to specific embodiments, the drug concentration and incubation time with the drug or drug combination results in a detectable effect on the tissue, as further described below.

[0190] The selection of drug concentrations to be used in ex vivo studies that will produce a detectable effect on tissue is well within the ability of one of ordinary skill in the art. Preferably, the concentrations used should be within the linear range of the selected parameter.

[0191] The number of drug concentrations tested can be at least 1, at least 2, at least 3, at least 5, at least 6, 1-10, 2-10, 3-10, 5-10, 1-5, 2-5 and 3-5 different concentrations in the same assay.

[0192] The number of sample replicates for each drug concentration tested can be 2, 3, 4, 5 or 6 replicates.

[0193] Following culturing, the effect of the drug or drug combination on the tissue can be determined, thereby determining the efficacy of the drug.

[0194] Thus according to another aspect of the invention there is provided a method for determining the potency of a drug or a batch of a drug, comprising the steps of: (i) culturing precision-cut tissue slices on tissue culture inserts in culture medium under an oxygen-enriched atmosphere containing at least 50% oxygen while agitating the culture with rotation facilitating intermittent immersion of the tissue slices in the culture medium; (ii) adding a drug or a batch of drug to said culture medium; and (iii) determining the effect of said drug or batch of said drug on said tissue; wherein the sensitivity of the composition to the drug indicates the efficacy of the drug.

[0195] According to specific embodiments, the determining step is carried out after a predetermined incubation time, which may vary, but determining an incubation time that will result in a detectable effect is well within the capabilities of one of ordinary skill in the art.

[0196] According to specific embodiments, the determination is performed within 2-10 days, within 2-7 days, within 2-5 days, within 3-10 days, within 3-7 days, within 3-5 days or within 4-5 days of culture.

[0197] According to a specific embodiment, the determination is carried out within 3 to 5 days of culture.

[0198] According to another specific embodiment, the determination is carried out within 4 to 5 days of culture.

[0199] Methods for determining the effects induced by a drug or drug combination are known in the art and include, for example: - viability assessment, for example using the MTT test, the WST assay or the ATP uptake assay, which is based on the selective ability of live cells to reduce the yellow salt MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) (Sigma, Aldrich, St. Louis, MO, USA) to an insoluble purple-blue formazan precipitate; - proliferation assessment, e.g. using the BrDu assay [Cell Proliferation ELISA BrdU Colorimetric Kit (Roche, Mannheim, Germany)] or Ki67 staining; cell death assessment, e.g. using the TUNEL assay [Roche, Mannheim, Germany], Annexin V assay [ApoAlert® Annexin V Apoptosis Kit (Clontech Laboratories, Inc., California, USA)], LDH assay, activated caspase 3 assay, activated caspase 8 assay and nitric oxide synthase assay; - aging assessment, e.g. using the senescence-associated β-galactosidase assay (Dimri GP, Lee X, et al. 1995. A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc Natl Acad Sci USA 92:9363-9367) and telomerase truncation assay; - Cellular metabolism assessment, e.g. using glucose uptake assays; - a variety of RNA and protein detection methods (detecting levels of expression and / or activity); and - Morphological assessment, for example using Hematoxylin and Eosin (H&E) staining.

[0200] According to specific embodiments, the determination is carried out by morphology assessment, viability assessment, proliferation assessment and / or cell death assessment.

[0201] According to a particular embodiment, the determination is performed by morphology evaluation.

[0202] Morphological assessment using H&E staining can provide details regarding, for example, cellular content, cell size and density, live / dead cell ratio, diseased (e.g., tumor) / healthy cell ratio, immune cell infiltration, desmoplasia, nuclear size and density and integrity, apoptotic bodies and mitotic figures, etc. According to specific embodiments, the effect of drugs on tissues is determined by morphological assessment, for example by a pathologist.

[0203] Typically, the results of each assay are expressed in a numerical format, with high scores correlating with drug sensitivity and low scores correlating with drug resistance.

[0204] As used herein, the phrase "sensitivity to a drug" or "sensitivity to a drug combination" refers to the ability of a drug or drug combination to induce cellular changes, such as cell viability, proliferation rate, differentiation, cell death, necrosis, apoptosis, senescence, changes in the transcription and / or translation rate of a specific gene, and / or protein state, such as phosphorylation, dephosphorylation, translocation, and any combination thereof. According to specific embodiments, the cellular changes are reflected in decreased cell viability, decreased proliferation rate, increased cell death, and / or abnormal morphology, compared to that in the absence of the drug.

[0205] According to a particular embodiment, the cellular alteration is reflected in a decrease in cell viability.

[0206] According to a particular embodiment, the cellular changes are reflected in an abnormal morphology.

[0207] According to specific embodiments, the change is at least 1.5-fold, at least 2-fold, at least 3-fold, at least 5-fold, at least 10-fold, or at least 20-fold compared to that in the absence of the drug.

[0208] According to another specific embodiment, the change is at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or at least 100% change compared to that in the absence of the drug.

[0209] According to a specific embodiment, the determined potency of a drug indicates the efficacy of a batch of drug, i.e. the drug susceptibility indicates that the batch is efficacious.

[0210] The term "potency" as used herein refers to a measure of a drug's biological activity based on the attributes of the drug linked to relevant biological properties (ie, drug susceptibility).

[0211] According to a specific embodiment of the invention, the method comprises comparing the susceptibility of the tissue to the drug with the susceptibility of the tissue to a reference batch of the drug to determine the relative efficacy of the batches.

[0212] As used herein, the term "relative potency" refers to a qualitative measure of the potency of a batch of drug compared to a standard reference (RS) of drug of known potency.

[0213] According to a specific embodiment, the potency of a batch of drug is determined relative to the known potency of a reference standard (RS).

[0214] As used herein, the phrase "reference standard" or "RS" refers to a standardized drug used as a standard for measuring a drug. The RS provides a calibrated level of biological effect against which new preparations of the drug can be compared.

[0215] According to a particular embodiment, the RS is characterized by optimal potency and quality of active components effective in the treatment of diseases (eg inflammatory diseases, cancer).

[0216] Calculation of potency and relative potency are known in the art. According to specific embodiments, relative potency is calculated using software suitable for biological assays, such as parallel line analysis software, e.g., PLA (Stegmann Systems GmbH) and Gen5 data analysis software (BioTek).

[0217] Implementation of the methods and / or systems of the present invention may involve performing or completing selected tasks manually, automatically, or a combination thereof. Furthermore, depending on the actual instrumentation and installation of the methods and / or systems of the present invention, selected tasks could be performed by hardware, software or firmware, or a combination thereof using an operating system.

[0218] According to specific embodiments, the determined efficacy of a drug or drug combination indicates the suitability of that drug for treating a disease.

