Methods and products for ex vivo modeling of immune tumor therapy

Abstract

The field of the invention is in vitro assays for the production of in vitro organ culture systems and the use of such in vitro organ culture systems for screening assays in drug screening, patient selection and personalized medical context. More specifically, the present invention relates to an in vitro method of establishing a tumor organoid from a cancer tissue sample obtained from a subject, an in vitro method of providing immune cells activated by an autologous tumor organoid, a method of preparing an in vitro organ culture system for mimicking interaction of a tumor with an immune system of a subject, and a method of preparing an in vitro organ culture system for mimicking interaction of a tumor with an immune system of a subject. The invention relates to an in vitro organ culture system, and to an in vitro organ culture system produced thereby, to a method for determining the reactivity of a tumor to at least one immunooncology drug therapy using said in vitro organ culture system, and to a kit for preparing an in vitro organ culture system that mimics the interaction of a tumor with the immune system of a subject, as disclosed herein.

Description

TECHNICAL FIELD

[0001] The field of the invention is in vitro assays for generating in vitro organ culture systems, and the use of such in vitro organ culture systems for screening assays in the context of drug screening, patient selection and personalized medicine. More specifically, as disclosed herein, the invention relates to an in vitro method of establishing a tumor organoid from a cancer tissue sample obtained from a subject, an in vitro method of providing immune cells activated by an autologous tumor organoid, a method of preparing an in vitro organ culture system for mimicking the interaction of a tumor with the immune system of a subject, and the in vitro organ culture system resulting therefrom, a method of applying said in vitro organ culture system for determining the responsiveness of a tumor to at least one immuno-oncology drug treatment, and a kit-of-parts for preparing an in vitro organ culture system for mimicking the interaction of a tumor with the immune system of a subject. BACKGROUND

[0002] Immunotherapy, including immune checkpoint inhibitors (ICIs), has revolutionized cancer treatment and changed the management of several cancer types. Anti-programmed death cell protein-1 (PD-1) or anti-cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) are immune checkpoint inhibitors (ICIs) that can promote anti-tumor activity, resulting in durable clinical benefits in various cancers. Despite the long-term efficacy of immunotherapy, the overall response rate (ORR) of anti-PD-1 therapy is 40-50% in first-line melanoma treatment and 18-40% in second-line non-small cell lung cancer (NSCLC) treatment.

[0003] Currently, the success of immuno-oncology (IO) drugs is hampered by the lack of reliable biomarkers, which makes it difficult to predict which patients will respond to treatment. Although tumor PD-L1 expression guided the use of anti-PD-1 therapy in NSCLC and other cancers, PD-L1 expression as a biomarker has been controversial due to its dynamic nature and poor predictive ability.

[0004] In addition to PD-1 / PD-L1 and CTLA-4, there are several other immune checkpoints, such as lymphocyte-activation gene-3 (LAG-3), T-cell immunoglobulin and mucin-domain containing-3 (TIM-3), and cytokines that contribute to immune escape and resistance to immunotherapy.

[0005] There is increasing interest in combining ICIs to overcome resistance to IO treatment. Combination therapy strategies with ICIs have shown better efficacy in some cases, but also led to higher toxicity in some cases. Several clinical studies of IO combination therapy are currently underway, but these studies have failed to keep up with the rapid development of new immunotherapeutic agents.

[0006] Therefore, there is an increasing need for complex preclinical platforms that closely mimic the complexity of the patient tumor microenvironment (TME). Such platforms would allow clinicians to make more informed decisions when deciding whether to use expensive IO drugs, while also more accurately representing what these drugs can do in the body.

[0007] Current preclinical models for assessing the efficacy of IO therapy have significant limitations. 3D organoids derived from patient tumors lack an immune context, but humanized mice are not ideal in terms of reproducibility cost.

[0008] Recently, microfluidic systems have been developed to utilize tumor fragments to assess response to ICIs (Jenkins et al., 2018). Although these platforms replicate some features of the patient TME, their application is limited due to the small size of tumor resections and lack of systemic immune effects.

[0009] Previous studies have established organ-on-chip technologies, but these technologies are not personalized or systemic and cannot be used to screen treatment regimens for different patients. Nguyen et al. (2018) developed a platform to screen targeted therapies for breast cancer and investigate the effects of fibroblasts on tumor growth, treatment efficacy, and immune cells. However, this platform consists of commercial tumor cell lines and fibroblast lines, while immune cells are derived from mismatched healthy donors. Similarly, Ayuso et al. (2019 and 2021) developed a microfluidic platform to investigate the systemic effects of NK-92 cell lines on breast cancer cell lines in the presence of different antibodies. Although these methods have successfully integrated the main features of the TME into their platforms, they are not personalized or systemic and cannot be used to screen treatment regimens for different patients.

[0010] Other studies on methods of response to IO drugs or cell-based therapies rely on tumor-infiltrating lymphocytes (TILs) as a source of immune cells. This strategy has been successful in melanoma, but is still limited in epithelial cancers (Rosenberg et al., 2015). It has been shown that not all TILs are tumor-specific (Simoni et al., 2018). In addition, some tumors have a small number of infiltrating T cells, or the patient specimen obtained through biopsy is small, so the number of immune cells (such as T cells) that can be obtained is limited.

[0011] Other microfluidic technology-based methods using inactivated or non-specifically activated peripheral blood mononuclear cells (PBMCs) or commercially available non-matched immune cell lines also lack personalized tumor-specific T cells (Aung et al., 2020; Al-Samadi et al., 2019; DeHaan et al., 2021).

[0012] Cattaneo et al. (2020) describe a method of activating PBMCs using tumor organoids. However, Cattaneo et al. do not provide tumor organoids containing cells under different culture conditions. Furthermore, Cattaneo et al. also state in the abstract that “tumor-reactive T cells were obtained from approximately 33-50% of non-small cell lung cancer (NSCLC) and microsatellite instability (MSI) colorectal cancer (CRC) patient samples,” which is significantly lower than the over 80% success rate found with the methods of the present disclosure. Moreover, Cattaneo et al. do not incorporate other cells, such as fibroblasts, into the organoids.

[0013] Schuth et al. (2020) describe a three-dimensional organoid-fibroblast co-culture system and discuss the importance of creating heterotypic tumor organoid models that more accurately reflect real-life tumors. Schuth et al. do not describe the use of 2D culture to grow additional tumor epithelial cells to overcome the difficulty of obtaining sufficient cells from tumor samples, and they do not combine their system with immune cells, whether systemic or otherwise, contained in a tumor organoid system.

