WO2025062181A1 - Ex vivo cancer tissue culture system - Google Patents

Abstract

Provided herein are methods of culturing ex-vivo tissue, comprising culturing a tissue slice under a highly oxygenated atmosphere in the presence of an amount of an agent and agitating the culture. Also provided are methods of determining a therapeutically effective dose of an agent in the treatment of a disease or disorder using the ex-vivo tissue culture method.

Description

EX VIVO CANCER TISSUE CULTURE SYSTEM

RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application Serial No. 63/539686 filed September 21, 2023, the entire contents of which are incorporated herein by this reference.

BACKGROUND

Despite the widespread acceptance of genomic sequencing as an integral part of cancer personalized medicine, highly accurate individual patient drug selection remains a major unsolved problem. Targeted genetic diagnostics are used as high-fidelity companion biomarkers for inhibitors of molecular pathways, yet broad-based genomic sequencing aimed at drug selection has proven inadequate for improving patient response and outcome. Friedman, A. A., et al., Precision medicine for cancer with next-generation functional diagnostics. Nat Rev Cancer, 2015. 15(12): p. 747-56; Ashley, E.A., Towards precision medicine. Nat Rev Genet, 2016. 17(9): p. 507-22. For example, the SHIVA study, a randomized trial of genomic -based precision medicine, did not show a benefit in progression- free survival for patients assigned to genome-based treatment when compared to physicians’ choice. Le Tourneau, C., et al., Molecularly targeted therapy based on tumour molecular profiling versus conventional therapy for advanced cancer (SHIVA): a multicentre, openlabel, proof-of-concept, randomised, controlled phase 2 trial. Lancet Oncol, 2015. 16(13): p. 1324-34; Le Tourneau, C. and R. Kurzrock, Targeted therapies: What have we learned from SHIVA? Nat Rev Clin Oncol, 2016. 13(12): p. 719-720.

Genomic -based drug selection may improve patient outcomes when combined with a functional platform capable of assessing the effect of specific drugs on a patient’s tumor sample. Initially, several approaches evaluated the predictive capacity of functional assays by dissociating the tumor and testing drugs on patient cancer cells (van den Tempel, N., et al., Ex vivo assays to predict enhanced chemo sensitization by hyperthermia in urothelial cancer of the bladder. PLoS One, 2018. 13(12): p. e0209101; Larsson, P., et al., Optimization of cell viability assays to improve replicability and reproducibility of cancer drug sensitivity screens. Sci Rep, 2020. 10(1): p. 5798) or by growing patient cancer avatars in patient- derived xenograft (PDX) mouse models. More recently several groups have examined the potential of organoids as a functional assay for modeling patient drug response. Friedman, A.A., et al., Precision medicine for cancer with next-generation functional diagnostics. Nat Rev Cancer, 2015. 15(12): p. 747-56; Meijer, T.G., et al., tumor culture systems for functional drug testing and therapy response prediction. Future Sei OA, 2017. 3(2): p. FSO190. These systems do not account for the complex role of the tumor microenvironment in regulating the response to therapy. Multiple stromal components such as immune cells, fibroblasts, blood vessels, and even bacteria have been shown to affect tumor response to treatment, suggesting the need for their inclusion in functional predictive assays. Straussman, R., et al., Tumour micro -environment elicits innate resistance to RAF inhibitors through HGF secretion. Nature, 2012. 487(7408): p. 500-4; Joyce, J.A., Therapeutic targeting of the tumor microenvironment. Cancer Cell, 2005. 7(6): p. 513-20; Joyce, J.A. and J.W. Pollard, Microenvironmental regulation of metastasis. Nat Rev Cancer, 2009. 9(4): p. 239-52.

The ability to accurately predict patient response prior to treatment holds significant promise for improving patient outcomes and survival. Thus, there is a need for new methods for determining the drugs and doses that are effective to be administered to individual patients.

SUMMARY

Provided herein are methods and compositions related to the culturing of cancer tissue slices (e.g., cancer biopsy slices) in the presence of defined concentrations of therapeutic agents. In certain aspects, the concentration of therapeutic agent included in the culture has been empirically determined such that the effect of the therapeutic agent on the cancer tissue slice in culture is predictive of the efficacy of the same therapeutic agent when administered to the subject from whom the cancer tissue slice was obtained. Thus, in certain embodiments, the methods provided herein facilitate the personalized selection of effective cancer therapy for subjects in need thereof.

In certain aspects, provided herein are methods of culturing a cancer tissue (e.g., a human cancer tissue), the method comprising culturing a precision-cut cancer tissue slice (e.g., a human cancer tissue slice, such as a biopsy slice) on a tissue culture insert in a culture medium under an atmosphere containing at least 60% oxygen (e.g., at least 70% oxygen, a least 75% oxygen, between 60% and 95% oxygen, between 70% and 90% oxygen, between 75% and 85% oxygen, about 80% oxygen, or 80% oxygen) in the presence of an amount (e.g., a concentration) of an agent or a combination of agents. In some embodiments, the amount of the cancer agent is such that the effect of the cancer agent on the tissue slice is predictive of the effect of the same cancer agent (e.g., a standard therapeutic dose of the same cancer agent) when administered to the subject from whom the tissue slice was obtained. In some embodiments, the cancer tissue slice is intermittently submersed in the culture medium. In some embodiments, the amount of the agent or combination of agents comprises one or more of: (i) 30-50 (e.g., about 40) pM cetuximab; (ii) 70-90 (e.g., about 80) pM alpelisib;

(iii) 40-60 (e.g., about 50) pM carboplatin; (iv) 40-60 (e.g., about 50) pM cisplatin; (v) 90- 110 (e.g., about 100) pM etoposide; (vi) 10-30 (e.g., about 20) pM everolimus; (vii) 30-50 (e.g., about 40) pM fluorouracil, 20-40 (e.g., about 30) pM oxaliplatin, and 40-60 (e.g., about 50) pM irinotecan; (viii) 30-50 (e.g., about 40) pM ifosfamide; (ix) 40-60 (e.g., about 50) pM gemcitabine; (x) 40-60 (e.g., about 50) pM paclitaxel; and (xi) 40-60 (e.g., about 50) pM pazopanib. In some embodiments, a plurality (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more) of precision-cut cancer tissue slices from a cancer tissue are cultured in parallel with a plurality of different cancer agents or combinations of cancer agents (e.g., to identify which agents or combinations of cancer agents are most effective for the treatment of cancer in the subject from whom the cancer tissue slice was obtained).

In another aspect, provided herein is a method of predicting efficacy of an agent and/or a combination of agents on a cancer tissue, the method comprising: (a) culturing a precision-cut slice of the cancer tissue according to the method of disclosed above; and (b) determining the effect of the amount of the agent on the slice of the cancer tissue, wherein sensitivity of cancer cells in the slice of the cancer tissue to the amount of agent indicates efficacy of the agent on the cancer tissue.

In another aspect, provided herein is a method of treating a cancer in a subject (e.g., a human subject), the method comprising: (a) predicting efficacy of an agent and/or a combination of agents on a cancer tissue from a subject according to the method disclosed above; and (b) administering to the subject the agent and/or combination of agents if it is predicted to be effective on the cancer tissue.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A shows representative images of tissue samples from colorectal cancer (CRC), transitional cell carcinoma (TCC), and breast cancer (BC) that were cultured in an ex vivo organ culture (EVOC) assay that preserves the tumor microenvironment. Day 0 is the day the tissue was received, and Day 5 is after 5 days in culture. Hematoxylin and eosin (H&E) and Ki67 staining are shown. FIG. IB shows the viability of the different cancer samples, compared between Day 0 and Day 5 (N=5 per cancer type). Cancer viability was assessed and quantified by a pathologist showing non-significant changes during the culture period (N=16).

FIG. 1C shows the proliferation of the different cancer samples, compared between Day 0 and Day 5 (N=5 per cancer type). Ki67% stain was assessed and quantified by a pathologist showing non-significant changes during the culture period (N=16).

FIG. ID shows the capacity for signal transduction modification, as assessed by adding pathway- specific inhibitors and determining their effect after 24 hours. Trametinib, a pERK inhibitor, palbociclib, a CDK4/6 inhibitor and NT219, a pStat3/insulin receptor substrate (IRS) inhibitor were added to the culture and their respective pathway targets were stained by immunohistochemistry showing downregulation of activity in the culture system.

FIG. 2A shows the association between EVOC scores and clinical response of patients with MIBC. EVOC scores were computed for each sample treated with cisplatin alone, gemcitabine alone and combined cisplatin and gemcitabine. The average is marked with an X while median is denoted by a horizontal line.

FIG. 2B shows the percentage response of MIBC patients. Using an EVOC score of 45 to differentiate between non-response and response found that 75.6% of patients with MIBC who had a high EVOC score were classified as responders while 24.4% were classified as non-responders.

FIG. 2C shows the clinical outcomes (pathology or RECIST) of patients who received a full course of cisplatin and gemcitabine as correlated to their EVOC score.. Patients who underwent cystectomy were listed as non-responders (NR), partial responder (PR), and complete responder (CR) based on pathology, while tumors of patients who were evaluated by imaging alone were designated a RECIST score (progressive disease [PD], stable disease [SD], PR, or CR).

FIG. 2D shows representative H&E staining from a responder and non-responder MIBC patient. EVOC samples were treated with vehicle or cisplatin and gemcitabine.