[0219] Since the inventors show that the developed EVOC system is capable of predicting tumor response to anti-cancer drugs and combination treatments (Example 2 in the Examples section below), the present invention also contemplates the use of the culture system and methods described herein to predict the response of a specific subject to a treatment regimen, thereby selecting an appropriate treatment regimen for this specific subject, and treating the subject according to that selection.

[0220] Thus, according to one aspect of the present invention there is provided a method of selecting a drug for treating a disease in a subject in need thereof, comprising the steps of: (i) culturing pathological precision-cut tissue sections obtained from a subject on tissue culture inserts in culture medium under an oxygen-enriched atmosphere containing at least 50% oxygen with agitation by rotation facilitating intermittent immersion of the tissue sections in the culture medium; (ii) adding a drug to the culture medium; and (iii) determining the effect of the drug on the tissue; wherein sensitivity of said tissue to said drug indicates the efficacy of said drug for treating said disease in said subject.

[0221] In another aspect of the invention, there is provided a method of treating a disease in a subject in need thereof, comprising: (a) selecting a drug or combination of drugs according to the methods described herein; and (b) administering to the subject a therapeutically effective amount of a drug or combination of drugs that is effective for treating the disease in the subject, thereby treating the disease in the subject. A method is provided that includes:

[0222] The term "subject" as used herein refers to a mammalian subject (e.g., a human) diagnosed with or at risk of developing a disease. Veterinary use is also contemplated. Subjects can be of any gender and age, including neonates, infants, children, juveniles, adolescents, adults, and the elderly.

[0223] The term "treat" refers to inhibiting or arresting the development of a pathology (disease, disorder, or condition) and / or causing a reduction, remission, or regression of a pathology. Those skilled in the art will appreciate that a variety of methodologies and assays can be used to assess the development of a pathology, and also to assess the reduction, remission, or regression of a pathology.

[0224] The determination of a therapeutically effective amount of a drug or drug combination is well within the capabilities of those skilled in the art. Dosage may vary depending on the drug selected, the dosage form used, and the route of administration utilized. Individual physicians can select the exact formulation, route of administration, and dosage in consideration of the patient's condition. (See, for example, Fingl, et al., 1975, "The Pharmacological Basis of Therapeutics", Ch. 1, p. 1).

[0225] According to a specific embodiment, the disease is an inflammatory disease, which can be a chronic inflammatory disease or an acute inflammatory disease.

[0226] Hypersensitivity-related inflammatory disorders Non-limiting examples of hypersensitivity include: Type II hypersensitivity, for example but not limited to, rheumatic-like diseases, rheumatic-like autoimmune diseases, rheumatoid arthritis (Krenn V. et al., Histol Histopathol 2000 Jul;15(3):791), spondylitis, ankylosing spondylitis (Jan Voswinkel et al., Arthritis Res 2001;3(3):189), systemic diseases, systemic autoimmune diseases, systemic lupus erythematosus (Erikson J. et al., Immunol Res 1998;17(1-2):49), sclerosis, systemic sclerosis (Renaudineau Y. et al., Clin Diagn Lab Immunol. 1999 Mar;6(2):156), Chan OT. et al., Immunol Rev 1999 Jun;169:107), glandular diseases, glandular autoimmune diseases, pancreatic autoimmune diseases, diabetes, type I diabetes (Zimmet P. Diabetes Res Clin Pract 1996 Oct;34 Suppl:S125), thyroid diseases, autoimmune thyroid diseases, Graves' disease (Orgiazzi J. Endocrinol Metab Clin North Am 2000 Jun;29(2):339), thyroiditis, idiopathic autoimmune thyroiditis (Braley-Mullen H. and Yu S, J Immunol 2000 Dec 15;165(12):7262), Hashimoto's thyroiditis (Toyoda N. et al., Nippon Rinsho 1999 Aug;57(8):1810), myxedema, idiopathic myxedema (Mitsuma T. Nippon Rinsho. 1999 Aug;57(8):1759), autoimmune genital diseases, ovarian diseases, ovarian autoimmunity (Garza KM. et al., J Reprod Immunol 1998 Feb;37(2):87), autoimmune antisperm infertility (Diekman AB. et al., Am J Reprod Immunol. 2000 Mar;43(3):134), recurrent miscarriage (Tincani A. et al., Lupus 1998;7 Suppl 2:S107-9), neurodegenerative diseases, nerve diseases, neurological autoimmune diseases, multiple sclerosis (Cross AH. et al.,J Neuroimmunol 2001 Jan 1;112(1-2):1), Alzheimer's disease (Oron L.et al.,J Neural Transm Suppl.1997;49:77), myasthenia gravis (Infante AJ.And Kraig E,Int Rev Immunol 1999;18(1-2):83), motor neuropathy (Kornberg AJ.J Clin Neurosci.2000 May;7(3):191), Guillain-Barre syndrome, neuropathy and autoimmune neuropathy (Kusunoki S.Am J Med Sci.2000 Apr;319(4):234), myasthenic diseases, Lambert-Eaton myasthenic syndrome (Takamori M.Am J Med Sci.2000 Apr;319(4):204), paraneoplastic neurological disorders, cerebellar atrophy, paraneoplastic cerebellar atrophy, non-paraneoplastic stiff-man syndrome, cerebellar atrophy, progressive cerebellar atrophy, encephalitis, Rasmussen's encephalitis, amyotrophic lateral sclerosis, Sydeham chorea, Gilles de la Tourette syndrome, polyendocrinopathy, autoimmune polyendocrinopathy (Antoine JC.and Honnorat J.Rev Neurol(Paris)2000 Jan;156(1):23), neuropathy, immune neuropathy (Nobile-Orazio E.et al.,Electroencephalogr Clin Neurophysiol Suppl 1999;50:419), neuromyotonia, acquired neuromyotonia, arthrogryposis multiplex congenita (Vincent A.et al.,Ann NY Acad Sci.1998 May 13;841:482), cardiovascular disease, cardiovascular autoimmune disease, atherosclerosis (Matsuura E.et al.,Lupus.1998;7 Suppl 2:S135), myocardial infarction (Vaarala O.Lupus.1998;7 Suppl 2:S132), thrombosis (Tincani A.et al.,Lupus 1998;7 Suppl 2:S107-9), granulomatosis, Wegener's granulomatosis, arteritis, Takayasu arteritis and Kawasaki syndrome (Praprotnik S. et al., Wien Klin Wochenschr 2000 Aug 25;112(15-16):660), anti-factor VIII autoimmune disease (Lacroix-Desmazes S.et al., Semin Thromb Hemost.2000;26(2):157), vasculitis, lethal small vasculitis, microscopic polyvasculitis, チャーグ·ストラウス syndrome, glomerulonephritis, microimmune type lethal glomerulonephritis, meniscus-forming glomerulonephritis (Noel LH. Ann Med Interne (Paris). 2000 May; 151 (3): 178), anti-lipid syndrome (Flamholz R. et al., J Clin Apheresis 1999;14(4):171), cardiac insufficiency, β-adrenocorticoid antibodies in patients with cardiac insufficiency (Wallukat G. et al., Am J Cardiol. 1999 Jun 17;83(12A):75H), thrombocytopenic purpura (Moccia F. Ann Ital Med Int. 1999 Apr-Jun;14(2):114), hemolytic anemia, autoimmune hemolytic anemia (Efremov DG. et al., Leuk Lymphoma 1998 Jan;28(3-4):285), gastrointestinal diseases, autoimmune diseases of the digestive tract, intestinal diseases, chronic inflammatory intestinal diseases (Garcia Herola A. et al. al., Gastroenterol Hepatol. 2000 Jan; 23(1): 16), cervical disease (Landau YE. and Shoenfeld Y. Harefuah 2000 Jan 16; 138(2): 122), autoimmune diseases of the tendon system, myofasciitis, autoimmune myofasciitis, schwann syndrome (Feist E. et al., Int Arch Allergy Immunol 2000 Sep; 123(1): 92), autoimmune diseases of smooth tendons (Zauli D. et al., Biomed Pharmacother 1999 Jun; 53(5-6): 234), liver disease, autoimmune liver disease, autoimmune hepatitis (Manns MP. J Hepatol 2000 Aug;33(2):326)およびPrimary biliary cirrhosis (Strassburg CP.et al., Eur J Gastroenterol Hepatol.1999 Jun;11(6):595);. Type IV or T cell-mediated hypersensitivity, such as, but not limited to, rheumatic-like diseases, rheumatoid arthritis (Tisch R, McDevitt HO. Proc Natl Acad Sci USA 1994 Jan 18;91(2):437), systemic diseases, systemic autoimmune diseases, systemic lupus erythematosus (Datta SK., Lupus 1998;7(9):591), glandular diseases, glandular autoimmune diseases, pancreatic diseases, pancreatic autoimmune diseases, type 1 diabetes (Castano L. and Eisenbarth GS. Ann.Rev.Immunol. 8:647), thyroid diseases, autoimmune thyroid diseases, Graves' disease (Sakata S. et al., Mol Cell Endocrinol 1993 Mar;92(1):77), ovarian diseases (Garza KM. et al., J Reprod Immunol 1998 Feb;37(2):87), prostatitis, autoimmune prostatitis (Alexander RB. et al., Urology 1997 Dec;50(6):893), polyglandular syndromes, autoimmune polyglandular syndromes, type I autoimmune polyglandular syndrome (Hara T. et al., Blood. 1991 Mar 1;77(5):1127), neurological diseases, autoimmune neurological diseases, multiple sclerosis, neuritis, optic neuritis (Soderstrom M. et al., J Neurol Neurosurg Psychiatry 1994 May;57(5):544), myasthenia gravis (Oshima M. et al., Eur J Immunol 1990 Dec;20(12):2563), stiff man syndrome (Hiemstra HS. et al., Proc Natl Acad Sci USA 2001 Mar 27;98(7):3988), cardiovascular diseases, cardiac autoimmunity in Chagas disease (Cunha-Neto E. et al., J Clin Invest 1996 Oct 15;98(8):1709), autoimmune thrombocytopenic purpura (Semple JW. et al., Blood 1996 May 15;87(10):4245), anti-helper T lymphocyte autoimmunity (Caporossi AP. et al., Viral Immunol 1998;11(1):9), hemolytic anemia (Sallah S. et al.Clin Sci(Colch)1996 Nov;91(5):551), renal disease, renal autoimmune disease, nephritis, interstitial nephritis (Kelly CJ.J Am Soc Nephrol 1990 Aug;1(2):140), connective tissue disease, ear disease, autoimmune connective tissue disease, autoimmune ear disease (Yoo TJ.et al.,Cell Immunol 1994) Aug;157(1):249), diseases of the inner ear (Gloddek B. et al., Ann NY Acad Sci 1997 Dec 29;830:266), skin diseases, cutaneous diseases, dermal diseases, bullous skin diseases, pemphigus vulgaris, bullous pemphigoid and pemphigus foliaceus;. Delayed type hypersensitivity, including but not limited to contact dermatitis and drug rash.