[0014] U.S. Patent No. 10,472,599 describes a 3D cell culture system in a microfluidic device that uses primary tumor cells isolated from a tumor sample by enzymatic digestion and embeds these cells in a matrix within a microfluidic chip to allow growth of spheroids of primary tumor origin that do not include fibroblasts or cells from 2D culture. The use of autologous primary tumor-activated systemic immune cells is not described.

[0015] Maulana et al. (2021) discuss an immune-competent chip cancer model to evaluate immune-oncology therapies. While Maulana et al. appear to summarize the desire to create a system to study drugs in a more realistic tumor microenvironment, they do not appear to describe a system that achieves this goal.

[0016] Gopal et al. (2021) describe a high-throughput microcolumn-microwell sandwich 3D cell culture platform. While the system described by Gopal et al. employs co-culture of tumor spheroids and NK cells, it does not disclose or suggest co-culture of tumor cells and autologous primary tumor-activated systemic immune cells.

[0017] Saraiva et al. in August 2020 described the establishment of 3D co-cultures with breast cancer cell lines and patient-derived immune cells. Saraiva et al. did not describe the use of a combination of 3D and 2D cultures nor the addition of other cells to the system. The system of Saraiva et al. also did not include the use of microfluidic channels or structures similar to blood vessels between the tumor organoids and the added patient-derived PBMCs.

[0018] Despite this, there is still an urgent need for a more complex chip-organ platform that mimics the patient’s TME and contains personalized tumor-specific immune cells, including tumor-specific T cells, which can be used to identify IO drug combinations that are effective in patients. SUMMARY

[0019] To address the above challenges in the art, the inventors have established an improved method for preparing tumor organoids. Due to the small amount of tissue available, the establishment of tumor organoids with patient material can be limited, and some epithelial tumor cells can prefer to grow in 2D or 3D cultures. The two-step tumor organoid establishment protocol disclosed herein, which combines 2D and 3D culture steps, allows for the successful generation of tumor and benign tissue organoids from more than 80% of specimens, including the isolation of cancer-associated fibroblast cells from 45% of patients. The disclosed method also allows for the establishment of cancer-associated fibroblast (CAF) cell lines that match the tumor.

[0020] Furthermore, such prepared tumor organoids can be used to trigger and activate autologous (i.e. the patient’s own) tumor-specific immune cells, such as tumor-specific T cells and NK cells.

[0021] A personalized ex vivo microfluidic platform, herein referred to as "Solid-IO", was then developed using these fully autologous tumor organoids, CAFs and tumor-specific immune cells. Solid-IO is a unique platform that utilizes tumor cells and immune cells matched to the patient to assess response to IO drugs. The platform allows for screening of different IO drug combinations while investigating the systemic effects of patient tumor-specific immune cells. The platform can be used to screen for immunotherapy response and to discover immunotherapy biomarkers in the tumor. The chip organoid platform consists of 3D tumor microenvironments (optionally separated by endothelial barriers or small vessels) and autologous tumor-specific immune cells, with or without IO drug stimulation. With this platform, the inventors are able to ex vivo mimic primary resistance of cancer to immune checkpoint inhibitors and identify individual ex vivo responders to checkpoint inhibitor and chemotherapy combination treatments. For example, the platform allows for screening and / or identification of IO-drug combinations that are able to overcome resistance in a given patient. Conversely, the platform can be used to identify which patients will benefit from a given IO-drug combination. Moreover, the platform also allows for identification of potential biomarkers of response or non-response to a given IO-drug combination.

[0022] The Solid-IO platform is a high-throughput, easy-to-use tool with the potential to significantly accelerate the discovery of new IO-drug and biomarkers. It has the potential to help clinical decision making in IO treatments, thereby improving patient treatment outcomes in an economic and efficient way.

[0023] The challenges in the prior art are overcome by the method and the in vitro organ culture system characterized by what is stated in the independent claims. Preferred embodiments of the invention are disclosed in the dependent claims.

[0024] More specifically, an in vitro method of establishing tumor organoids from a cancer tissue sample obtained from a subject is provided, the method comprising the following steps:

[0025] (a) dissociating the cancer tissue sample into cells and fragments;

[0026] (b) propagating a first portion of the cells and / or fragments obtained in step (a) in a first 3D culture step;

[0027] (c) propagating a second portion of the cells and / or fragments obtained in step (a), for example to enrich for epithelial tumor cells and optionally fibroblasts, in a first 2D culture step;

[0028] (d) combining the propagated cells and fragments obtained in step (b) with the propagated cells and fragments obtained in step (c) and propagating the combined cells and fragments in a second 3D culture step;

[0029] thereby establishing a tumor organoid from the cancer tissue sample, as further defined in the claims.

[0030] Further, there is provided an in vitro method of providing autologous tumor organoid-activated immune cells, the method comprising:

[0031] (a) establishing a tumor organoid from a cancer tissue sample obtained from a subject according to the methods disclosed herein; and

[0032] (b) co-culturing the tumor organoid with immune cells obtained from the subject;

[0033] thereby providing autologous tumor organoid-activated immune cells, as further defined in the claims.

[0034] Further, there is provided a method of preparing an in vitro organ culture system to mimic tumor interaction with the immune system of a subject, the method comprising:

[0035] (i) providing a microfluidic device having a microfluidic channel with at least a first compartment and a second compartment separated by a physical boundary that allows cells to move from the first compartment to the second compartment;

[0036] (ii) incorporating a three-dimensional tumor organoid established from a cancer tissue sample obtained from a subject and a suitable tumor cell growth medium in the first compartment; and

[0037] (iii) introducing autologous immune cells from the subject that have been activated by co-culturing with the established tumor organoid and a suitable immune cell medium into the second compartment;

[0038] thereby providing the in vitro organ culture system, as further defined in the claims.

[0039] The above method results in providing an in vitro organ culture system for mimicking tumor interaction with the immune system of a subject, comprising:

[0040] (i) a microfluidic device having a microfluidic channel with at least a first compartment and a second compartment separated by a physical boundary that allows cells to move from the first compartment to the second compartment,

[0041] (ii) a three-dimensional tumor organoid established from a cancer tissue sample obtained from the subject and a suitable tumor cell growth medium in the first compartment; and

[0042] (iii) in the second compartment autologous immune cells from the subject that have been activated by co-culturing with the established tumor organoid and a suitable immune cell medium, as further defined in the claims.

[0043] Further provided is the use of the in vitro organ culture system, for example a method for determining the responsiveness of a tumor to treatment with at least one immuno-oncology drug, comprising the following steps:

[0044] (a) preparing an in vitro organ culture system according to the methods disclosed herein, or providing an in vitro organ culture system according to the disclosure;

[0045] (b) adding one or more, preferably at least two, immuno-oncology drugs to the second compartment; and

[0046] (c) determining the responsiveness of the in vitro organ culture system to the at least two immuno-oncology drugs, as further defined in the claims.