FIG. 2E shows representative H&E images from a responder and non-responder patient with bladder cancer. Vehicle samples were compared to samples treated with cisplatin and gemcitabine for 5 days.

FIG. 3A shows biopsies of breast cancer (BC), pancreatic ductal adenocarcinoma (PDAC), and sarcoma that were obtained prior to initiation of patient treatment and maintained in EVOC for 5 days. Representative images of the biopsies on day 0 and day 5 show high viability and preservation during the culture assay of several prominent tissue types.

FIG. 3B shows representative images of a pancreatic cancer biopsy stained with H&E comparing treatment with vehicle and treatment with combined paclitaxel and gemcitabine after 5 days. The sample treated with paclitaxel and gemcitabine shows cell-death representing a response and an EVOC score of 64.

FIG. 3C shows a graph demonstrating the percent of each cancer type obtained in the clinical trial evaluating EVOC predictive capabilities on biopsies.

FIG. 3D shows the clinical outcomes (pathology or RECIST) of 18 patients with available data correlated to their EVOC score.

FIG. 4A shows the distribution of responders and non-responders based on an EVOC score of 45.

FIG. 4B shows the statistical calculations for sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV).

FIG. 4C shows the correlation between EVOC scores and clinical outcomes of patients (determined by pathology or RECIST) with resected or biopsy samples (N=34).

DETAILED DESCRIPTION

Provided herein are methods of culturing ex-vivo tissue, comprising culturing a tissue slice under a highly oxygenated atmosphere in the presence of an amount of an agent or combination of agents and agitating the culture. Also provided are methods of determining a therapeutically effective agent or combination of agents in the treatment of cancer in a subject using the ex-vivo tissue culture method. In some embodiments, these methods can be used to predict patient treatment outcomes on both resected samples and core biopsies from solid tumors. Additionally, in some embodiments, these methods can be used to determine side effects of anti-cancer treatments on healthy tissue. The instant methods differentiate between samples that are responders or non-responders to a given therapy with high sensitivity.

In certain aspects, provided herein are methods of culturing a cancer tissue (e.g., a human cancer tissue), the method comprising culturing a precision-cut cancer tissue slice (e.g., a human cancer tissue slice, such as a biopsy slice) on a tissue culture insert in a culture medium under an atmosphere containing at least 60% oxygen (e.g., at least 70% oxygen, a least 75% oxygen, between 60% and 95% oxygen, between 70% and 90% oxygen, between 75% and 85% oxygen, about 80% oxygen, or 80% oxygen) in the presence of an amount (e.g., a concentration) of an agent or a combination of agents. In some embodiments, the amount of the cancer agent is such that the effect of the cancer agent on the tissue slice is predictive of the effect of the same cancer agent (e.g., a standard therapeutic dose of the same cancer agent) when administered to the subject from whom the tissue slice was obtained. In some embodiments, the cancer tissue slice is intermittently submersed in the culture medium. In some embodiments, the amount of the agent or combination of agents comprises one or more of: (i) 10 to 100 pM cetuximab; (ii) 10 to 100 pM alpelisib; (iii) 10 to 100 pM carboplatin; (iv) 10 to 100 pM cisplatin; (v) 10 to 100 pM etoposide; (vi) 10 to 100 pM everolimus; (vii) 10 to 100 pM fluorouracil, 10 to 100 pM oxaliplatin, and 10 to 100 pM irinotecan; (viii) 10 to 100 pM ifosfamide; (ix) 10 to 100 pM gemcitabine; (x) 10 to 100 pM paclitaxel; and (xi) 10 to 100 pM pazopanib. In some embodiments, a plurality (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more) of precision-cut cancer tissue slices from a cancer tissue are cultured in parallel with a plurality of different cancer agents or combinations of cancer agents (e.g., to identify which agents or combinations of cancer agents are most effective for the treatment of cancer in the subject from whom the cancer tissue slice was obtained).

In another aspect, provided herein is a method of predicting efficacy of an agent and/or a combination of agents on a cancer tissue, the method comprising: (a) culturing a precision-cut slice of the cancer tissue according to the method of disclosed above; and (b) determining the effect of the amount of the agent on the slice of the cancer tissue, wherein sensitivity of cancer cells in the slice of the cancer tissue to the amount of agent indicates efficacy of the agent on the cancer tissue.

In another aspect, provided herein is a method of treating a cancer in a subject (e.g., a human subject), the method comprising: (a) predicting efficacy of an agent and/or a combination of agents on a cancer tissue from a subject according to the method disclosed above; and (b) administering to the subject the agent and/or combination of agents if it is predicted to be effective on the cancer tissue.

In certain embodiments, the determining step comprises morphology evaluation, viability evaluation, proliferation evaluation, and/or cell death evaluation. In certain preferred embodiments, the determining step comprises morphology evaluation. In certain embodiments, the determining step is performed within 3 to 5 days of culturing.

In certain preferred embodiments, the cancer tissue is a sarcoma. In some embodiments, the cancer tissue is ovarian, breast, pancreatic, colorectal, esophageal, liver, lung, skin, cartilage, bone, or gastric tissue. In some embodiments, the cancer tissue is not liver tissue. In certain preferred embodiments, the precision-cut cancer tissue slice is from a biopsy. In certain preferred embodiments, the precision-cut tissue slice is 200 to 300 pm thick.

In certain embodiments, the precision-cut tissue slice is in direct contact with the tissue culture insert. In certain embodiments, the tissue culture insert is a titanium grid insert.

In certain embodiments, the culture is rotationally agitated to facilitate the intermittent submersion. In certain preferred embodiments, the rotational agitation is performed at an agitation frequency of 50 to 100 rotations per minute (rpm).

In certain preferred embodiments, the culture medium is DMEM/F12 culture medium.

In certain embodiments, the tissue slice is cultured for at least 4 days. In some embodiments, the tissue culture slice is cultured for at least 5 days.

In certain embodiments, the agent comprises a monoclonal antibody that functions as an epidermal growth factor receptor inhibitor (i.e., cetuximab, panitumumab, nimotuzumab, or necitumumab). In certain preferred embodiments, the amount of agent is 40 pM cetuximab.

In certain embodiments, the agent comprises alpelisib. In certain embodiments, the amount of agent is 80 pM alpelisib.

In certain embodiments, the agent comprises carboplatin. In certain embodiments, the amount of agent is 50 pM carboplatin.

In certain embodiments, the agent comprises cisplatin. In certain embodiments, the amount of agent is between 40 and 60 pM cisplatin (e.g., about 50 pM cisplatin).

In certain embodiments, the agent comprises etoposide. In certain embodiments, the amount of agent is between 90 and 110 pM etoposide (e.g., about 100 pM etoposide).

In certain embodiments, the agent comprises everolimus. In certain embodiments, the amount of agent is between 10 and 30 pM everolimus (e.g., about 20 pM everolimus).

In certain embodiments, the agent comprises one or more of fluorouracil, oxaliplatin, and irinotecan. In certain embodiments, the amount of agent is between 30 and 50 pM fluorocuracil (e.g., about 40 pM fluorouracil), between 10 and 40 pM oxaliplatin (e.g., about 30 pM oxaliplatin), and between 10 and 40 pM irinotecal (e.g., about 30 pM irinotecan).

In certain embodiments, the agent comprises gemcitabine. In certain embodiments, the amount of agent is 50 pM gemcitabine.

In certain embodiments, the agent comprises ifosfamide. In certain embodiments, the amount of agent is 40 pM ifosfamide. In certain embodiments, the agent comprises paclitaxel. In certain embodiments, the amount of agent is 50 pM paclitaxel.

In certain embodiments, the agent comprises pazopanib. In certain embodiments, the amount of agent is 50 pM pazopanib.

It is appreciated that certain features of the invention, which 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, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements. Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

Definitions

Unless specifically stated or obvious from context, as used herein, the term "or" is understood to be inclusive. Unless specifically stated or obvious from context, as used herein, the terms “a,” “an,” and “the” are understood to be singular or plural.

“Administration” broadly refers to a route of administration of a composition (e.g., a therapeutic composition) to a subject. Examples of routes of administration include oral administration, rectal administration, topical administration, inhalation (nasal) or injection. Administration by injection includes intravenous (IV), intramuscular (IM), and subcutaneous (SC) administration. A therapeutic composition described herein can be administered in any form by any effective route, including but not limited to oral, parenteral, enteral, intravenous, intraperitoneal, topical, transdermal (e.g., using any standard patch), intradermal, ophthalmic, (intra)nasally, local, non-oral, such as aerosol, inhalation, subcutaneous, intramuscular, buccal, sublingual, (trans)rectal, vaginal, intra-arterial, and intrathecal, transmucosal (e.g., sublingual, lingual, (trans)buccal, (trans)urethral, vaginal (e.g., trans- and perivaginally), implanted, intravesical, intrapulmonary, intraduodenal, intragastrical, and intrabronchial. In preferred embodiments, a therapeutic composition described herein is administered orally, rectally, topically, by injection into or adjacent to a draining lymph node, intravenously, by inhalation or aerosol, or subcutaneously. In another preferred embodiment, a therapeutic composition described herein is administered orally or intravenously. As used herein, the phrases “anti-cancer drug” or “cancer therapeutic agent” refer to an agent that has an anti-tumor effect including chemotherapy, small molecules, biological drugs, hormonal therapy, antibodies and targeted therapy.