[0227] autoimmune disease Autoimmune diseases include, but are not limited to, cardiovascular diseases, rheumatic diseases, glandular diseases, gastrointestinal diseases, skin diseases, liver diseases, neurological diseases, muscular diseases, renal diseases, diseases related to reproduction, connective tissue diseases, and systemic diseases.

[0228] Examples of autoimmune cardiovascular diseases include atherosclerosis (Matsuura E.et al.,Lupus.1998;7 Suppl 2:S135), myocardial infarction (Vaarala O.Lupus.1998;7 Suppl 2:S132), and thrombosis (Tincani A.et al.,Lupus 1998;7 Suppl 2:S107-9), Wegener's granulomatosis, Takayasu's arteritis, Kawasaki syndrome (Praprotnik S. et al., Wien Klin Wochenschr 2000 Aug 25;112(15-16):660), anti-factor VIII autoimmune disease (Lacroix-Desmazes S. et al., Semin Thromb Hemost.2000;26(2):157), necrotizing small vasculitis, microscopic polyangiitis, Churg-Strauss syndrome, microimmune focal necrotizing and crescentic glomerulonephritis (Noel LH. Ann Med Interne(Paris).2000 May;151(3):178), antiphospholipid syndrome (Flamholz R. et al., J Clin Apheresis) 1999;14(4):171), antibody-induced heart failure (Wallukat G.et al.,Am J Cardiol.1999 Jun 17;83(12A):75H), thrombocytopenic purpura (Moccia F.Ann Ital Med Int.1999 Apr-Jun;14(2):114, Semple JW.et al.,Blood 1996 May 15;87(10):4245), autoimmune hemolytic anemia (Efremov DG.et al.,Leuk Lymphoma 1998 Jan;28(3-4):285; Sallah S.et al.,Ann Hematol 1997 Mar;74(3):139), cardiac autoimmunity in Chagas disease (Cunha-Neto E.et al.,J Clin Invest 1996 Oct 15;98(8):1709) and anti-helper T lymphocyte autoimmunity (Caporossi AP.et al.,Viral Immunol 1998;11(1):9).

[0229] Examples of autoimmune rheumatoid diseases include, but are not limited to, rheumatoid arthritis (Krenn V. et al., Histol Histopathol 2000 Jul;15(3):791; Tisch R, McDevitt HO. Proc Natl Acad Sci units SA 1994 Jan 18;91(2):437) and ankylosing spondylitis (Jan Voswinkel et al., Arthritis Res 2001;3(3):189).