[0047] Finally, also contemplated is a kit for preparing an in vitro organ culture system that mimics the interaction of a tumor with the immune system of a subject, the kit comprising at least two, preferably at least three, more preferably at least four, even more preferably at least five, most preferably all of:

[0048] (a) a microfluidic device having a microfluidic channel with at least a first compartment and a second compartment separated by a physical boundary that allows cells to move from the first compartment to the second compartment, in particular wherein the physical boundary is selected from a membrane, a mechanical barrier, a membraneless phase-directed boundary, a gel-based boundary, a basement membrane-based boundary, or a mesh-based boundary;

[0049] (b) a tumor cell growth medium comprising at least two, preferably all of:

[0050] (i) a WNT / beta catenin signaling pathway inducer, for example R-spondin and / or WNT, preferably R-spondin 1 ;

[0051] (ii) FGF, preferably FGF-7 and / or FGF-10, more preferably FGF-7 and FGF-10;

[0052] (iii) a bone morphogenetic protein (BMP) antagonist; preferably wherein the BMP antagonist is noggin;

[0053] (iv) an ALK5 inhibitor; preferably wherein the ALK5 inhibitor is A83-01 ;

[0054] (v) a Rho kinase inhibitor; preferably wherein the Rho kinase inhibitor is Y-27632;

[0055] (vi) a p38 MAPK inhibitor; preferably wherein the p38 MAPK inhibitor is SB202190;

[0056] (c) an immune cell culture medium, a medium comprising

[0057] (i) a T cell growth factor, preferably interleukin 2 (IL-2);

[0058] (ii) an immune checkpoint inhibitor, preferably an immune checkpoint selected from the group consisting of an anti-PD-1 / PD-L1 antibody and an anti-CTLA4 antibody, more preferably an anti-PD-1 / PD-L1 antibody, in particular an anti-PD-1 / PD-L1 antibody selected from the group consisting of nivolumab, pembrolizumab, cemiplimab, dostarlimab, retifanlimab, vopratelimab, spartalizumab, camrelizumab, sintyrozumab, tislelizumab, toripalimab, INCMGA00012, AMP-224, AMP-514, aklieshantibody, atezolizumab, durvalumab, and avelumab, more preferably wherein the anti-PD-1 antibody is nivolumab;

[0059] (iii) nicotinamide;

[0060] and optionally a B27 supplement and N-acetyl cysteine, preferably about 1.25 mM N-acetyl cysteine.

[0061] (d) a cell culture dish or cell culture vessel that has been pre-coated with an anti-CD28 antibody;

[0062] (e) a container with IFN-gamma; and / or

[0063] (f) a container with an extracellular matrix gel comprising extracellular matrix proteins, such as laminin and collagen. BRIEF DESCRIPTION OF DRAWINGS

[0064] The application will be described in more detail below by preferred embodiments with reference to the attached drawings, in which

[0065] Figure 1 A schematic is provided for activating patient immune cells, including T cells, by co-culturing with a tumor organoid established from the patient’s tumor.

[0066] Figure 2: Co-culture did not significantly change T cell subpopulations. Immunological analysis by flow cytometry comparing the overall percentage of CD3+ cells and CD4+ and CD8+ T cell subpopulations before (week 0) and after (week 2) two weeks of stimulation with patient-matched tumor organoids. Two scatter plot points of the same color represent biological replicates. Scatter plot points of different colors represent different patients. Black solid line represents the mean. N / A = no mutation found with clinical significance. Student's t-test (unpaired).

[0067] Figure 3 : (A) Circulating T cells were activated upon matched tumor organoid stimulation. Example flow cytometry plots show CD8+ / IFNy+ cells from 3 example patients. (B) Quantification of IFNy production by reactive CD8+ T cells after two weeks of co-culture of PBMCs with matched tumor organoids. Columns in the graph represent the percentage of anti-IFNy APCs out of controls in CD8+ T cells. Columns in the graph represent values from one replicate experiment. For ORG-35, ORG-54 and Lung-19, each column shows the mean of two biological replicates.

[0068] Figure 4 : IFNy production by reactive CD8+ T cells from two patients at baseline (week 0), one day after stimulation with the new organoid (week 1) and after two weeks of co-culture with the organoid (week 2).

[0069] Figure 5 : Killing of tumor organoids (ORG-55) by baseline PBMCs or stimulated immune cells in three conditions (control, nivolumab, nivolumab and chemotherapy combined). Columns represent log2 fold change in luminescent signal of cleaved caspase-3 / 7 after 48 hours of co-culture.

[0070] Figure 6 : AIM or Solid-IO design in Mimetas 3-channel microfluidics. Each device contains a middle channel (tumor site) where tumor organoids are seeded in Matrigel (extracellular matrix) and separated from two flanking medium channels (circulation) where activated immune cells and selected drugs are added. Brightfield images (from AIM chip) taken with a Nikon Eclipse microscope show that after 24 hours of seeding, immune cells migrate and infiltrate the tumor site around the organoid.

[0071] Figure 7 : Expression of tumor cell surface immune checkpoint PD-L1 in tumor resections or biopsies provided by the HUS pathology lab. Columns in the graph represent the percentage of tumor cells expressing PD-L1.

[0072] Figure 8Quantification of live / dead cells images after 48h of co-culture in the Solid-IO platform. Scatter plot points of the same color represent technical replicates (lung-19 excepted, showing two technical replicates from each of the two biological replicates). Non-paired t-test was performed to assess differences in response with and without immune cells. P values are shown in the graph. On the right of the graph, the mutation status of each patient is shown; N / A = not available. Black solid lines represent the mean of the values in each case.

[0073] Figure 9 Quantification of live (AO = white boxes) and dead (PI = gray boxes) cells of lung-19 after 48h of co-culture in the Solid-IO platform. Columns represent the mean of the two biological replicates while error bars represent their variance. Non-paired t-test was performed, *= P<0.05, **= P<0.01, ***= P<0.001, ns = not statistically significant. Columns represent the percentage of live / dead areas.

[0074] Figure 10 Advanced setup of the Solid-IO showing the chip tumor comprising a tumor microenvironment (TME) middle channel containing a tumor organoid and a matched tumor-associated fibroblast separated from the circulation side channel by an endothelial tube. In the upper side channel, an endothelial tube is formed. Immune cells and drugs are injected into the endothelial tube and then the immune cells infiltrate the TME channel. Brightfield images acquired with a Nikon Eclipse microscope (from Mimetas chip) showing the upper channel as the endothelial tube and the lower channel as the TME.