As used herein, the term “culture system” refers to at least a precision-cut tissue slice, insert and medium in an ex-vivo environment.

As used herein, term “effective dose” is the amount of the therapeutic composition that is effective to achieve the desired therapeutic response for a particular subject, composition, and mode of administration, with the least toxicity to the subject.

As used herein, the phrase “optimal area of viability” refers to a microscopic field of the tissue (e.g. in 20X magnification) in which the highest number of live cells per unit area are present, as assessed by a pathologist, in comparison to the immediate pre-EVOC sample of the same species.

As used herein the phrase “patient-derived xenograft (PDX)"” refers to tissue generated by the implantation of a primary tissue into an animal from a different species relative to the donor of the primary tissue.

As used herein, the term “potency” refers to the measure of the biological activity of the drug, based on the attribute of the drug which is linked to the relevant biological properties (i.e.; drug sensitivity).

As used herein, the phrase “precision-cut tissue slice” refers to a viable slice obtained from an isolated solid tissue with reproducible, well defined thickness (e.g. ± 5 % variation in thickness between slices).

As used herein, the term “relative potency” refers to a qualitative measure of potency of a batch of the drug, relatively to a standard reference (RS) of the drug, having a known potency.

As used herein the phrases “rotationally agitated facilitating intermittent submersion of the tissue slice in the culture medium” or “agitating in a rotation facilitating intermittent submersion of the tissue slice in the culture medium” refers to agitation which allows periodic submersion of the tissue slice in the medium such that facilitates nutrients and gas diffusion throughout the medium and through the tissue slice.

As used herein the phrase “sensitivity to a drug” or “sensitivity to drug combination” refers to the ability of a drug or drug combination to induce cellular changes such as changes in cell viability, proliferation rate, differentiation, cell death, necrosis, apoptosis, senescence, transcription and/or translation rate of specific genes and/or changes in protein states e.g. phosphorylation, dephosphorylation, translocation and any combinations thereof. Cellular changes can be reflected by decreased cell viability, decreased proliferation rate, increased cell death and/or aberrant morphology as compared to same in the absence of the drug.

As used herein, terms “subject” or “patient” refers to any mammal. A subject or a patient described as “in need thereof’ refers to one in need of a treatment (or prevention) for a disease. Mammals (i.e., mammalian animals) include humans, laboratory animals (e.g., primates, rats, mice), livestock (e.g., cows, sheep, goats, pigs), and household pets (e.g., dogs, cats, rodents). The subject may be a human. The subject may be a non-human mammal including but not limited to of a dog, a cat, a cow, a horse, a pig, a donkey, a goat, a camel, a mouse, a rat, a guinea pig, a sheep, a llama, a monkey, a gorilla or a chimpanzee. The subject may be healthy or may be suffering from a disease or disorder at any developmental stage.

As used herein, the term “therapeutic agent” refers to an agent for therapeutic use.

As used herein, the term “therapeutic composition” or “pharmaceutical composition” refers to a composition that comprises a therapeutically effective amount of a therapeutic agent.

As used herein the term “tissue” refers to part of a solid organ (i.e., not blood) of an organism having some vascularization that includes more than one cell type and maintains at least some macro structure of the in-vivo tissue from which it was excised. Examples include, but are not limited to, ovarian tissue, colorectal tissue, lung tissue, pancreatic tissue, breast tissue, brain tissue, retina, skin tissue, bone, cardiac tissue and renal tissue.

As used herein, the phrase “tissue culture insert” refers to a porous membrane suspended in a vessel for tissue culture and is compatible with subsequent ex-vivo culturing of a tissue slice. The pore size is capable of supporting the tissue slice while it is permeable to the culture medium enabling the passage of nutrients and metabolic waste to and from the slice, respectively.

As used herein, the term “treating” a disease in a subject or “treating” a subject having or suspected of having a disease refers to administering to the subject to a pharmaceutical treatment, e.g., the administration of one or more agents, such that at least one symptom of the disease is decreased or prevented from worsening. Thus, in one embodiment, “treating” refers inter alia to delaying progression, expediting remission, inducing remission, augmenting remission, speeding recovery, increasing efficacy of or decreasing resistance to alternative therapeutics, or a combination thereof. As used herein, the term “preventing” a disease in a subject refers to administering to the subject to a pharmaceutical treatment, e.g., the administration of one or more agents, such that onset of at least one symptom of the disease is delayed or prevented. Exemplary Cancer Therapeutic Agents

In certain embodiments, the methods provided herein relate to methods of culturing ex vivo precision-cut tissue culture slices (e.g., biopsy slices) in the presence of defined amounts of cancer therapeutic agents and/or combinations of cancer therapeutic agents. In some embodiments, a single cancer therapeutic agent is included in the culture. In some embodiments, a combination of cancer therapeutic agents (e.g., 2, 3, 4, 5, or more agents) is included in the culture.

In certain embodiments, the cancer therapeutic agent is alpelisib. As used herein, term “alpelisib” refers to the chemotherapy drug:

or a pharmaceutically acceptable salt thereof. In certain embodiments, 10-100 pM alpelisib is included in the culture. In some embodiments, 80 pM alpelisib is included in the culture.

In certain embodiments, the cancer therapeutic is carboplatin. As used herein, term “carboplatin” refers to the chemotherapy drug:

or a pharmaceutically acceptable salt thereof. In certain embodiments, 10-100 pM carboplatin is included in the culture. In some embodiments, 50 pM carboplatin is included in the culture.

In certain embodiments, the cancer therapeutic is cetuximab. As used herein, term “cetuximab” refers to a chimeric human/mouse IgGl monoclonal antibody that targets epidermal growth factor receptor (EGFR). In certain embodiments, 10-100 uM cetuximab is included in the culture. In some embodiments, 40 pM cetuximab is included in the culture. In some embodiments, 50 pM cetuximab is included in the culture.

In certain embodiments, the cancer therapeutic is cisplatin. As used herein, term “cisplatin” refers to the chemotherapy drug:

or a pharmaceutically acceptable salt thereof. In certain embodiments, 10-100 pM cetuximab is included in the culture. In some embodiments, 50 pM cisplatin is included in the culture. In certain embodiments, the cancer therapeutic is etoposide. As used herein, term

“etoposide” refers to the chemotherapy drug:

or a pharmaceutically acceptable salt thereof. In certain embodiments, 10-100 pM etoposide is included in the culture. In some embodiments, 100 pM etoposide is included in the culture.

In certain embodiments, the cancer therapeutic is everolimus. As used herein, term “everolimus” refers to the chemotherapy drug:

or a pharmaceutically acceptable salt thereof. In certain embodiments, 10-100 pM everolimus is included in the culture. In some embodiments, 20 pM everolimus is included in the culture.

In certain embodiments, the cancer therapeutic is fluorouracil. As used herein, term “fluorouracil” refers to the chemotherapy drug:

or a pharmaceutically acceptable salt thereof. In certain embodiments, 10-100 pM fluorouracil is included in the culture. In some embodiments, 40 pM fluorouracil is included in the culture.

In certain embodiments, the cancer therapeutic is FOLFIRINOX. As used herein, term “FOLFIRINOX” refers to a combination of folinic acid, fluorouracil, irinotecan, and oxaliplatin. In certain embodiments, 40 pM fluorouracil, 30 pM oxaliplatin, and 30 pM irinotecan are included in the culture.

In certain embodiments, the cancer therapeutic is FOLFOX. As used herein, term “FOLFOX” refers to a combination of folinic acid, fluorouracil, and oxaliplatin.

In certain embodiments, the cancer therapeutic comprises folinic acid. As used herein, term “folinic acid” refers to the medication:

or a pharmaceutically acceptable salt thereof.

In certain embodiments, the cancer therapeutic is gemcitabine. As used herein, terms “gemcitabine” and “Gemzar” refer to the chemotherapy drug:

or a pharmaceutically acceptable salt thereof. In certain embodiments, 10-100 pM gemcitabine is included in the culture. In some embodiments, 50 pM gemcitabine is included in the culture.

In certain embodiments, the cancer therapeutic is ifosfamide. As used herein, term “ifosfamide” refers to the chemotherapy drug:

or a pharmaceutically acceptable salt thereof. In certain embodiments, 10-100 pM ifosfamide is included in the culture. In some embodiments, 40 pM ifosfamide is included in the culture.

In certain embodiments, the cancer therapeutic is irinotecan. As used herein, term “irinotecan” refers to the chemotherapy drug:

or a pharmaceutically acceptable salt thereof. In certain embodiments, 10-100 pM irinotecan is included in the culture. In some embodiments, 30pM irinotecan is included in the culture.

In certain embodiments, the cancer therapeutic is NT219. As used herein, term “NT219” refers to the chemotherapy drug:

or a pharmaceutically acceptable salt thereof. In certain embodiments, 20 pM NT219 is included in the culture.

In certain embodiments, the cancer therapeutic is oxaliplatin. As used herein, term “oxaliplatin” refers to the chemotherapy drug:

or a pharmaceutically acceptable salt thereof. In certain embodiments, 10-100 pM oxaliplatin is included in the culture. In some embodiments, 30 pM oxaliplatin is included in the culture.