[0230] Examples of autoimmune glandular diseases include, but are not limited to, pancreatic disease, type I diabetes, thyroid disease, Graves' disease, thyroiditis, idiopathic autoimmune thyroiditis, Hashimoto's thyroiditis, idiopathic myxedema, ovarian autoimmunity, autoimmune antispermia, autoimmune prostatitis, and autoimmune polyglandular syndrome type I. Diseases include pancreatic autoimmune disease, type 1 diabetes (Castano L.and Eisenbarth GS.Ann.Rev.Immunol.8:647, Zimmet P.Diabetes Res Clin Pract 1996 Oct;34 Suppl:S125), autoimmune thyroid disease, Graves' disease (Orgiazzi J.Endocrinol Metab Clin North Am 2000). Jun;29(2):339, Sakata S.et al., Mol Cell Endocrinol 1993 Mar;92(1):77), idiopathic autoimmune thyroiditis (Braley-Mullen H.and Yu S, J Immunol 2000 Dec 15;165(12):7262), Hashimoto's thyroiditis (Toyoda N.et al., Nippon Rinsho 1999 Aug;57(8):1810), idiopathic myxedema (Mitsuma T. Nippon Rinsho. 1999 Aug;57(8):1759), ovarian autoimmunity (Garza KM.et al., J Reprod Immunol 1998 Feb;37(2):87), autoimmune antizoospermia (Diekman AB.et al., Am J Reprod Immunol. 2000 Mar;43(3):134), autoimmune prostatitis (Alexander RB.et al., Urology 1997 Dec;50(6):893) and type I autoimmune polyglandular syndrome (Hara T.et al., Blood. 1991 Mar 1;77(5):1127).

[0231] Examples of autoimmune gastrointestinal diseases include, but are not limited to, chronic inflammatory bowel disease (Garcia Herola A. et al., Gastroenterol Hepatol. 2000 Jan;23(1):16), celiac disease (Landau Y E. and Shoenfeld Y. Harefuah 2000 Jan 16;138(2):122), colitis, ileitis and Crohn's disease.

[0232] Examples of autoimmune skin diseases include, but are not limited to, autoimmune bullous skin diseases such as, but not limited to, pemphigus vulgaris, bullous pemphigoid, and pemphigus foliaceus.

[0233] Examples of autoimmune liver diseases include, but are not limited to, hepatitis, autoimmune chronic active hepatitis (Franco A. et al., Clin Immunol Immunopathol 1990 Mar;54(3):382), primary biliary cirrhosis (Jones DE. Clin Sci (Colch) 1996 Nov;91(5):551; Strassburg CP. et al., Eur J Gastroenterol Hepatol. 1999 Jun;11(6):595) and autoimmune hepatitis (Manns MP. J Hepatol 2000 Aug;33(2):326).

[0234] Autoimmune neurological diseases include multiple sclerosis (Cross AH.et al.,J Neuroimmunol 2001 Jan 1;112(1-2):1), Alzheimer's disease (Oron L.et al.,J Neural Transm Suppl.1997;49:77), myasthenia gravis (Infante AJ.And Kraig E,Int Rev Immunol 1999;18(1-2):83, Oshima M.et al.,Eur J Immunol 1990 Dec;20(12):2563), neuropathy, motor neuropathy (Kornberg AJ.J Clin Neurosci.2000 May;7(3):191), Guillain-Barre syndrome and autoimmune neuropathy (Kusunoki S.Am J Med Sci.2000 Apr;319(4):234), myasthenia, Lambert-Eaton myasthenic syndrome (Takamori M.Am J Med Sci.2000 Apr;319(4):204), paraneoplastic neurological diseases, cerebellar atrophy, paraneoplastic cerebellar atrophy and stiff-man syndrome (Hiemstra HS.et al.,Proc Natl Acad Sci units SA 2001 Mar 27;98(7):3988), non-paraneoplastic stiff-man syndrome, progressive cerebellar atrophy, encephalitis, Rasmussen encephalitis, amyotrophic lateral sclerosis, Sydeham chorea, Gilles de la Tourette syndrome and autoimmune polyendocrinopathy (Antoine JC.and Honnorat J.Rev Neurol(Paris)2000 Jan;156(1):23), immune neuropathy (Nobile-Orazio E. et al., Electroencephalogr Clin Neurophysiol Suppl 1999;50:419), acquired neuromyotonia, arthrogryposis multiplex congenita (Vincent A. et al., Ann NY Acad Sci. 1998 May 13;841:482), neuritis, optic neuritis (Soderstrom M. et al., J Neurol Neurosurg Psychiatry 1994 May;57(5):544) and neurodegenerative diseases, but are not limited to these.

[0235] Examples of autoimmune muscle diseases include, but are not limited to, myositis, autoimmune myositis and primary Sjogren's syndrome (Feist E. et al., Int Arch Allergy Immunol 2000 Sep;123(1):92) and smooth muscle autoimmune disease (Zauli D. et al., Biomed Pharmacother 1999 Jun;53(5-6):234).

[0236] Examples of autoimmune renal diseases include, but are not limited to, nephritis and autoimmune interstitial nephritis (Kelly CJ. J Am Soc Nephrol 1990 Aug;1(2):140).

[0237] Examples of autoimmune diseases related to reproduction include, but are not limited to, recurrent miscarriage (Tincani A. et al., Lupus 1998;7 Suppl 2:S107-9).

[0238] Examples of autoimmune connective tissue diseases include, but are not limited to, ear diseases, autoimmune ear diseases (Yoo TJ. et al., Cell Immunol 1994 Aug;157(1):249) and autoimmune diseases of the inner ear (Gloddek B. et al., Ann NY Acad Sci 1997 Dec 29;830:266).

[0239] Examples of autoimmune systemic diseases include, but are not limited to, systemic lupus erythematosus (Erikson J. et al., Immunol Res 1998;17(1-2):49) and systemic sclerosis (Renaudineau Y. et al., Clin Diagn Lab Immunol. 1999 Mar;6(2):156), Chan OT. et al., Immunol Rev 1999 Jun;169:107).

[0240] infectious disease Examples of infectious diseases include, but are not limited to, chronic infectious diseases, subacute infectious diseases, acute infectious diseases, viral diseases, bacterial diseases, protozoal diseases, parasitic diseases, fungal diseases, mycoplasmal diseases, and prion diseases.

[0241] According to a specific embodiment, the disease is cancer.

[0242] The terms "cancer" and "cancerous" refer to the physiological condition in mammals that is typically characterized by unregulated cell growth. As used herein, the terms "cancer" and "cancerous" refer to any solid tumor, cancer metastasis, and / or solid pre-cancer.