[0075] Figure 11 (A) Quantification of live (AO = left column) and dead (PI = right column) cells of tumor organoids after 48h of co-culture in advanced Solid-IO where the tumor site is separated from the adjacent channel by an endothelial tube. Quantification was performed in two conditions: control and chemotherapy, which were injected into the tube and needed to diffuse to the tumor site. Columns represent the percentage of live / dead areas of the tumor organoids. (B) Quantification of live (AO = left column) and dead (PI = right column) cells of HUVEC cells forming an endothelial tube. Quantification was performed to assess the viability of HUVEC cells after 4 days of tube formation and after more than two days of injection of control or chemotherapeutic agents in the tube structure. Columns represent the percentage of live / dead areas of HUVEC cells in the tube channel.

[0076] Figure 12 Alternative design of the Solid-IO showing the chip tumor comprising a tumor microenvironment (TME) middle channel containing a tumor organoid and a matched tumor-associated fibroblast separated from the circulation side channel by an endothelial tube.

[0077] Figure 13 Multiple common and individual genes were upregulated in CD4+ (helper) and CD8+ (cytotoxic) T cells from different patients after the PBMC / tumor co-culture protocol. DETAILED DESCRIPTION

[0078] ICI targeting PD-1 / PD-L1 shows promise in cancer treatment, but currently only a small number of patients benefit from ICI. Therefore, there is an urgent need for more efficient and precise IO drug testing, and there is also a lack of preclinical models that can accurately predict which patients will benefit from IO treatment. Current 2D and 3D cell models lack the immune architecture and tumor microenvironment and cannot accurately capture the patient’s anti-tumor immunity and complex tumor microenvironment. To solve this problem, methods and devices were developed that contain patient-matched tumor and immune cells, with the possibility of adding personalized TME components such as fibroblasts and endothelial cells. The design of this platform aims to be easy to use and compatible with high-throughput testing.

[0079] Due to the small amount of tissue available, the establishment of tumor organoids from patient material can be limited, and some epithelial tumor cells can prefer to grow in 2D or 3D culture. Nonetheless, the presently disclosed two-step organoid establishment protocol allows for the successful generation of tumor and benign tissue organoids from over 80% of specimens, including the isolation of cancer-associated fibroblasts from 45% of patients. This straightforward approach relies primarily on the ratio of epithelial cells to fibroblasts, thereby enriching either cell type without the need for bead- or flow cytometry-based separation, which often results in cell loss and poor cell viability.

[0080] In addition, the new 3D / 2D culture system for growing tumor organoids from patient samples enables a more reliable method of cell proliferation and isolation, whether from resection samples or biopsy samples, to obtain both tumor cells and fibroblasts from a patient, compared to the use of 3D culture systems alone to grow tumor organoids in prior art references. Heterotypic tumor organoids provide an improved tumor model for screening systems that not only contain tumor cells but also tumor-associated fibroblasts.

[0081] In summary, the presently disclosed method overcomes the shortcomings of the prior art that not all samples can be grown directly from 3D organoids. The presently disclosed method generates more cells faster than the prior art method, allowing for faster drug efficacy testing, and the method also allows for the growth of a matched fibroblast population. In addition, the inventors’ preliminary data show that the presently disclosed method is particularly useful in preserving cancer mutations compared to the prior art.

[0082] More broadly, there is provided an in vitro method of establishing tumor organoids from a cancer tissue sample obtained from a subject, the method comprising the steps of:

[0083] (a) dissociating the cancer tissue sample into cells and fragments;

[0084] (b) propagating a first portion of the cells and / or fragments obtained in step (a) in a first 3D culture step;

[0085] (c) propagating a second portion of the cells and / or fragments obtained in step (a) in a first 2D culture step, for example to enrich for epithelial tumor cells and optionally fibroblasts;

[0086] (d) combining the propagated cells and fragments obtained in step (b) with the propagated cells and fragments obtained in step (c) and propagating the combined cells and fragments in a second 3D culture step;

[0087] thereby establishing tumor organoids from the cancer tissue sample.

[0088] The cancer tissue sample can be a tissue sample obtained from a tissue selected from the group comprising or preferably consisting of lung, bronchus, colon, rectum, prostate, breast, bladder, thyroid, kidney, renal pelvis, uterine corpus, oral cavity or ovarian tissue. However, the sample can also be obtained from a tissue other than the above examples.

[0089] The cancer tissue sample obtained from said tissue can be derived from the same type of tissue or can be a cancer tissue derived from another type of tissue. The cancer tissue sample can be a cancer tissue sample from a benign tumor or a malignant cancer tumor. The term "malignant" here refers to a tumor whose cells grow uncontrolled and can spread locally and / or distantly. Thus, a malignant cancer tumor also includes a metastatic cancer tumor.

[0090] For a malignant tumor, the cancer can for example be selected from lung cancer, colon cancer, rectal cancer, prostate cancer, breast cancer, bladder cancer, thyroid cancer, kidney cancer, renal cancer, epithelial cancer or ovarian cancer. Alternatively or additionally, the cancer tissue sample is a cancer tissue sample from an adenocarcinoma, an epidermoid carcinoma and / or a colorectal cancer. However, the sample can also be obtained from a cancer other than the above examples.

[0091] In certain embodiments, the method can also be performed in parallel using a non-tumor tissue sample or a healthy sample from the subject or a healthy donor, preferably from the subject, with the aim of providing "non-tumor" organoids which can be used as controls in the methods described hereinafter. For optimal comparison, the non-tumor tissue or healthy tissue is from the same type of tissue as the cancer tissue sample.

[0092] Although the method is illustrated below with reference to a human body, the system can also be used for subjects other than humans. This can be related to animal cancer models, including animal models in which human cancer cells have been injected into animals. Accordingly, the subject can be a mammal; preferably a primate, such as a human; or, without limitation, a rodent, such as a mouse, rat, hamster or guinea pig; or a cat, dog, sheep, horse or cow. However, since "personalized medicine" is currently mainly developed for humans, the subject of the study is preferably a human.

[0093] One particular advantage of the disclosed method is that it enables a better preservation of the genetic identity information of the cancer tissue in the tumor organoid. This is particularly relevant when the test aims to overcome the resistance to a drug monotherapy with a personalized immuno-oncology drug combination. The presently disclosed method is therefore particularly advantageous in case the cancer tissue sample is from a cancer comprising at least one genetic mutation, optionally of a gene associated with tumor resistance. Some typical examples of such mutations are cancers in which at least one mutation is a mutation of a gene selected from KRAS, EGFR, KEAP1, ALK, STK11, MET, TP53, ROS1, RB1, NOTCH1, NOTCH2, BRAF, NTRK1 / 2 / 3, RET, ERBB2, HRAS, NRAS, ASCL1, NF1, PI3CA, FGFR1 / 2, MAP2K1, in particular a mutation of a gene selected from KRAS, EGFR, KEAP1, ALK and STK11.