In certain embodiments, the cancer therapeutic is paclitaxel. As used herein, terms “paclitaxel” and “Taxol” refer to the chemotherapy drug:

or a pharmaceutically acceptable salt thereof. In certain embodiments, 10-100 pM paclitaxel is included in the culture. In some embodiments, 50 pM paclitaxel is included in the culture. In certain embodiments, the cancer therapeutic is palbociclib. As used herein, term

“palbociclib” refers to the chemotherapy drug:

or a pharmaceutically acceptable salt thereof. In certain embodiments, 10 pM palbociclib is included in the culture. In certain embodiments, the cancer therapeutic is pazopanib. As used herein, term

“pazopanib” refers to the chemotherapy drug:

or a pharmaceutically acceptable salt thereof. In certain embodiments, 10-100 pM pazopanib is included in the culture. In some embodiments, 50 pM pazopanib is included in the culture.

In certain embodiments, the cancer therapeutic is trametinib. As used herein, term “trametinib” refers to the chemotherapy drug:

or a pharmaceutically acceptable salt thereof. In certain embodiments, 10 nM trametinib is included in the culture.

Culture Systems and Methods

In certain embodiments, the methods provided herein relate to methods of culturing ex vivo precision-cut tissue culture slices (e.g., biopsy slices) in the presence of defined amounts of cancer therapeutic agents.

In certain embodiments, the culture system systems provided herein preserve the tissue microenvironment, architecture, viability and genetic heterogeneity of the tissue that is being cultured. This system enables studying a human tissue (e.g. cancer tissue) response in a fast, reliable and cost effective manner. Consequently, the present teachings further suggest the use of such culture systems for qualifying efficacy of a drug for cancer treatment in general and for personalized cancer therapy in particular.

According to specific embodiments, the culture system maintains structure and viability of the precision-cut tissue slice for at least 2-10, 2-7, 2-5, 4-7, 5-7 or 4-5 days in culture. According to a specific embodiment, the precision-cut tissue slice maintains viability for at least 5 days, 6 days, 7 days or even 10 days, unless such viability is reduced by a test agent added to the culture. According to a specific embodiment, the precision-cut tissue slice maintains viability for at least 5 days, unless such viability is reduced by an test agent added to the culture.

According to specific embodiments, in the absence of an effective test agent, at least 60%, at least 70%, at least 80% of the cells in the precision-cut tissue maintain viability following 4-5 days in culture as determined by e.g. morphology analysis of an optimal area of viability. According to a specific embodiment, the culturing is effected for at least 4 days. According to a specific embodiment, the culturing is effected for at least 5 days. According to a specific embodiment, the culturing is effected for up to 7 days. According to specific embodiments, the tissue is selected from the group consisting of ovarian, colorectal, lung, pancreas, gastric, gastro esophageal and breast. According to specific embodiments, the tissue is selected from the group consisting of ovarian, colorectal, lung, pancreas gastric, gastro esophageal, breast, liver, cartilage and bone. According to specific embodiments the tissue is a metastatic cancer tissue obtained from sites such as, but not limited to the liver, the bone, the lung and the peritoneum. According to specific embodiments, the tissue is not a liver tissue. According to specific embodiments, the tissue is not a prostate tissue.

According to specific embodiments, the tissue is a mammalian tissue. According to a specific embodiment, the tissue is a human tissue. According to specific embodiments, the tissue is a healthy tissue.

According to other specific embodiments, the tissue is a cancer (e.g., tumor) tissue. The method may employ a plurality of screened precision-cut tissue slices (e.g., each on a separate insert), all of which can be from a cancer (e.g., tumor) tissue(s), healthy tissue(s) or a combination of same (e.g., when the healthy tissue serves as control when taken from the same tissue origin as the cancer/tumor tissue). According to specific embodiments the tissue is a cancer (e.g., tumor) tissue.

According to specific embodiments the tissue is obtained surgically or by biopsy, laparoscopy, endoscopy or as xenograft or any combinations thereof. The tissue may be cut and cultured directly following tissue extraction (i.e. primary tissue) or following implantation in an animal model (i.e. a patient-derived xenograft (PDX)), each possibility represents a separate embodiment of the present invention. The tissue or the tissue slice to some embodiments of the present invention can be freshly isolated or stored e.g., at 4 °C or cryopreserved (i.e. frozen) at e.g. liquid nitrogen.

According to specific embodiments, the tissue or the tissue slice is freshly isolated (e.g., not more than 24 hours after retrieval from the subject and/or not subjected to preservation processes).

According to specific embodiments, the tissue is cryopreserved following tissue retrieval and prior to cutting. According to specific embodiments, the tissue is thawed prior to cutting. According to specific embodiments, the tissue slice is cryopreserved following cutting.

According to specific embodiments, the tissue is preserved at 4 °C in e.g. medium following tissue retrieval and prior to cutting. According to specific embodiments the tissue slice is preserved at 4 °C in e.g. medium following cutting and prior to culturing. According to specific embodiments, the preservation at 4 °C is effected for up to 120 hours, up to 96 hours, up to 72 hours or up to 48 hours. According to specific embodiments, the preservation at 4 °C is effected for 24-48 hours. According to specific embodiments the PDX is a tissue generated by implantation of a human primary tissue (e.g. cancerous tissue) into an immunodeficient mouse. Following tissue extraction the tissue is sliced to precision-cut slices.

Typically, the tissue slice is a mini-model of the tissue which contains the cells of the tissue in their natural environment and retains the three-dimensional connectivity such as intercellular and cell-matrix interactions of the intact tissue with no selection of a particular cell type among the different cell type that constitutes the tissue or the organ. Precisioncutting reduces sources of error due to variations in slice thickness and damage to cut surfaces, which both contribute to uneven gas and nutrient exchange throughout tissue slices; it enhances reproducibility; and allows adjacent slices to be evaluated for histology and compared pair- wise under different experimental conditions.

The slice section can be cut in different orientations (e.g. anterior-posterior, dorsal- ventral, or nasal-temporal) and thickness. The size/thickness of the tissue section is based on the tissue source and the method used for sectioning. According to specific embodiment the thickness of the precision-cut slice allows maintaining tissue structure in culture. According to specific embodiments the thickness of the precision-cut slice allows full access of the inner cell layers to oxygen and nutrients, such that the inner cell layers are exposed to sufficient oxygen and nutrients concentrations.

According to specific embodiments the thickness of the precision-cut slice allows full access of the inner cell layers to oxygen and nutrients, such that the inner cell layers are exposed to the same oxygen and nutrients concentrations as the outer cell layers. According to specific embodiments, the precision-cut slice is between 50-1200 pm, between 100-1000 pm, between 100-500 pm, between 100-300 pm, or between 200-300 pm. According to a specific embodiment, the precision-cut slice is 200-300 pm.

Methods of obtaining tissue slices are known in the art and described for examples in the Examples section which follows and 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 fully incorporated herein by reference. Such methods include, but are not limited to slicing using a vibratome, agarose embedding followed by sectioning by a microtome, or slicing using a matrix. As a non-limiting example, the tissue is isolated and immediately placed in a physiological dissection media (e.g. ice cold PBS) which may be supplemented with antibiotics.

According to specific embodiments, the warm ischemic time is less than 2 hours, less than 1.5 hours or less than 1 hour. 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 hour, less than 5 hours or less than 2 hours.

Prior to slicing, the tissue is attached to the tissue slicer using e.g. contact glue followed by embedding in e.g. low melting agarose gel. Subsequently, the tissue is sectioned into precision-cut slices. Numerous suitable tissue sectioning devices are commercially available, such as, but not limited to Compresstome™ VF-300 (Precisionary Instruments Inc. NC, USA), Brendel- Vitron tissue slicer (Tucson, AZ), Krumdieck precision tissue slicer (model no. MD4000-01; Alabama R&D) and Leica VT1200S vibrating blade microtome (Leica, Wetzlar, Germany). According to specific embodiments, the sectioning devise is filled with ice cold medium such as Williams Medium E or Krebs-Henseleit buffer (KHB). The skilled artisan would know which medium and conditions for dissection and for preserving the tissue and the tissue slice prior to culturing for each type of tissue.

Following, the tissue slice is 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 specific embodiments, one slice is placed on a single tissue culture insert.

According to specific embodiments, the culture vessel is filled with culture medium up to the bottom of the tissue slice (e.g. 4 ml of medium in a 6-well plate containing an insert). The culture may be in a glass, plastic or metal vessel that can provide an aseptic environment for tissue culturing. According to specific embodiments, the culture vessel includes dishes, plates, flasks, bottles and vials. Culture vessels such as COSTAR®, NUNC® and FALCON® are commercially available from various manufacturers.

According to specific embodiments, the culture vessel is a tissue culture plate such as a 6-wells plate, 24-wells plate, 48-wells plate and 96-wells plate. According to a specific embodiment, the culture vessel is a tissue culture 6-wells plate.

According to specific embodiments, the culture vessel is not pre-coated with proteins extracted from a matched tissue (e.g. when the tissue is a cancerous tissue, then the culture vessel is not pre-coated with stage and grade-matched tumor extracted proteins). Non limiting examples for such proteins include ECM proteins such as collagen, fibronectin, laminin, vibronectin, cadherin, filamin A, vimentin, osteopontin, Decorin, tenascin X, basement membrane proteins, cytoskeletal proteins and matrix proteins; and growth factors. The culture medium used by the present invention can be a water-based medium which includes a combination of substances such as salts, nutrients, minerals, vitamins, amino acids, nucleic acids and/or proteins such as cytokines, growth factors and hormones, all of which are needed for cell proliferation and are capable of maintaining structure and viability of the tissue.