[0243] Examples of cancer include, but are not limited to, carcinoma, blastoma, sarcoma and lymphoma.More specific examples of such cancer include squamous cell carcinoma, lung cancer (small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma and lung squamous cell carcinoma), glioma, melanoma cancer, peritoneal cancer, hepatocellular carcinoma, gastric cancer, gastroesophageal or stomach cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, soft tissue sarcoma, kidney or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, Kaposi's sarcoma carcinoid cancer, and various types of head and neck cancer.

[0244] Precancers are well characterized and known in the art (see, e.g., Berman JJ. and Henson DE., 2003. Classifying the precancers: a metadata approach. BMC Med Inform Decis Mak. 3:8). Examples of precancers include, but are not limited to, acquired small precancers, acquired large lesions with nuclear atypia, precursor lesions occurring with hereditary hyperplastic syndromes that progress to cancer, and acquired diffuse hyperplasia and diffuse dysplasia. Non-limiting examples of small precancers include HGSIL (high grade squamous intraepithelial lesions of the cervix), AIN (anal intraepithelial neoplasia), vocal cord dysplasia, aberrant crypts (of the colon), and PIN (prostatic intraepithelial neoplasia).

[0245] Non-limiting examples of acquired large lesions with nuclear atypia include tubular adenoma, angioimmunoblastic lymphadenopathy with dysproteinemia (AILD), atypical meningiomas, gastric polyps, large plaque psoriasis, myelodysplasia, papillary transitional intraepithelial carcinoma, refractory anemia with excess blasts, and Schneider's papilloma. Non-limiting examples of precursor lesions occurring with inherited hyperplastic syndromes that progress to cancer include atypical nevus syndrome, C-cell adenomatosis, and MEA. Non-limiting examples of acquired diffuse hyperplasia and diffuse dysplasia include Paget's disease of bone and ulcerative colitis.

[0246] According to specific embodiments, the cancer is selected from the group consisting of ovarian cancer, colorectal cancer, lung cancer, pancreatic cancer, gastric cancer, gastroesophageal cancer and breast cancer.

[0247] According to a specific embodiment, the cancer is a metastatic cancer.

[0248] Because there are a variety of other techniques available for predicting a patient's response to a treatment regimen, the methods of the present invention can be combined with other methods, including but not limited to, genetic profiling, tumor-derived cell lines generated from a patient's tumor, and patient-derived xenograft (PDX) models, as described in the Examples section below.

[0249] According to a specific embodiment, the method is carried out in combination with genetic profiling.

[0250] As used herein, the term "gene profiling" refers to the molecular signature of a set of genes in a biological sample using any suitable profiling technique, including but not limited to DNA sequencing, RNA sequencing and microarray techniques.

[0251] As used herein, the term "about" refers to ±10%.

[0252] The terms "comprises," "comprising," "includes," "including," and "having" and their conjugations mean "including but not limited to."

[0253] The term "consisting of" means "including and limited to."

[0254] The term "consisting essentially of" means that a composition, method, or structure may include additional ingredients, steps, and / or components, but only if the additional ingredients, steps, and / or components do not materially alter the essential and novel characteristics of the claimed compositions, methods, and structures.

[0255] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, "a compound" or "at least one compound" can include multiple compounds, including mixtures thereof.

[0256] Throughout this application, various embodiments of the invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Thus, the description of a range should be considered to have specifically disclosed all possible subranges and individual numerical values ​​within that range. For example, the description of a range such as 1-6 should be considered to have specifically disclosed subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc., as well as individual numerical values ​​within that range, e.g., 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0257] Whenever a numerical range is given herein, it is intended to include any stated numerical value (fractional or integer) that is within the stated range. The phrases "ranging between" a first denoted number and a second denoted number and "ranging from" a first denoted number to a second denoted number may be used interchangeably herein and are intended to include the first and second denoted numbers and all fractional and integer values ​​therebetween.

[0258] As used herein, the term "method" refers to means, means, techniques, and procedures for accomplishing a given task, and includes, but is not limited to, means, means, techniques, and procedures that are known to or are readily developed from known means, means, techniques, and procedures by those of skill in the art of chemistry, pharmacology, biology, biochemistry, and medicine.

[0259] When reference is made to a particular sequence listing, such reference should be understood to include minor sequence variations due, for example, to sequencing errors, cloning errors, or other modifications resulting in base substitutions, deletions, or additions, as well as to encompass sequences which substantially correspond to their complementary sequences, provided that the frequency of such variations is less than 1 per 50 nucleotides, alternatively less than 1 per 100 nucleotides, alternatively less than 1 per 200 nucleotides, alternatively less than 1 per 500 nucleotides, alternatively less than 1 per 1000 nucleotides, alternatively less than 1 per 5,000 nucleotides, or alternatively less than 1 per 10,000 nucleotides.

[0260] It is understood that certain features of the invention that are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention that are, for clarity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination, or in any other described embodiment of the invention, as appropriate. Certain features described in the context of various embodiments are not to be regarded as essential features of those embodiments, unless the embodiment cannot function without those elements.

[0261] Various embodiments and aspects of the present invention as delineated above and as claimed below are found to be experimentally supported in the following examples. Working Example

[0262] The following examples, together with the above description, illustrate some embodiments of the invention in a non-limiting manner.

[0263] In general, the terminology used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are fully explained in the literature. See, for example, Molecular Cloning: A laboratory Manual, Sambrook et al. (1989); Current Protocols in Molecular Biology, Vols. I-III, Ausubel, RM ed. (1994); Current Protocols in Molecular Biology, Ausubel et al., John Wiley and Sons, Baltimore, MD (1989); Perbal, A Practical Guide to Molecular Cloning, John Wiley & Sons, New York (1988); Watson et al., Recombinant DNA, Scientific American Books, New York; Birren et al. (eds.), Genome Analysis: A Laboratory Manual Series, Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); the methodology described in U.S. Patent Nos. 4,666,828, 4,683,202, 4,801,531, 5,192,659, and 5,272,057; Cell Biology: A Laboratory Handbook, Vols. I-III, Cellis, JE ed. (1994); Culture of Animal Cells-A Manual of Basic Technique, Freshney, Wiley-Liss, New York (1994), 3rd ed.; Current Protocols in Immunology, Vols. I-III, Coligan JE ed. (1994); Basic and Clinical Immunology, Stites et al. (eds.), 8th ed., Appleton & Lange, Norwalk, CT (1994); Selected Methods in Cellular Immunology, W. Mishell and Shiigi (eds.).H. Freeman and Co., New York (1980); available immunoassays have been described extensively in the patent and scientific literature, see, e.g., U.S. Pat. Nos. 3,791,932, 3,839,153, 3,850,752, 3,850,578, 3,853,987, 3,867,517, 3,879,262, 3,901,654, 3,935,074, 3,984,533, 3,996,345, 4,034,074, 4,098,876, 4,879,219, 5,011,771 and 5,281,521; Synthesis" Gait, MJ et al. (1984); "Nucleic Acid Hybiridaization" edited by Hames, BD and Higgins SJ (1985); "Transcription and Translation" edited by Hames, BD and Higgins SJ (1984); "Animal Cell Culture" edited by Freshney, RI (1986); "Immobilized Cells and Enzymes" IRL Press (1986); "A Practical Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology", Vols. 1-317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, Calif. (1990); Marshak et al., "Strategies for Protein Purification and Characterization--A Laboratory Course Manual", CSHL Press (1996); all of which are incorporated herein by reference as if fully set forth herein. Other general references are listed throughout the specification. The procedures described therein are believed to be well known in the art and are provided for the convenience of the reader. The information contained therein is incorporated herein in its entirety by reference. material and method