[0094] As shown in the examples, the at least one mutation can be, for example, a mutation in KRAS. In particular, in embodiments in which the subject is a human, the at least one mutation in KRAS can be, for example, a mutation at position Gly 12 and / or Gln 61, more particularly at least one KRAS mutation selected from GL12Ala, Gly 12ASP, Gly 12Val, Gly 12Cys and Gln 61His.

[0095] In step (a), the cancer tissue sample is dissociated into cells and fragments. That is, unlike the organoid construction protocols of the prior art, the present protocol does not filter out undigested tissue fragments, thereby limiting the possibility of loss of tumor cell clumps or rare tumor cell populations. Having a reliable source of tumor organoid cells is crucial, since the tumor organoids in the system are both for activating immune cells and for screening the system.

[0096] Step (a) can comprise using mechanical and / or enzymatic methods to dissociate the cancer tissue sample. For example, the cancer tissue sample is minced, for example into 1-2 mm 3Additionally or alternatively, the cancer tissue sample can also be treated with enzymes, for example with enzymes selected from the group consisting of collagenase, pronase, trypsin, hyaluronidase and papain. In particular, the use of collagenase has proven to be effective in this respect. These enzymes are commercially available and are usually distributed by commercial manufacturers together with their protocols for use.

[0097] In comparison to prior art methods, the system employing its two-stage (2D / 3D) cell proliferation method is able to produce a sufficient number of tumor cells from a patient's tumor sample, whether the sample is a biopsy or a resection, more reliably, to establish a tumor organoid structure.

[0098] In contrast to the 2D culture step (c), the 3D culture step (b) is usually performed in an extracellular matrix gel comprising extracellular matrix proteins, for example laminin and collagen. Commercial examples of such extracellular matrix gels are Matrigel or Cultrex or Collagen. As shown in the experimental section below, step (b) usually also comprises proliferating the cells and the fragments within the extracellular matrix gel in a culture medium comprising at least two or more, preferably all of (i)-(vii) below:

[0099] (i) an inducer of the WNT / beta catenin signaling pathway, for example R-spondin and / or WNT, for example R-spondin 1. R-spondin, for example R-spondin-1, can be present, for example, in an effective amount in the typical range of 1-10000 ng / ml. Preferably, it can be present in the range of 10-5000 ng / ml, more preferably in the range of 100-2000 ng / ml, and even more preferably in the range of 250-1000 ng / ml. As shown in the experimental section, it can be present, for example, in an amount of about 500 ng / ml.

[0100] (ii) FGF, for example FGF-7 and / or FGF-10, preferably both FGF-7 and FGF-10. Effective amounts of FGF-7 can be readily determined and are typically in the range of 0.1-1000 ng / ml, in particular in the range of 1-500 ng / ml, more in particular in the range of 5-200 ng / ml, even more in particular in the range of 10-100 ng / ml, for example in the range of 15-50 ng / ml. As shown in the experimental section, FGF-7 can be present, for example, in an amount of about 25 ng / ml. Similarly, typical effective amounts of FGF-10 are in the range of 1-10000 ng / ml, in particular in the range of 10-5000 ng / ml, more in particular in the range of 25-1000 ng / ml, for example in the range of 50-500 ng / ml. As shown in the experimental section, FGF-10 can be present, for example, in an amount of about 100 ng / ml.

[0101] (iii) Bone morphogenetic protein (BMP) antagonist. An example of such an antagonist is Noggin, and an effective amount of Noggin is typically in the range of 10-1000 ng / ml, in particular in the range of 25-500 ng / ml, more in particular in the range of 50-250 ng / ml. As shown in the experimental section, an effective amount can be for example about 100 ng / ml.

[0102] (iv) ALK5 inhibitor. An example of an ALK5 inhibitor is A83-01, which is typically effective in the range of 1-10000 nM, in particular in the range of 10-5000 nM, more in particular in the range of 50-2500 nM, for example in the range of 100-1000 nM. As shown in the experimental section, an effective amount can be for example about 500 nM.

[0103] (v) Rho kinase inhibitor. An example of a suitable Rho kinase inhibitor is Y-27632. Typically, Y-27632 is applied effective in the range of 0.01-50 µM, in particular in the range of 0.1-25 µM, more in particular in the range of 1-10 µM. As shown in the experimental section, an effective amount can be for example about 5 μΜ.

[0104] (vi) p38 MAPK inhibitor. An example of a suitable p38 MAPK inhibitor is SB202190, which can be applied effective in the range of 1-10000 nM, in particular in the range of 10-5000 nM, more in particular in the range of 50-2500 nM, for example in the range of 100-1000 nM. As shown in the experimental section, an effective amount can be for example about 500 nM.

[0105] (vii) Nicotinamide, in particular in the range of 0.1-100 mM, preferably in the range of 0.5 - 50 mM, more preferably in the range of 1-25 mM, even more preferably in the range of 2-10 mM. As shown in the experimental section, a suitable amount can be for example about 5 mM.

[0106] Optionally, the culture medium can further contain B27 supplement and N-acetyl cysteine, for example about 1.25 mM N-acetyl cysteine.

[0107] Similarly, step (c) of the above method typically comprises propagating in a culture medium comprising at least two or more, preferably all, of the following (i)-(iii):

[0108] (i) FGF, such as FGF-basic. Typically, an effective amount of FGF-basic is in the range of 0.1-10000 ng / ml, in particular in the range of 0.5-5000 ng / ml, more particularly in the range of 1-1000 ng / ml, even more particularly in the range of 5-500 ng / ml, such as in the range of 10-50 ng / ml. As shown in the experimental section, a suitable amount can be e.g. about 20 ng / ml.

[0109] (ii) EGF, wherein an effective amount of EGF is typically in the range of 0.1-10000 ng / ml, in particular in the range of 0.5-5000 ng / ml, even more particularly in the range of 1-2000 ng / ml, even more particularly in the range of 2-1000 ng / ml, even more particularly in the range of 5-500 ng / ml, such as in the range of 10-100 mg / ml. As shown in the experimental section, a suitable amount can be e.g. about 50 ng / ml.