For example, a culture medium can be a synthetic tissue culture medium such as DMEM/F12 (can be obtained from e.g. Biological Industries), M199 (can be obtained from e.g. Biological Industries), RPMI (can be obtained from e.g. Gibco-Invitrogen Corporation products), M199 (can be obtained from e.g. Sigma- Aldrich), Ko-DMEM (can be obtained from e.g. Gibco- Invitrogen Corporation products), supplemented with the necessary additives as is further described hereinunder. Preferably, all ingredients included in the culture medium of the present invention are substantially pure, with a tissue culture grade. The skilled artisan would know to select the culture medium for each type of tissue contemplated.

According to specific embodiments of the invention, the culture medium comprises serum e.g. fetal calf serum (FCS, can be obtained e.g. from Gibco-Invitrogen Corporation products). According to specific embodiments, the culture medium comprises less than 10% serum. According to specific embodiments, the culture medium comprises less than 2% serum obtained from the same species as the cultured tissue. According to specific embodiments, the culture medium is devoid of serum obtained from the same species as the cultured tissue. According to specific embodiments, the culture medium comprises less than 2% serum autologous to the cultured tissue (i.e. from the same subject). According to specific embodiments, the culture medium is devoid of serum autologous to the cultured tissue (i.e. from the same subject). According to specific embodiments, the culture medium comprises less than 2% human serum. According to some embodiments of the invention, the culture medium is devoid of human serum. According to some embodiments of the invention, the culture medium is devoid of any animal contaminants, i.e., animal cells, fluid or pathogens (e.g., viruses infecting animal cells), i.e., being xeno-free.

According to some embodiments of the invention, the culture medium can further include antibiotics (e.g., penicillin, streptomycin, gentamycin), anti-fungal agents (e.g. amphotericin B), L-glutamine or NEAA (non-essential amino acids). According to a specific embodiment, the medium comprises serum and antibiotics. According to a specific embodiment, the medium comprises DMEM/F12, 5 % FCS, glutamine, penicillin, streptomycin, gentamycin and amphotericin B.

It should be noted that the culture medium may be periodically refreshed to maintain sufficient levels of supplements and to remove metabolic waste products that can 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. According to specific embodiments, the culture medium is refreshed every 12-48 hours. According to a specific embodiment, the culture medium is refreshed once after 12-24 hours and then every about 48 hours.

According to specific embodiments, the tissue slice is placed on the tissue culture insert, thereby allowing access of the culture medium to both the apical and basal surf aces of the tissue slice. According to specific embodiments the pore size is 0.1 pm - 20 pm, 0.1 pm - 15 pm, 0.1 pm- 10 pm, 0.1pm-5 pm, 0.4 pm-20 pm, 0.4 pm- 10 pm or 0.4 pm- 5 pm. According to specific embodiments the pore size is 0.4 mm - 4 mm, 0.4 mm - 1 mm, 1 mm - 4 mm, 1 mm - 3 mm or 1 mm - 2 mm. According to specific embodiments, the tissue culture insert is sterile. According to some embodiments, the tissue culture inset is disposable. According to other embodiments, the tissue culture insert is re-usable and autoclavable. The cell culture insert may be synthetic or natural, it can be inorganic or polymeric e.g. titanium, alumina, Polytetrafluoroethylene (PTFE), Teflon, stainless steel, polycarbonate, nitrocellulose and cellulose esters. According to specific embodiments, the cell culture insert is a titanium insert. Cell culture inserts that can be used with specific embodiments of the invention are commercially available from e.g. Alabama R&D, Millipore Corporation, Costar, Coming Incorporated, Nunc, Vitron Inc. and SEFAR and include, but not limited to MA0036 Well plate Inserts, BIOCOAT™, Transwell®, Millicell®, Falcon®-Cyclopore, Nunc® Anapore, titanium- screen and Teflon-screen. According to specific embodiments, the tissue culture insert is a titanium grid insert, such as but not limited to Titanium MA0036 Well plate Inserts (Alabama R&D).

According to specific embodiments, the tissue culture insert is not coated with an organic material such as collagen, fibronectin or polyethylene glycol (PEG). According to specific embodiments, the tissue culture insert is not coated with proteins extracted from a matched tissue (e.g. when the tissue is a cancerous tissue, then the tissue culture insert is not pre-coated with stage and grade-matched tumor extracted proteins). According to specific embodiments, the tissue slice is in direct contact with the tissue culture insert.

According to specific embodiments, the tissue slice is not separated from the culture medium by a heterologous organic material such as collagen, fibronectin or synthetic polymer (which is not part of the culture vessel) e.g., polyethylene glycol (PEG).

According to specific embodiments, the tissue slice is in direct contact with the culture medium.

According to specific embodiments, the tissue slice is placed in the middle of the cell culture insert. Thus, for example, when a titanium grid such as the Titanium MA0036 Well plate Inserts is applied the tissue slice is placed in the concavity located at the middle of the insert.

In certain embodiments, the tissue slice is cultured (or maintained) at a physiological temperature, e.g. 37 °C, in a highly oxygenated humidified atmosphere containing, e.g., at least 60% oxygen and e.g. 5% CO2. According to specific embodiments the highly oxygenated atmosphere contains at least 60%, at least 70% or at least 80% oxygen. According to specific embodiments, the highly oxygenated atmosphere contains at least 70% oxygen. According to other specific embodiments, the highly oxygenated atmosphere contains less than 95% oxygen. According to a specific embodiment, the highly oxygenated atmosphere contains about 80% oxygen.

According to a specific embodiment, during the culturing process, the culture is agitated in a rotation facilitating intermittent submersion of the tissue slice in the culture medium. According to specific embodiments, the agitation is orbital agitation. According to specific embodiments the agitation is an angled agitation, e.g. an angle of 30 ⁰ - 45 °. According to specific embodiments, the agitation is effected by an inclined rotator (such as the MD2500 incubation unit commercially available from Alabama Research and Development). According to specific embodiments the agitation frequency is 50 - 200 rpm, 50 - 150 rpm or 50 - 100 rpm. According to specific embodiment the agitation frequency is about 70 rpm.

The tissue culture and the methods of the present invention can be adapted to many applications, including, but not limited to predicting patient's response to drugs (e.g. anticancer drugs) and drug combinations and consequently using this prediction to tailor the specific treatment regime for the patient (i.e. personalized medicine). As the tissue slice maintains the structure and presents high viability following 5 days of culture, this system allows modeling of chronic as well as acute toxicity studies, including the metabolic activity of the tissue.

According to other specific embodiments, the method described hereinabove comprises adding a drug or a drug combination, as further described herein below. According to specific embodiments, the drug is an anti-cancer drug.