[0264] Tissue sections - healthy and cancerous tissues (liver, lung, colon, breast, pancreas, or metastatic tissues of cancer originating from lung, colon, breast or pancreas) were obtained from mouse PDX models (Nod Scid gamma mice, Jackson Laboratories USA and / or Harlan Laboratories), human biopsies and surgically removed tissues, and human-derived xenografts (ovarian cancer, pancreatic PDX). Immediately after collection, fresh tissues were placed in ice-cold phosphate buffered saline (PBS, Biological Industries, Kbbutz Beit Hamek) or ice-cold culture medium (RPMI, Biological Industries). The precision tissue sectioning instrument (Compresstome™ VF-300: Precision Instruments Inc., North Carolina, USA) was cleaned with 70% ethanol, rinsed twice with sterile water and fitted with a new blade (double-edged blade 32017759, Belgar, Jerusalem, Israel) and correctly positioned.

[0265] The tissue was kept on ice until it was transferred to a previously cleaned tissue sectioner, then it was removed from the PBS, gently attached to the plunger using contact adhesive, and stabilized in 3% low gelling temperature agarose warmed to 50°C, then cooled to solidify before use (agarose, low gelling temperature, A0701, Sigma-Aldrich, 3% in 0.9 g NaCl). The plunger with tissue was inserted into the designated location in the tissue sectioner, and the bath was filled with ice-cold Williams medium E supplemented with penicillin and streptomycin. To keep the medium in the bath at a low temperature, a hollow cooling coil was assembled that was placed in the bath. Ice-cold water was then passed through the coil using a standard immersion pump. The tissue was sectioned into approximately 250 μM sections. 1 cm 3 Approximately 30-40 sections were prepared from each tissue piece. Two or three sections were immediately placed into disposable plastic tissue cassettes and fixed in 4% PFA.

[0266] Culture procedure. Tissue slices were individually placed directly into 6-well plates (Corning CC-3516, Getter, Petah Tikva, Israel), placed on titanium inserts (MA0036-well inserts, Alabama R&D, Alabama, USA), placed on PEG, or placed on PEG placed on titanium inserts. Each well contained 4.5 ml of DMEM / F12 or M199 medium supplemented with penicillin (100 IU / ml), streptomycin (100 μg / ml), gentamicin (G1397, 50 μg / ml, Sigma-Aldrich), 5% fetal calf serum (FCS, 10270106, Gibco Sigma-Aldrich, Rehoboth, Israel), amphotericin B (A-4888, 2.5 μg / ml, Sigma-Aldrich), and glutamine (L-glutamine, 03-020-1B, 100 μg / ml, Biological Industries). Plates were incubated in a humidified incubator at 37°C with 5% CO2 and 21% or 80% O2 (oxygen chamber: BioSpherix C274, NY, USA; oxygen controller: Biospherix ProOx C21, NY, USA; and oxygen 98%, Maxxima Air Innovations, Ashdod, Israel).

[0267] The entire incubator was placed on an orbital shaker agitation (TOU-120N, MRC, Holon, Israel) at 70 rpm. After overnight incubation, the medium was replaced with fresh medium. In the indicated wells, fresh medium was added to the cells cultured with anticancer drugs [4-hydroperoxycyclophosphamide (sc-206885, ENCO, Petah Tikva, Israel), 5-flurourisyl (5FU, A10042, Adooq Biotag, Kfar Yona, Israel), oxaliplatin (O9512, Sigma-Aldrich), irinotecan (A10479-200, Adooq Biotag, Kfar Yona, Israel), docetaxel (01885, Sigma-Aldrich), cisplatin (Sigma-Aldrich), trametinib (A11029, Adooq Biotag, Kfar Yona, Israel), CDK4 / 6 inhibitor palbociclib (PZ0199, Sigma-Aldrich) or FGFR inhibitor AZD4547 (A11075-5, Adooq Biotag, Kfar Yona, Israel)]. Biotag) at the indicated concentrations. Media was replaced with fresh drug / DMSO once every 48 h for 4-6 days.

[0268] Functional assays - 18-24 hours before the end of the culture, BrdU was added to the medium (final concentration: 10 μm / ml) for later staining with anti-BrdU antibodies (BRDU+anti-BRDU B5002, Sigma-Aldrich and 94-MS-1058-P, Eldan, Petah Tikva, Israel) and / or anti-Ki67 (94-RM-9106-S, Eldan, Petah Tikva, Israel). At the end of the culture, the tissues were placed between two cover glasses, placed in disposable plastic tissue cassettes, and fixed overnight in 4% paraformaldehyde (PFA). Sections were then paraffinized, blocked, and cut into 5 μM sections for hematoxylin and eosin (H&E) staining as well as other IHC staining, e.g. Ki67 staining. EXAMPLES

[0269] Ex vivo organotypic culture conditions for maintaining structure and function of healthy and tumor tissue slices Several conditions were examined to obtain tissue structure and function in ex vivo organ culture (EVOC) for at least 5 days. Conditions included: 1. To culture tissue slices on different surfaces, such as titanium inserts, and compare them to immersing the slices directly in culture medium; 2. Incubating tissues under standard tissue culture conditions of 21% O2 and 5% CO2 and comparing with a hyperoxic atmosphere containing 80% O2; 3. Cultivating one slice per well compared with several slices per well; 4. Using different media, such as DMEM / F12 and M199; 5. Culture on PEG with or without titanium inserts Including.

[0270] The results are summarized in Table 1 below.

[0271] To this end, incubation of tissues in a high oxygen atmosphere containing 80% O2 significantly increased tissue viability compared to standard tissue culture conditions of 21% O2 (representative cultures of human ovarian cancer xenografts are shown in Figure 1). In addition, tissues survive better when supported on titanium inserts that provide a medium / air interface compared to when the tissues are inserted directly into tissue culture wells such that the slices are fully submerged in medium (representative cultures of human ovarian cancer xenografts are shown in Figure 1). Furthermore, DMEM / F12 was a better medium than M199 (representative cultures of human ovarian cancer xenografts are shown in Figure 2).