[0110] (iii) a Rho kinase inhibitor. One example of a suitable Rho kinase inhibitor is Y-27632. An effective amount of Y-27632 is typically in the range of 0.01-50 µM, in particular in the range of 0.1-25 µM, more particularly in the range of 1-10 µM, e.g. applied at about 5 µM effective.

[0111] Typically, step (c) is performed for at least 2-6 days, e.g. at least 4-6 days. Additionally or alternatively, step (d) is performed for no more than passage 3 of the epithelial tumor cells, in particular no more than passage 2, more in particular no more than passage 1, e.g. at passage 0.

[0112] Step (c) can further comprise a step of collecting and propagating cancer-associated fibroblasts, preferably in the same medium as used in step (c). This allows culturing autologous fibroblasts and incorporating them into the tumor organoid structure, thereby providing a more accurate heterotypic tumor organoid reflecting the real tumor.

[0113] Similar to step (b), step (d) is typically performed in an extracellular matrix gel comprising extracellular matrix proteins, such as laminin and collagen. For example, step (d) can be performed in Matrigel or Cultrex. As shown in the experimental section below, step (d) typically further comprises propagating in the same or similar medium as used in step (b). Accordingly, in embodiments it comprises at least two or more, preferably all, of the following (i)-(vii):

[0114] (i) WNT / beta catenin signaling pathway inducer, such as R-spondin and / or WNT, such as R-spondin 1. R-spondin, such as R-spondin-1, can be present, for example, in an effective amount in the typical range of 1-10000 ng / ml. Preferably, it can be present in the range of 10-5000 ng / ml, more preferably in the range of 100-2000 ng / ml, and even more preferably in the range of 250-1000 ng / ml. As shown in the experimental section, it can be present, for example, in an amount of about 500 ng / ml.

[0115] (ii) FGF, such as FGF-7 and / or FGF-10, preferably both FGF-7 and FGF-10. An effective amount of FGF-7 can be readily determined and is typically in the range of 0.1-1000 ng / ml, in particular in the range of 1-500 ng / ml, more in particular in the range of 5-200 ng / ml, even more in particular in the range of 10-100 ng / ml, such as in the range of 15-50 ng / ml. As shown in the experimental section, FGF-7 can be present, for example, in an amount of about 25 ng / ml. Similarly, a typical effective amount of FGF-10 is in the range of 1-10000 ng / ml, in particular in the range of 10-5000 ng / ml, more in particular in the range of 25-1000 ng / ml, such as in the range of 50-500 ng / ml. As shown in the experimental section, FGF-10 can be present, for example, in an amount of about 100 ng / ml.

[0116] (iii) Bone morphogenetic protein (BMP) antagonist. An example of such an antagonist is noggin, and an effective amount of noggin is typically in the range of 10-1000 ng / ml, in particular in the range of 25-500 ng / ml, more in particular in the range of 50-250 ng / ml. As shown in the experimental section, an effective amount can be, for example, about 100 ng / ml.

[0117] (iv) ALK5 inhibitor. An example of an ALK5 inhibitor is A83-01, which is typically effective in the range of 1-10000 nM, in particular in the range of 10-5000 nM, more in particular in the range of 50-2500 nM, such as in the range of 100-1000 nM. As shown in the experimental section, an effective amount can be, for example, about 500 nM.

[0118] (v) Rho kinase inhibitors. One example of a suitable Rho kinase inhibitor is Y-27632. Typically, Y-27632 is applied effectively in the range of 0.01-50 µM, in particular in the range of 0.1-25 µM, more particularly in the range of 1-10 µM. As shown in the experimental section, an effective amount can be, for example, about 5 μΜ.

[0119] (vi) p38 MAPK inhibitors. One example of a suitable p38 MAPK inhibitor is SB202190, which can be applied effectively in the range of 1-10000 nM, in particular in the range of 10-5000 nM, more particularly in the range of 50-2500 nM, for example in the range of 100-1000 nM. As shown in the experimental section, an effective amount can be, for example, about 500 nM.

[0120] (vii) nicotinamide, in particular in the range of 0.1-100 mM, preferably in the range of 0.5 - 50 mM, more preferably in the range of 1-25 mM, even more preferably in the range of 2-10 mM. As shown in the experimental section, a suitable amount can be, for example, about 5 mM.

[0121] Optionally, the culture medium can also contain B27 supplement and N-acetyl cysteine, for example about 1.25 mM N-acetyl cysteine. Due to the culture medium used for culturing the tumor organoids, almost all tumor-associated immune cells are removed from the organoids.

[0122] Further contemplated is the direct product of the above method, i.e. the ex vivo tumor organoid obtained by the above method. Thus, this tumor organoid is characterized in that it is established from a cancer tissue sample obtained from a tissue selected from lung, bronchus, colon, rectum, prostate, breast, urinary bladder, thyroid, kidney, renal pelvis, uterine corpus, oral cavity or ovary tissue. The tumor organoid can be established from a benign tumor or a malignant cancer tumor. Where the tumor organoid is established from a malignant cancer tumor, for example, the cancer can be selected from lung cancer, colon cancer, rectal cancer, prostate cancer, breast cancer, urinary bladder cancer, thyroid cancer, kidney cancer, renal epithelial cancer or ovarian cancer. In embodiments, the tumor organoid can be established from an adenocarcinoma, epidermoid carcinoma and / or colorectal cancer. However, the sample can also be obtained from a cancer or tissue other than the above examples. In embodiments, the tumor organoid is established from a subject, wherein the subject is a mammal; preferably a primate, such as a human; or a rodent, such as a mouse, rat, hamster or guinea pig; or a cat, dog, sheep, horse or cow. In embodiments, the tumor organoid comprises at least one mutated gene, which gene is optionally a gene associated with tumor drug resistance, in particular wherein the at least one mutation is a mutation in a gene selected from KRAS, EGFR, KEAP1, ALK, STK11, MET, TP53, ROS1, RB1, NOTCH1, NOTCH2, BRAF, NTRFK1 / 2 / 3, RET, ERBB2, HRAS, NRAS, ASCL1, NF1, PI3CA, FGFR1 / 2, MAP2K1, in particular a mutation in a gene selected from KRAS, EGFR, KEAP1, ALK and STK11. For example, the at least one mutation can be a mutation in KRAS. Where the subject is a human, the at least one mutation in KRAS can be a mutation in position Gly12 and / or Gln61, more particularly, the at least one mutation can be at least one KRAS mutation selected from GL12Ala, Gly12ASP, Gly12Val, Gly12Cys and Gln61His.