Anti-cancer drugs that can be used with specific embodiments of the invention include, but are not limited to: Acivicin; Aclarubicin; Acodazole Hydrochloride; Acronine; Adriamycin; Adozelesin; Aldesleukin; Altretamine; Ambomycin; Ametantrone Acetate; Aminoglutethimide; Amsacrine; Anastrozole; Anthramycin; Asparaginase; Asperlin; Azacitidine; Azetepa; Azotomycin; Batimastat; Benzodepa; Bicalutamide; Bisantrene Hydrochloride; Bisnafide Dimesylate; Bizelesin; Bleomycin Sulfate; Brequinar Sodium; Bropirimine; Busulfan; Cactinomycin; Calusterone; Caracemide; Carbetimer; Carboplatin; Carmustine; Carubicin Hydrochloride; Carzelesin; Cedefingol; Chlorambucil; Cirolemycin; Cisplatin; Cladribine; Crisnatol Mesylate; Cyclophosphamide; Cytarabine; Dacarbazine; Dactinomycin; Daunorubicin Hydrochloride; Decitabine; Dexormaplatin; Dezaguanine; Dezaguanine Mesylate; Diaziquone; Docetaxel; Doxorubicin; Doxorubicin Hydrochloride; Droloxifene; Droloxifene Citrate; Dromostanolone Propionate; Duazomycin; Edatrexate; Eflomithine Hydrochloride; Elsamitrucin; Enloplatin; Enpromate; Epipropidine; Epirubicin Hydrochloride; Erbulozole; Esorubicin Hydrochloride; Estramustine; Estramustine Phosphate Sodium; Etanidazole; Etoposide; Etoposide Phosphate; Etoprine; Fadrozole Hydrochloride; Fazarabine; Fenretinide; Floxuridine; Fludarabine Phosphate; Fluorouracil; Flurocitabine; Fosquidone; Fostriecin Sodium; Gemcitabine; Gemcitabine Hydrochloride; Hydroxyurea; Idarubicin Hydrochloride; Ifosfamide; Ilmofosine; Interferon Alfa-2a; Interferon Alfa- 2b; Interferon Alfa-nl; Interferon Alfa-n3; Interferon Beta- 1 a; Interferon Gamma- 1 b; Iproplatin; Irinotecan Hydrochloride; Lanreotide Acetate; Letrozole; Leuprolide Acetate; Liarozole Hydrochloride; Lometrexol Sodium; Lomustine; Losoxantrone Hydrochloride; Masoprocol; Maytansine; Mechlorethamine Hydrochloride; Megestrol Acetate; Melengestrol Acetate; Melphalan; Menogaril; Mercaptopurine; Methotrexate; Methotrexate Sodium; Metoprine; Meturedepa; Mitindomide; Mitocarcin; Mitocromin; Mitogillin; Mitomalcin; Mitomycin; Mitosper; Mitotane; Mitoxantrone Hydrochloride; Mycophenolic Acid; Nocodazole; Nogalamycin; Ormaplatin; Oxisuran; Paclitaxel; Pegaspargase; Peliomycin; Pentamustine; Peplomycin Sulfate; Perfosfamide; Pipobroman; Piposulfan; Piroxantrone Hydrochloride; Plicamycin; Plomestane; Porfimer Sodium; Porfiromycin; Prednimustine; Procarbazine Hydrochloride; Puromycin; Puromycin Hydrochloride; Pyrazofurin; Riboprine; Rogletimide; Safingol; Safingol Hydrochloride; Semustine; Simtrazene; Sparfosate Sodium; Sparsomycin; Spirogermanium Hydrochloride; Spiromustine; Spiroplatin; Streptonigrin; Streptozocin; Sulofenur; Talisomycin; Taxol; Tecogalan Sodium; Tegafur; Teloxantrone Hydrochloride; Temoporfin; Teniposide; Teroxirone; Testolactone; Thiamiprine; Thioguanine; Thiotepa; Tiazofuirin; Tirapazamine; Topotecan Hydrochloride; Toremifene Citrate; Trestolone Acetate; Triciribine Phosphate; Trimetrexate; Trimetrexate Glucuronate; Triptorelin; Tubulozole Hydrochloride; Uracil Mustard; Uredepa; Vapreotide; Verteporfin; Vinblastine Sulfate; Vincristine Sulfate; Vindesine; Vindesine Sulfate; Vinepidine Sulfate; Vinglycinate Sulfate; Vinleurosine Sulfate; Vinorelbine Tartrate; Vinrosidine Sulfate; Vinzolidine Sulfate; Vorozole; Zeniplatin; Zinostatin; Zorubicin Hydrochloride. Additional antineoplastic agents include those disclosed in Chapter 52, Antineoplastic Agents (Paul Calabresi and Bruce A. Chabner), and the introduction thereto, 1202-1263, of Goodman and Gilman's "The Pharmacological Basis of Therapeutics", Eighth Edition, 1990, McGraw-Hill, Inc. (Health Professions Division).

Non-limiting examples for anti-cancer approved drugs include: abarelix, aldesleukin, aldesleukin, alemtuzumab, alitretinoin, allopurinol, altretamine, amifostine, anastrozole, arsenic trioxide, asparaginase, azacitidine, AZD9291, AZD4547, AZD2281, bevacuzimab, bexarotene, bleomycin, bortezomib, busulfan, calusterone, 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, dexrazoxane, docetaxel, doxorubicin, dromostanolone propionate, Elliott's B 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, 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, Oprelvekin, 5 oxaliplatin, paclitaxel, palbociclib palifermin, pamidronate, pegademase, pegaspargase, Pegfilgrastim, pemetrexed disodium, pentostatin, pipobroman, plicamycin mithramycin, 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.

It will be appreciated that the culture system and methods described herein can be used to test the differential effect of drug combinations. Thus, according to specific embodiments, the drug is a drug combination.

According to specific embodiments, several tissue slices from one tissue are prepared and cultured in several culture vessels (e.g. wells of a plate) allowing testing of a number of drugs and drug combinations.

In some embodiments, the methods described herein can be used to determine the effect induced by the drug combination in comparison to the effect induced by each of the drugs in the combination, which may by an additive effect or a synergistic effect. According to specific embodiments, the effect is a synergistic effect.

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 as compared to the effect induced by each of the drugs in the combination. According to other specific embodiments the increase is by at least 5 %, by at least a 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 more than 100 % as compared to the effect induced by each of the drugs in the combination.

As mentioned, according to specific embodiments, the tissue or the tissue slices may be stored i.e. at 4 °C or cryopreserved and thawed as desired allowing serial testing of several drugs and drugs combinations sequentially.

The drug or the drug combination may be added to the culture at various time points. According to specific embodiments, the drug is added to the culture 2-48 hours, 2-36, 2-24, 12-48, 12-36 or 12-24 hours following the beginning of the culture. According to a specific embodiment, the drug or the drug combination is added to the culture 12-24 hours following the beginning of the culture. According to a specific embodiment, the drug or the drug combination is added to the culture 12-36 hours following the beginning of the culture. According to a specific embodiment, the drug or the drug combination is added to the culture 12-48 hours following the beginning of the culture. Culturing in the presence of the drug or the drug combination may be effected throughout the whole culturing period from first drug addition or can be limited in time. Alternatively, or additionally, the drug or the drug combination may be added to the culture multiple times e.g. when the culture medium is refreshed. According to specific embodiments, the drug concentration and incubation time with the drug or the drug combination results in detectable effect on the tissue as further described herein.

The number of tested drug concentration 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.

The number of samples repeats for each of the tested drug concentration can be 2, 3, 4, 5 or 6 repeats.

Following culturing the effect of the drug or the drug combination on the tissue can be determined to thereby determine efficacy of a drug.

According to specific embodiments, the determining step is effected following predetermined culturing time. The culturing time may vary and determination of the culturing time that will result in detectable effect is well within the capabilities of those skilled in the art.

According to specific embodiments, the determining is effected within 2-10, 2-7, 2-5, 3-10, 3-7, 3-5 or 4-5 days of culturing. According to a specific embodiment, the determining is effected within 3-5 days of culturing. According to another specific embodiment, the determining is effected within 4-5 days of culturing.

Methods of determining effects induced by the drug or the drug combination are known in the art and include for example:

(i) Viability evaluation using e.g. the MTT test which is based on the selective ability of living cells to reduce the yellow salt MTT (3-(4, 5- dimethylthiazolyl-2)-2, 5- diphenyltetrazolium bromide) (Sigma, Aldrich St Louis, MO, USA) to a purple-blue insoluble formazan precipitate; the WST assay or the ATP uptake assay;

(ii) Proliferation evaluation using e.g. the BrDu assay [Cell Proliferation ELISA BrdU colorimetric kit (Roche, Mannheim, Germany] or Ki67 staining; Cell death evaluation using e.g. the TUNEL assay [Roche, Mannheim, Germany] the Annexin V assay [ApoAlert® Annexin V Apoptosis Kit (Clontech Laboratories, Inc., CA, USA)], the LDH assay, the Activated Caspase 3 assay, the Activated Caspase 8 assay and the Nitric Oxide Synthase assay;

(iii) Senescence evaluation using e.g. the Senescence associated— -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 US A 92:9363-9367) and telomerase shortening assay; Cell metabolism evaluation using e.g. the glucose uptake assay;

(iv) Various RNA and protein detection methods (which detect level of expression and/or activity); and

(v) Morphology evaluation using e.g. the Haemaotxylin & Eosin (H&E) staining;

According to specific embodiments, the determining is effected by morphology evaluation, viability evaluation, proliferation evaluation and/or cell death evaluation.

According to specific embodiments, the determining is effected by morphology evaluation.

Morphology evaluation using H&E staining can provide details on e.g. cell content, size and density, ratio of viable cells/dead cells, ratio of diseased (e.g. tumor) cells/healthy cells, immune cells infiltration, fibrosis, nuclear size and density and integrity, apoptotic bodies and mitotic figures. According to specific embodiments effect of the drug on the tissue is determined by morphology evaluation by e.g. a pathologist.

Typically, results from each of the assays are expressed in a numeric form wherein a high score correlates with drug sensitivity and a low score correlated with drug resistance.

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 as compared to same in the absence of the drug. According to other specific embodiments the change is by at least 5 %, by at least a 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 % as compared to same in the absence of the drug.

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

Implementation of the method and/or system of embodiments of the invention can involve performing or completing selected tasks manually, automatically, or a combination thereof. Moreover, according to actual instrumentation and equipment of embodiments of the method and/or system of the invention, several selected tasks could be implemented by hardware, by software or by firmware or by a combination thereof using an operating system.

According to specific embodiments, the determined efficacy of the drug or the drug combination indicates suitability of the drug for the treatment of a disease. As the present inventors show that the developed EVOC system can predict responses of tumors to anti-cancer drugs, the present invention also contemplates the use of the culture systems and methods described herein for predicting response of a specific subject to a treatment regime, thereby selecting the suitable treatment regime for this specific subject and treating the subject according to this selection.

EXAMPLES

Example 1: A clinical evaluation of an ex-vivo organ culture system to predict patient response to cancer therapy

A multicenter, prospective, single-arm observational trial has been conducted to examine the ability of this EVOC platform to predict patient response to therapy in the clinic. First, the previously shown capacity of the system for preserving resected patient cancer tissue with its microenvironment over several days has been recapitulated. Next, a predictive clinical trial in muscle-invasive bladder cancer (MIBC) was established to evaluate the accuracy of the platform’s prediction of drug response in patients receiving neoadjuvant or induction chemotherapy where up to 30% of patients are not expected to respond to therapy. MIBC was chosen as a first indication, since significant quantities of resected tissue are frequently available for EVOC profiling, and the patient’s response can be followed throughout their treatment. To further validate the capacity of the platform to preserve tissue and predict patient response based on core biopsy samples, a cohort of patients with different metastatic cancers who underwent biopsies prior to treatment was assembled. Finally, the platform’s results were compared to the patients’ clinical response using pathology scores or Response Evaluation Criteria in Solid Tumors (RECIST).