[0272] [Table 1]

[0273] All tissues for EVOC listed in Table 1 above were obtained from surgical procedures. However, the developed assay can be adapted to small biopsy tissues taken by core biopsy from tumor tissues (Figure 9). Specifically, tissues are embedded in agarose in two steps: first, agarose liquid is placed in a small metal tray, the core biopsy is placed on the agarose, and more agarose is added on top of the biopsy. The tray is cooled, and the agarose containing the biopsy is excised en bloc. In a second step, the block of agarose is fixed to a plunger with a contact clue and surrounded by agarose using a similar technique to that used for tissue pieces.

[0274] Overall, EVOCs were established from different sources representing numerous tissues that maintained tissue architecture and viability for at least 5 days in culture (Figures 1, 2, and 11). Importantly, all tissues were examined by a pathologist and deemed to be fully viable and with the same morphology as typical pathological tissue from patients.

[0275] Taken together, when single tissue slices were cultured directly in the center of titanium inserts in DMEM / F12 medium in a highly oxygenated atmosphere containing 80% O2, with the cultures agitated by orbital shaking facilitating intermittent immersion of the tissue slices in the culture medium, tissue structure and viability were maintained during at least 5 days of culture. This newly developed EVOC system was used in the following examples. EXAMPLES

[0276] The developed EVOCs can be used to predict tumor response to anticancer drugs The developed EVOC can predict the in vivo response of patient-derived xenografts to treatment: Several mouse patient-derived xenograft (PDX) models with known in vivo responses to anticancer drugs were purchased from Jackson Laboratories. Tumors were excised from mice and cultured as described in Materials and Methods and Example 1 above, and the sensitivity of tissue sections to anticancer drugs was tested. A total of seven PDX models derived from three tumor types (breast cancer, lung cancer, and colorectal cancer) were tested, and the developed EVOC system was able to correctly predict the tumor sensitivity to drug treatment in all models tested (Table 2 and Figures 3-4 below). In addition, a clear dose-dependent response was observed in the EVOC system.

[0277] Thus, as can be seen in Figures 3-4 and Table 2 below, tumors that were resistant to in vivo treatments were also resistant to ex vivo treatments, even when very high doses were applied. In contrast, when treatments to which tumors were sensitive in vivo were applied, cell death was detected ex vivo at low drug concentrations, and drug sensitivity increased with increasing drug concentration.

[0278] [Table 2]

[0279] The developed EVOCs show that human tumors exhibit differential sensitivity to different anticancer treatments: patient tumor tissues excised from patient tumors at the time of surgical resection were directly cultured as described in the Materials and Methods and in Example 1 above, and the sensitivity of tissue sections to anticancer drugs was tested. The most common anticancer regimens for each tumor type were used; i.e., for example, in the case of human colorectal cancer, the standard treatment is 5-fluoruricil, oxaliplatin and / or irinotecan. Figure 5 shows an example of a tumor that was found to be resistant to high doses of 5-fluorouracil, but relatively sensitive to irinotecan and oxaplatin. Cell proliferation determination, for example by Ki67 staining, can also be used to assess the viability of tissue sections and their sensitivity to anticancer drugs. Thus, Figure 8 shows an example of a colorectal cancer that was found to be partially sensitive to 5-fluoracil, but highly sensitive to oxaliplatin treatment by Ki67 staining.

[0280] Taken together, the developed EVOC system clearly demonstrated that patient tumors can be sensitive to one anticancer drug but resistant to another. Furthermore, EVOCs generated from individual different patients responded differentially to the same anticancer treatment (FIG. 6 and Tables 3-4 below). In addition, a dose-dependent response was observed for each anticancer treatment (e.g., Tables 3-4).

[0281] [Table 3]

[0282] [Table 4] 0-complete response, 1-strong partial response, 2-moderate partial response, 3-weak partial response, and 4-no response.

[0283] The developed EVOCs can predict sensitivity to targeted therapies predicted by molecular profiling: As a means of personalized medicine, the patient's tumor tissue can be tested for possible actionable genetic alterations, for example by exome sequencing. One of the tumors obtained was from a patient with triple-negative breast cancer that was found to harbor potentially targetable somatic genetic alterations, including amplification of FGFR1 and amplification of CDK6. As can be seen in Figure 7, the tumor obtained from this patient (patient 1) was resistant ex vivo to the CDK4 / 6 inhibitor palbociclib, but highly sensitive to the FGFR inhibitor AZD4547. Most importantly, a breast cancer obtained from another patient (patient 2) without FGFR1 amplification was resistant to FGFR inhibition in the developed EVOC system.

[0284] Tissues can be stored for 48 hours before culturing: Patient tumor tissues excised from patient tumors at the time of surgical resection were either directly cultured as described above in Materials and Methods and in Example 1, or cultured after 48 hours of storage in medium at 4° C. to test the sensitivity of the tissue sections to anticancer drugs. Figure 10 shows an example of colon cancer tissue that was found to be equally partially sensitive to 5-fluorouracil under both conditions (i.e., directly cultured and after 48 hours at 4° C.).

[0285] Taken together, storing the tissue in culture medium for at least 48 hours before initiating the experiment has no detrimental effect on the tissue or drug sensitivity.

[0286] Taken together, the newly developed EVOCs make the process of studying human cancer tissue responses rapid, cost-effective, and universal across many cancer tissues. All cancerous tissues tested, including ovarian, colorectal, lung, pancreatic, and breast tissues, exhibited high viability after 4–7 days of culture.

[0287] While the present invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

[0288] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated herein by reference.In addition, citation or identification of any reference in this specification should not be considered as an admission that such reference is available as prior art to the present invention.When section headings are used, they should not necessarily be construed as limiting. References