[0123] While tumor infiltrating lymphocytes (TILs) directly from surgical tissue have been used previously to study anti-tumor immunity, this does not reflect the systemic immune response and can miss critical interactions between tumor cells and the immune system, thus not fully reflecting the systemic immunotherapy response. This hurdle is overcome in that the tumor organoid obtained after step (d) is typically substantially free of tumor infiltrating leukocytes. Moreover, the tumor organoid can be used to mimic the activation of the systemic immune response. At the same time, the method uses tumor-activated peripheral immune cells in order to provide a robust and reproducible screening system with sufficient relevant immune cells.

[0124] Accordingly, also provided is an in vitro method of providing autologous tumor organoid-activated immune cells, the method comprising:

[0125] (a) establishing a tumor organoid from a cancer tissue sample obtained from a subject according to the methods disclosed above; and

[0126] (b) co-culturing the tumor organoid with immune cells obtained from the subject;

[0127] thereby providing autologous tumor organoid-activated immune cells.

[0128] Step (a) typically comprises isolating the tumor organoid from an extracellular matrix gel comprising extracellular matrix proteins, such as laminin and collagen, e.g. as used in step (d) of the above method. The tumor organoid is typically cultured in a culture medium which is similar or identical to the culture medium used in the above disclosed methods for establishing a tumor organoid. Accordingly, the culture medium preferably comprises at least two or more, preferably all of (i)-(vii) below:

[0129] (i) a WNT / beta catenin signaling pathway inducer, such as R-spondin and / or WNT, e.g. R-spondin 1. R-spondin, such as R-spondin-1, can be present, e.g. in an effective amount in the typical range of 1-10000 ng / ml. Preferably, it can be present in the range of 10-5000 ng / ml, more preferably in the range of 100-2000 ng / ml, and even more preferably in the range of 250-1000 ng / ml. As shown in the experimental section, it can be present in an amount of about 500 ng / ml.

[0130] (ii) FGF, such as FGF-7 and / or FGF-10, preferably both FGF-7 and FGF-10. Effective amounts of FGF-7 can be readily determined and are typically in the range of 0.1-1000 ng / ml, in particular in the range of 1-500 ng / ml, more in particular in the range of 5-200 ng / ml, even more in particular in the range of 10-100 ng / ml, such as in the range of 15-50 ng / ml. As shown in the experimental section, FGF-7 can be present, e.g. in an amount of about 25 ng / ml. Similarly, typical effective amounts of FGF-10 are in the range of 1-10000 ng / ml, in particular in the range of 10-5000 ng / ml, more in particular in the range of 25-1000 ng / ml, such as in the range of 50-500 ng / ml. As shown in the experimental section, FGF-10 can be present, e.g. in an amount of about 100 ng / ml.

[0131] (iii) Bone morphogenetic protein (BMP) antagonist. An example of such an antagonist is Noggin, and an effective amount of Noggin is typically in the range of 10-1000 ng / ml, in particular in the range of 25-500 ng / ml, more in particular in the range of 50-250 ng / ml. As shown in the experimental section, an effective amount can be for example about 100 ng / ml.

[0132] (iv) ALK5 inhibitor. An example of an ALK5 inhibitor is A83-01, which is typically effective in the range of 1-10000 nM, in particular in the range of 10-5000 nM, more in particular in the range of 50-2500 nM, for example in the range of 100-1000 nM. As shown in the experimental section, an effective amount can be for example about 500 nM.

[0133] (v) Rho kinase inhibitor. An example of a suitable Rho kinase inhibitor is Y-27632. Typically, Y-27632 is effective in the range of 0.01-50 µM, in particular in the range of 0.1-25 µM, more in particular in the range of 1-10 µM. As shown in the experimental section, an effective amount can be for example about 5 μΜ.

[0134] (vi) p38 MAPK inhibitor. An example of a suitable p38 MAPK inhibitor is SB202190, which can be effective in the range of 1-10000 nM, in particular in the range of 10-5000 nM, more in particular in the range of 50-2500 nM, for example in the range of 100-1000 nM. As shown in the experimental section, an effective amount can be for example about 500 nM.

[0135] (vii) Nicotinamide, in particular in the range of 0.1-100 mM, preferably in the range of 0.5 - 50 mM, more preferably in the range of 1-25 mM, even more preferably in the range of 2-10 mM. As shown in the experimental section, a suitable amount can be for example about 5 mM.

[0136] Optionally, the culture medium can also contain B27 supplement and N-acetyl cysteine, for example about 1.25 mM N-acetyl cysteine.

[0137] An important aspect of the disclosed method compared to the prior art is that the immune cells applied in step (b) are obtained from the same subject from which the tumor organoid was established. Thus, the tumor organoid and the immune cells are autologous to each other. For example, the immune cells are isolated from whole blood, peripheral blood mononuclear cells (PBMCs), spleen, lymph nodes, buffy coat, pleural effusion, bone marrow aspirate, tumor, and / or from induced pluripotent stem cells. In a specific embodiment, the immune cells are peripheral immune cells; and in a preferred embodiment, the immune cells are PBMCs.

[0138] Advantageously, prior to step (b), the tumor organoid of step (a) is stimulated, for example to enhance antigen presentation. This can be achieved, for example, by pre-culturing in a culture medium comprising a type II interferon, preferably IFN-gamma. Typically, the type II interferon, e.g. IFN-gamma, is added in an amount in the range of 1-100000 ng / ml, in particular in the range of 10-10000 ng / ml, more in particular in the range of 50-5000 ng / ml, even more in particular in the range of 100-1000 ng / ml, such as in the range of 150-500 ng / ml. As shown in the experimental section, a suitable amount can be, for example, about 200 ng / ml.

[0139] The co-culturing in step (b) can comprise culturing in a culture medium comprising:

[0140] (i) a T cell growth factor, preferably interleukin 2 (IL-2). The T cell growth factor, such as IL-2, is typically added in an amount in the range of 1-100000 U / ml, in particular in the range of 10-50000 U / ml, more in particular in the range of 50-10000 U / ml, even more in particular in the range of 100-1000 U / ml, such as in the range of 200-500 U / ml. As shown in the experimental section, a suitable amount can be, for example, about 300 U / ml.