Methods

Bladder Cancer

To determine the predictive capacity of the EVOC system, a clinical trial in patients with bladder cancer who were candidates for neoadjuvant or induction chemotherapy was established. Patients with newly diagnosed bladder cancer, who were referred for transurethral resection of bladder tumor (TURBT) at four medical centers, were considered eligible for this study. All patients underwent axial imaging (either computed tomography [CT] or positron-emission tomography [PET]/CT) for staging at diagnosis. At the beginning of the TURBT, a 1-3 mL sample was obtained from the tumor’s most superficial area and far from its base to avoid any impact on local histological staging. The sample was immediately placed in ice-cold Dulbecco’s Modified Eagle’s Medium (DMEM) and transferred to the lab. The final referral to chemotherapy was at the urologist's and oncologist’s discretion.

Core biopsies of metastatic tumors

Patients with highly suspected metastatic cancer, or those previously diagnosed with metastatic cancer, who underwent axial imaging (either CT or PET/CT) for staging of pancreatic, breast, liver, colon, sarcoma, or esophageal tumors (at least 2 cm in diameter) and referred for diagnostic needle core biopsy at three medical centers, were eligible for participating in the study. During the procedure 2-4 cores of 16-gauge or 18-gauge needles were removed and transferred to the lab in ice-cold DMEM.

Preparation of the EVOC

As previously described, tumors were cut into 250-pM slices using a vibratome (VF300, Precisionary Instruments). A sample of the tissue was fixed immediately in 4% paraformaldehyde (PFA) as a reference and analyzed for viability within 24 hours. The rest of the slices were placed in 12 or 24 well plates on titanium grids with 4 mL of DMEM/F12 medium (supplemented with 5% fetal calf serum [FCS], penicillin 100 lU/mL with streptomycin 100 pg/mL, amphotericin B 2.5 pg/mL, gentamicin sulfate 50 mg/mL, and L- glutamine 100 pL/mL). The tissue slices were then cultured at 70 rpm on an orbital shaker (TOU-120N, MRC) at 37°C, 5% CO2, and 80% O2. One day after sectioning, bladder tumor sections were treated with cisplatin 30 pM and/or gemcitabine 30 pM for 96 hours, with media and drug change after 48 hours.

Core biopsies obtained from patients with metastatic cancer study (2-5 core biopsies/patient) were sectioned as described above. One tissue section was fixed immediately as a reference and assessed by rapid histology within 24 hours. The remaining tissue was prepared as EVOC and cultured with drugs that were likely to be used in the upcoming clinical treatment as suggested by the treating oncologist. Drug concentrations were used as previously described and are listed in Table 1 unless otherwise noted with culture for 96 hours and medium change after 48 hours. At the end of the incubation period the tissue sections were fixed overnight with 4% PFA followed by formalin-fixed paraffin embedding (FFPE).

Table 1. Drugs and concentrations used in culture media.

To monitor pathway regulation in response to drug treatment, tissue samples were cultured with small molecule inhibitors of several oncogenic pathways for 24 hours: trametinib 10 nM (a pERK inhibitor), palbociclib 10 pM (a CDK4/6 inhibitor) and NT219 20 pM (a pStat3/insulin receptor substrate [IRS] inhibitor). The samples were then stained by immunohistochemistry.

EVOC Scoring

Tissue immunohistochemistry was performed on 4-pm sections from the FFPE tissue samples. Hematoxylin and eosin (H&E) staining was performed using an automated Stainer (Eeica Biosystems). Ki67 staining (Thermo Fischer Antibody (RM-9106); 1:500 dilution) was performed using an automated Stainer (BOND RX, Eeica Biosystems). The pathologists were shown the tissue fixed immediately after it was resected from the patient and sliced (time 0) and an untreated tissue obtained after 5 days (control) as reference samples. All other treated EVOC samples were evaluated blindly. The pathologists assessed the viability (Vz) of live treated tumor cells on a scale of 0-100% (compared to the immediately fixed and control samples), the level of damage (2) on a scale of 0-4, and Ki67 proliferation (K) factored as a percentage of replicating cells. To account for tissue heterogeneity, the scores are an average of the treated tissue with a particular drug from 3 different tumor slices taken from the first third, middle third and final third of the biopsied specimen. A final score on a scale of 0-100, accounting for all parameters, was obtained using the formula:

A score of 0 represents completely viable cancer cells, suggesting no response, and a score of 100 represents no viable cancer cells, suggesting complete response. The weighted output of the evaluation was based on coefficients X, Y, Z corresponding to 0.7, 0.2, 0.1, respectively. A threshold score of 45 was used to differentiate between non-responders and responders based on the complete training set applied using the above coefficients on MIBC bladder samples correlated to historical clinical response for neoadjuvant treatment.

Standard of clinical reference

In patients with MIBC treated with neoadjuvant gemcitabine and cisplatin, followed by radical cystectomy (RC), the pathological evaluation of the surgical specimen served as the standard of reference. Response to treatment by pathology was defined as 1. complete response (no malignancy present - pTO), 2. partial response (histological downstaging), or 3. no response (no change of histological staging or upstaging). In patients with bladder cancer who received chemotherapy without undergoing surgery, radiological follow-up data was used according to the RECIST criteria. In the core biopsy cohort, RECIST 1.1 criteria were based on imaging.

Genomic sequencing

Patients from the metastatic biopsy study underwent genomic sequencing on gDNA extracted from the tumor sample using the Qiagen DNeasy DNA purification kit. The test is based on next generation sequencing (NGS) using iSeqlOO of a panel containing 286 amplicons covering hotspots of 57 genes relevant to cancer and treatment (Swift Biosciences, Inc.). Genomic analysis for mutation frequencies greater than 5% is presented.

Statistical analysis

Data were analyzed by descriptive statistics. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of the EVOC tumor response scores were calculated. Differences in tissue viability, Ki67 staining, or EVOC scores and correlations to different clinical outcomes were evaluated using t-test or analysis of variance. P values <0.05 were considered statistically significant. All statistical analyses were conducted using SPSS 20 (IBM Corp.).

Results

The EVOC system preserves the tumor microenvironment

To validate the robustness of the EVOC technology, which maintains thick sections of different cancer types in culture for 5 days, the survival and cell dynamics of colorectal cancer, urothelial carcinoma and breast cancer were examined (FIG. 1A). All tissue types showed a mean 95% viability on day 5 when compared to the tissue sampled immediately after surgical removal on day 0 (FIG. IB). Additionally, no statistically significant difference was noted in the average percentage of Ki67-positive cancer cells on day 0 (5.8%) versus day 5 (5.5%), p=0.92, among samples of all tissue types (n=16) of breast cancer, urothelial carcinoma and colorectal cancer (FIG. 1C).

To assess signaling pathway dynamics in the tissue, the EVOC was cultured with small molecule inhibitors of several oncogenic pathways for 24 hours and evaluated the ability to suppress signaling using phosphorylated markers of signaling dynamics. Corresponding to their respective pathways, trametinib downregulated pERK, palbociclib lowered pRB and NT219 blocked pStat3 staining (FIG. ID). These results illustrate the sensitivity of the tissue to specific drug inhibition and the corresponding response shown by the signaling pathway.

EVOC of samples from patients with MIBC

In total, 111 patients with newly diagnosed bladder tumors provided 101 samples that were processed in the EVOC system. Fifty-one of these samples were non-muscle-invasive urothelial carcinomas (Ta/Tl), and 50 were muscle-invasive urothelial cancers (T2). The clinical characteristics of all patients are shown in Table 2.

Table 2. Characteristics of patients at recruitment evaluated with EVOC characteristics

MIBC EVOC score were obtained from 46 samples (FIG. 2A). Samples treated with cisplatin showed a median score of 64 (mean 59) while those treated with gemcitabine scored a median of 27 (mean 34), indicating a significantly greater effect of cisplatin therapy. Combined treatment with cisplatin and gemcitabine showed a median score of 87 (mean 70), indicating a combinatorial effect of the treatments. Notably, analysis of all cisplatingemcitabine scores showed high response scores (>45) in 75.6% (35/46) of samples compared to low scores (<45) in 24.4% (9/46) corresponding to values found in the literature for response to neoadjuvant bladder cancer therapy (FIG. 2B).

EVOC score predicts clinical response to therapy in MIBC

EVOC scores were computed for each MIBC sample treated with cisplatin alone, gemcitabine alone and combined cisplatin and gemcitabine (FIG. 2A). Among the 50 patients with MIBC, 16 (32%) received a full course of chemotherapy with cisplatin and gemcitabine, and their clinical response was correlated with the EVOC scores. Based on the previously noted distinction between the high and low EVOC scores, an EVOC score of 45 was applied to differentiate between responders and non-responders (FIG. 2B). The clinical outcomes (pathology or RECIST) of patients who received a full course of cisplatin and gemcitabine were correlated to their EVOC score. Among the 16 patients who completed therapy, 12 were responders while 4 were non-responders based on final pathology or RECIST (FIG. 2C). A correlation was observed between clinical response and EVOC scores (p=0.04 by t test comparing PD/NR scores to SD/PR/CR scores). Higher median EVOC scores were correlated with better clinical response category: a median EVOC score of 8 for PD (mean 27.5), 59.5 for SD (mean 59.5), 100 for PR (mean 92.4) and 100 for CR (mean 100) (FIG. 2D).