[0289] 1. Dancey, JE, Bedard, PL, Onetto, N. & Hudson, TJ The genetic basis for cancer treatment decisions. Cell 148, 409-420 (2012). 2. Garraway, LA Genomics-driven oncology: framework for an emerging paradigm. J. Clin. Oncol. Off. J. Am. Soc. Clin. Oncol. 31, 1806-1814 (2013). 3. Crystal,ASet al.Patient-derived models of acquired resistance can identify effective drug combinations for cancer.Science 346,1480-1486(2014). 4. Liu,X.et al.ROCK inhibitor and feeder cells induce the conditional reprogramming of epithelial cells.Am.J.Pathol.180,599-607(2012). 5. Daniel,V.C.et al.A primary xenograft model of small-cell lung cancer reveals irreversible changes in gene expression imposed by culture in vitro.Cancer Res.69,3364-3373(2009). 6. Clevers,H.Modeling Development and Disease with Organoids.Cell 165,1586-1597(2016). 7. Hidalgo,M.et al.Patient-derived xenograft models:an emerging platform for translational cancer research.Cancer Discov.4,998-1013(2014). 8. Straussman,R.et al.Tumour micro-environment elicits innate resistance to RAF inhibitors through HGF secretion.Nature 487,500-504(2012). 9. Olson,O.C.& Joyce,J.A.Microenvironment-mediated resistance to anticancer therapies.Cell Res.23,179-181(2013). 10. Vaira,V.et al.Preclinical model of organotypic culture for pharmacodynamic profiling of human tumors.Proc.Natl.Acad.Sci.U.S.A.107,8352-8356(2010). 11. Vickers,A.E.M.& Fisher,R.L.Organ slices for the evaluation of human drug toxicity.Chem.Biol.Interact.150,87-96(2004). 12. de Kanter,R.,Monshouwer,M.,Meijer,D.K.F.& Groothuis,G.M.M.Precision-cut organ slices as a tool to study toxicity and metabolism of xenobiotics with special reference to non-hepatic tissues.Curr.Drug Metab.3,39-59(2002). 13. Stoff-Khalili,M.A.et al.Preclinical evaluation of transcriptional targeting strategies for carcinoma of the breast in a tissue slice model system.Breast Cancer Res.BCR 7,R1141-1152(2005). 14. Merz,F.et al.Organotypic slice cultures of human glioblastoma reveal different susceptibilities to treatments.Neuro-Oncol.15,670-681(2013). 15. Gerlach,M.M.et al.Slice cultures from head and neck squamous cell carcinoma:a novel test system for drug susceptibility and mechanisms of resistance.Br.J.Cancer 110,479-488(2014). 16. Meijer,A.et al.Nutlin-3 preferentially sensitises wild-type p53-expressing cancer cells to DR5-selective TRAIL over rhTRAIL.Br.J.Cancer 109,2685-2695(2013). 17. Grosso,S.H.G.et al.Breast cancer tissue slices as a model for evaluation of response to rapamycin.Cell Tissue Res.352,671-684(2013). 18. Majumder,B.et al.Predicting clinical response to anticancer drugs using an ex vivo platform that captures tumour heterogeneity.Nat.Commun.6,6169(2015). 19. Roife,D.et al.Ex Vivo Testing of Patient-Derived Xenografts Mirrors the Clinical Outcome of Patients with Pancreatic Ductal Adenocarcinoma.Clin.Cancer Res.June 3,2016;DOI:10.1158 / 1078-0432.CCR-15-2936. 20. Maund,SL.et al.Optimization and comprehensive characterization of a faithful tissue culture model of the benign and malignant human prostate.Lab.Invest.94,208-221(2014). 21. Siolas,D.,Hannon,G.Patient Dervided Tumor Xengrafts:transforming clinical samples into mouse models.Cancer Res.73:5315-5319(2013). 22. Vaira,V.et al.Preclinical model of organotypic culture for pharmacodynamic profiling of human tumors.PNAS 107,8352-8356(2010). 23. van der Kuip,H.,et al.Short term culture of breast cancer tissues to study the activity of the anticancer drug taxol in an intact tumor environment.BMC Cancer 6,86-90. 24. Salmon,E.,et al.Matrix architecture defines the preferential localization and migration of T-cells into the stroma of human lung tumors.J.Clin.Invest.122,899-910(2012).

Claims

1. 1. A method of culturing tissue, comprising culturing precision-cut slices of cancerous tissue selected from the group consisting of lung cancer tissue, colorectal cancer tissue and pancreatic cancer tissue on tissue culture inserts in culture medium under a high oxygen atmosphere containing about 80% oxygen, and agitating the culture with rotation facilitating intermittent immersion of the tissue slices in the culture medium, wherein "about" refers to ±10%.

2. The method of claim 1, further comprising adding a drug to the culture medium.

3. 1. A method for determining the potency of a drug or a batch of a drug, comprising: (i) culturing a tissue according to the method of claim 1; (ii) adding the drug or a batch of the drug to the culture medium; and (iii) determining the effect of said drug or batch of said drug on said tissue; wherein sensitivity of the tissue to the drug indicates efficacy of the drug.

4. 1. A method for selecting a drug for treating a cancer selected from the group consisting of lung cancer, colorectal cancer and pancreatic cancer in a subject in need of treatment, comprising: (i) culturing the subject's cancerous tissue according to the method of claim 1; (ii) adding the drug to the culture medium; and (iii) determining the effect of the drug on the tissue. wherein sensitivity of the tissue to the drug indicates the efficacy of the drug for treating the cancer in the subject.

5. The method of claim 2 , wherein the drug is a drug combination.

6. The method according to any one of claims 3 and 4, wherein the determining is carried out by morphology assessment, viability assessment, proliferation assessment and / or cell death assessment.

7. The method according to any one of claims 3, 4 and 6, wherein said determining is performed by morphological evaluation.

8. The method according to any one of claims 3, 4 and 6, wherein said determining is carried out within 3 to 5 days of culture.

9. The method according to any one of claims 2 to 8, wherein the addition is carried out 12 to 24 hours after the initiation of the culture.

10. The method of any one of claims 2 and 5, wherein the drug is an anti-inflammatory drug or an anti-cancer drug.

11. 11. The method of any one of claims 2, 5 and 10, wherein the drug is selected from the group consisting of 5-Fluorouracil (5FU), oxaliplatin, irinotecan, cisplatin, 4-hydroperoxycyclophosphamide, docetaxel, doxorubicin, navalbine, gemcitabine, gefitinib, tamoxifen, olaparib, trametinib, everolimus and palbociclib.

12. The method of any one of claims 2, 5 and 11, wherein the drug concentration is higher than the IC50 of the drug in the cell line.

13. 13. The method of any one of claims 2, 5 and 12, wherein the drug concentrations are derived from dose-ranging experiments in which a dose-dependent response is observed.

14. The method according to any one of claims 1 to 13, wherein the culturing is carried out for at least 4 days.

15. The method according to any one of claims 1 to 14, wherein the culturing is carried out for at least 5 days.

16. The method according to any one of claims 1 to 15, wherein the culturing is carried out for up to 7 days.

17. The method according to any one of claims 1 to 16, wherein the culturing is carried out in a tissue culture plate.

18. The method of any one of claims 1 to 17, wherein the precision cut sections are 200 to 300 μm.

19. The method of any one of claims 1 to 18, wherein the section is placed in the center of the tissue culture insert.

20. The method of any one of claims 1 to 19, wherein the section is a single section.

21. The method of any of claims 1 to 20, wherein the section is in direct contact with the tissue culture insert.

22. The method of any one of claims 1 to 21, wherein the tissue culture insert is a titanium grid insert.

23. The method of any one of claims 1 to 22, wherein the tissue is human tissue.

24. The method according to any one of claims 1 to 23, carried out in combination with gene profiling.

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