Representative images of tissue response show significant cell death of urothelial carcinoma cells following treatment with cisplatin and gemcitabine, compared to control, in a patient with clinically confirmed response. In contrast, tissue samples obtained from a clinically confirmed non-responder show that urothelial carcinomas cells remain highly viable and refractory to treatment (FIG. 2E).

EVOC scores in metastatic tumor specimens obtained by needle core biopsy

To further validate the application of the EVOC platform for predicting patient response, an additional cohort of patients with solid tumors who underwent core needle biopsy samples known to be more challenging to maintain ex vivo was established. EVOC from core-needle biopsies of patients highly suspected of metastatic cancer or those previously diagnosed with metastatic cancer were compared to the clinical response of these patients.

In total, 94 patients with metastatic tumors were recruited to the study. Fifty of these patients had at least 90% viable cancer tissue in the immediate sample taken from their core biopsy, and their samples were prepared as EVOC. Forty samples completed the process with at least 80% cancer cell viability in the vehicle sample, which was determined as the cut-off for a successful assay. Representative images of biopsy samples of breast cancer, pancreatic ductal adenocarcinoma, and sarcoma, maintain tissue architecture and viability after 5 days in culture (FIG. 3A). Biopsy samples clinically identified as responders showed significant tissue death which corresponded to higher scores in the EVOC platform (FIG. 3B).

Among the 40 patients with EVOC results, a total of 18 completed their prescribed course of treatment and had follow-up clinical data for correlation. The largest group of samples was pancreatic cancer (38.9%). Additional samples were breast, colorectal, esophageal, sarcoma, and liver cancers (FIG. 3C). Genomic analysis was performed to identify notable genomic mutations, as shown in Table 3, and demonstrates mutational heterogeneity among the tumor types. EVOC scores were then correlated with their corresponding clinical outcome (FIG. 3D). Plotting the EVOC scores of each of the patients in their respective clinical response categories showed a significant difference between the scores of responders and non-responders (t-test p<0.001 comparing PD/NR scores to SD/PR/CR scores). Moreover, higher median EVOC scores were correlated with better clinical response categories: a median EVOC score of 31 for PD (mean 22.6), 50 for SD (mean 56), and 83.1 for PR (mean 83.1) (FIG. 3D).

Table 3. Genomic analysis of selected samples

Correlation of EVOC scores with clinical response of patients for clinical prediction

Overall, EVOC results were correlated with clinical data in 34 patients (16 MIBC samples from TURBT and 18 other cancers from core needle biopsy). A threshold EVOC score of 45 provided an optimal distinction between responders and non-responders, correlating closely with clinical results (FIG. 4A). Nine patients (26.4%) were non- responders to therapy (defined as PD in RECIST or NR on pathology) while 25 (73.5%) showed a response (SD, PR, CR in RECIST or PR or pathological CR in pathology). The EVOC system showed a predictive specificity of 77.7% (7/9, 95% CI 0.4-0.97), a sensitivity of 96% (24/25, 95% CI 0.80-0.99), a PPV of 92.3% (24/26, 95% CI 0.77-0.99) and NPV of 87.5% (7/8, 95% CI 0.47-0.99) (FIG. 4B). Comparison of EVOC scores of clinical responders with non-responders showed a statistically significant difference (p<0.01). Moreover, comparison of scores from each clinical response category yielded median EVOC scores of 17 for PD (mean 32.88), 56 for SD (mean 57.16), 86 for PR (mean 84), and 100 for CR (mean 100), indicating that higher EVOC scores predict better clinical response (ANOVA p<0.01, F=15.1453) (FIG. 4C).

Incorporation by Reference

All publications and patent applications mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

Equivalents

Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

What is claimed is:

1. A method of culturing a cancer tissue, the method comprising culturing a precisioncut cancer tissue slice on a tissue culture insert in a culture medium under an atmosphere containing at least 60% oxygen in the presence of an amount of an agent; wherein the cancer tissue slice is intermittently submersed in the culture medium; wherein the amount of the agent comprises:

(i) 30-50 pM cetuximab;

(ii) 70-90 pM alpelisib;

(iii) 40-60 pM carboplatin;

(iv) 40-60 pM cisplatin;

(v) 90-110 pM etoposide;

(vi) 10-30 pM everolimus;

(vii) 30-50 pM fluorouracil, 20-40 pM oxaliplatin, and 40-60 pM irinotecan;

(viii) 30-50 pM ifosfamide;

(ix) 40-60 pM gemcitabine;

(x) 40-60 pM paclitaxel; or

(xi) 40-60 pM pazopanib.

2. A method of predicting efficacy of an agent on a cancer tissue, the method comprising:

(a) culturing a precision-cut slice of the cancer tissue according to the method of claim 1; and

(b) determining the effect of the amount of the agent on the slice of the cancer tissue, wherein sensitivity of cancer cells in the slice of the cancer tissue to the amount of agent indicates efficacy of the agent on the cancer tissue.

3. A method of treating a cancer in a subject, the method comprising:

(a) predicting efficacy of an agent on a cancer tissue from a subject according to the method of claim 2; and

(b) administering to the subject the agent if it is predicted to be effective on the cancer tissue.

4. The method of claim 2 or 3, wherein the determining step comprises morphology evaluation, viability evaluation, proliferation evaluation, and/or cell death evaluation.

5. The method of claim 4, wherein the determining step comprises morphology evaluation.

6. The method of any one of claims 2 to 5, wherein the determining step is performed within 3 to 5 days of culturing.

7. The method of any one of claims 1 to 6, wherein the cancer tissue is a sarcoma.

8. The method of any one of claims 1 to 7, wherein the cancer tissue is ovarian, breast, pancreatic, colorectal, esophageal, liver, lung, skin, cartilage, bone, or gastric tissue.

9. The method of any one of claims 1 to 8, wherein the precision-cut cancer tissue slice is from a biopsy.

10. The method of any one of claims 1 to 9, wherein the precision-cut tissue slice is 200 to 300 pm thick.

11. The method of any one of claims 1 to 10, wherein the precision-cut tissue slice is in direct contact with the tissue culture insert.

12. The method of any one of claims 1 to 11, wherein the tissue culture insert is a titanium grid insert.

13. The method of any one of claims 1 to 12, wherein the tissue is a human tissue.

14. The method of any one of claims 1 to 13, wherein the atmosphere contains about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, or about 90% oxygen.

15. The method of any one of claims 1 to 14, wherein the atmosphere contains between 80% and 95% oxygen.

16. The method of any one of claims 1 to 15, wherein the culture is rotationally agitated to facilitate the intermittent submersion.

17. The method of claim 16, wherein the rotational agitation is performed at an agitation frequency of 50 to 100 rotations per minute (rpm).

18. The method of any one of claims 1 to 17, wherein the culture medium is DMEM/F12 culture medium.

19. The method of any one of claims 1 to 18, wherein the tissue slice is cultured for at least 4 days.

20. The method of any one of claims 1 to 19, wherein the tissue culture slice is cultured for at least 5 days.

21. The method of any one of claims 1 to 20, wherein the agent comprises a monoclonal antibody that functions as an epidermal growth factor receptor inhibitor (i.e., cetuximab, panitumumab, nimotuzumab, or necitumumab).

22. The method of claim 21, wherein the amount of agent is 40 pM cetuximab.

23. The method of any one of claims 1 to 20, wherein the agent comprises alpelisib.

24. The method of claim 23, wherein the amount of agent is 80 pM alpelisib.

25. The method of any one of claims 1 to 20, wherein the agent comprises carboplatin.

26. The method of claim 25, wherein the amount of agent is 50 pM carboplatin.

27. The method of any one of claims 1 to 20, wherein the agent comprises cisplatin.

28. The method of claim 27, wherein the amount of agent is 50 pM cisplatin.

29. The method of any one of claims 1 to 20, wherein the agent comprises etoposide.

30. The method of claim 29, wherein the amount of agent is 100 pM etoposide.

31. The method of any one of claims 1 to 20, wherein the agent comprises everolimus.

32. The method claim 31, wherein the amount of agent is 20 pM everolimus.

33. The method of any one of claims 1 to 20, wherein the agent comprises one or more of fluorouracil, oxaliplatin, and irinotecan.

34. The method of claim 33, wherein the amount of agent is 40 pM fluorouracil, 30 pM oxaliplatin, and 30 pM irinotecan.

35. The method of any one of claims 1 to 20, wherein the agent comprises gemcitabine.

36. The method of claim 35, wherein the amount of agent is 50 pM gemcitabine.

37. The method of any one of claims 1 to 20, wherein the agent comprises ifosfamide.

38. The method of claim 37, wherein the amount of agent is 40 pM ifosfamide.

39. The method of any one of claims 1 to 20, wherein the agent comprises paclitaxel.

40. The method of claim 39, wherein the amount of agent is 50 pM paclitaxel.

41. The method of any one of claims 1 to 20, wherein the agent comprises pazopanib.

42. The method claim 41, wherein the amount of agent is 50 pM pazopanib.