[0141] (ii) an immune checkpoint inhibitor, preferably selected from the group consisting of an anti-PD-1 / PD-L1 antibody and an anti-CTLA4 antibody, more preferably an anti-PD-1 antibody. In embodiments, the anti-PD-1 / PD-L1 antibody is selected from the group consisting of nivolumab, pembrolizumab, cemiplimab, dostarlimab, retifanlimab, vopratelimab, spartalizumab, camrelizumab, sindiizumab, tislelizumab, toripalimab, INCMGA00012, AMP-224, AMP-514, akliesimusab, atezolizumab, durvalumab, and avelumab. In specific embodiments shown below, the anti-PD-1 antibody is nivolumab. Typically, the effective amount of such antibody can be in the range of 0.1-1000 ng / ml, in particular in the range of 1-500 pg / ml, more in particular in the range of 5-250 pg / ml, even more in particular in the range of 10-100 pg / ml, such as in the range of 20-50 pg / ml. As shown in the experimental section, a suitable amount can be, for example, about 40 pg / ml; and / or

[0142] (iii) a T cell activator that binds to CD28. For example, the culture medium can comprise an anti-CD28 antibody. Alternatively, the stimulation by an anti-CD28 antibody can also be provided by pre-coating the surface of a cell culture vessel, such as a cell culture dish, cell culture flask or cell culture bag, in which the culture medium is received, i.e. in which the co-culturing step is performed, with an anti-CD28 antibody.

[0143] In step (b), the dissociated tumor organoids and immune cells are typically combined in a ratio of 20:1 tumor organoids:immune cells, preferably added in equal volumes. Typically, step (b) is performed for at least 5 days, preferably at least 7 days, more preferably at least 10 days. In specific embodiments, step (b) can be performed for 14 days or more. In particular in the latter embodiments, the tumor organoids are replaced by fresh tumor organoids after 5-7 days of co-culturing. Thus, the method of the present disclosure allows for the generation of autologously activated immune cells, i.e. immune cells derived from the same tumor patient, in amounts sufficient for large-scale IO drug screening systems.

[0144] As an intermediate product of the above method, there is also provided an in vitro cell co-culture composition comprising a tumor organoid established from a cancer tissue sample obtained from a subject as further described above and immune cells obtained from said subject.

[0145] The immune cells can be immune cells isolated from peripheral blood mononuclear cells (PBMCs), spleen, buffy coat, pleural effusion, bone marrow aspirate, tumor, and / or from stem cells, preferably wherein the immune cells are peripheral immune cells, more preferably wherein the immune cells are PBMCs. The co-culture typically further comprises a culture medium comprising at least two or more, preferably all, of the following (i)-(iii):

[0146] (i) a T cell growth factor, preferably interleukin 2 (IL-2). The T cell growth factor, e.g. IL-2, is typically added in an amount in the range of 1-100000 U / ml, in particular in the range of 10-50000 U / ml, more in particular in the range of 50-10000 U / ml, even more in particular in the range of 100-1000 U / ml, such as in the range of 200-500 U / ml. As shown in the experimental section, a suitable amount can be e.g. about 300 U / ml.

[0147] (ii) an immune checkpoint inhibitor, preferably selected from the group consisting of an anti-PD-1 / PD-L1 antibody and an anti-CTLA4 antibody, more preferably an anti-PD-1 antibody. In embodiments, the anti-PD-1 / PD-L1 antibody is selected from the group consisting of nivolumab, pembrolizumab, cemiplimab, dostarlimab, retifanlimab, vopratelimab, spartalizumab, camrelizumab, sindiizumab, tislelizumab, toripalimab, INCMGA00012, AMP-224, AMP-514, akliesimus, atezolizumab, durvalumab, and avelumab. In specific embodiments shown below, the anti-PD-1 antibody is nivolumab. Typically, an effective amount of such antibody can be in the range of 0.1-1000 ng / ml, in particular in the range of 1-500 µg / ml, more in particular in the range of 5-250 µg / ml, even more in particular in the range of 10-100 µg / ml, such as in the range of 20-50 µg / ml. As shown in the experimental section, a suitable amount can be e.g. about 40 µg / ml.

[0148] (iii) a T cell activator that binds to CD28, preferably an anti-CD28 antibody. Alternatively, stimulation by an anti-CD28 antibody can also be provided by pre-coating the surface of a cell culture vessel, such as a cell culture dish, cell culture flask or cell culture bag, in which the culture medium, i.e. in which the co-culture, is received, with an anti-CD28 antibody.

[0149] The cell co-culture composition can comprise dissociated tumor organoids and immune cells at a tumor organoid:immune cell ratio of 20:1.

[0150] The present disclosure aims to meet the need for a more realistic world, more able to reflect the individualized human systemic immune cell environment. Most existing models rely on tumor cell organoids without other cells, tumor cell organoids with tumor-associated immune cells or non-autologous immune cells, mouse models that are not human-derived and are costly as screening tools, and do not incorporate the vascular barrier between immune cells and tumor organoids into the system. In contrast, the system described herein comprises autologous immune cells activated by tumor organoids to mimic the patient's systemic immune system; and allows the incorporation of a blood vessel-like structure between the organoids and immune cells. To the best of the inventors' knowledge, the present disclosure is the first to combine tumor organoid cultures (e.g. heterotypic tumor organoids comprising autologous fibroblasts) with autologous immune cells activated by tumor organoids, wherein the system can also comprise a simulated blood vessel barrier grown between the tumor organoids and activated peripheral immune cells. In certain embodiments, the physical boundary can also be composed of autologous cells. The system is incorporated into a microfluidic environment that allows easy and parallel measurement of chemotaxis, tumor cell killing and immune cell activity status (e.g. by assaying cytokines or flow cytometry).

[0151] Accordingly, there is also provided a method of preparing an in vitro organ culture system to mimic tumor interaction with a subject's immune system, the method comprising:

[0152] (i) providing a microfluidic device having a microfluidic channel with at least a first compartment and a second compartment separated by a physical boundary, the physical boundary allowing cells to move from the first compartment to the second compartment;

[0153] (ii) incorporating into the first compartment a three-dimensional tumor organoid established from a cancer tissue sample obtained from a subject and a suitable tumor cell growth medium; and

[0154] (iii) introducing into the second compartment autologous immune cells from the subject that have been activated by co-culturing with the established tumor organoid and a suitable immune cell medium;

[0155] thereby providing an in vitro organ culture system. The physical boundary can for example be selected from a membrane, a mechanical barrier, a membraneless phase-directed boundary, a gel-based boundary, a basement membrane-based boundary or a mesh-based boundary. Suitable examples of microfluidic chips providing such boundaries are described for example in WO 2022 / 167647. In preferred embodiments, the tumor organoid is established from a cancer tissue sample obtained from a subject according to a method as specifically described above. Likewise, in preferred embodiments, the autologous immune cells from the subject have been activated by co-culturing with the established tumor organoid according to a method as described above.

[0156] The suitable tumor cell growth medium in step (ii) can be any suitable tumor cell growth medium. However, in preferred embodiments it is the medium described above which is suitable for culturing tumor organoids. Thus, the medium comprises at least two or more, preferably all of the following (i)-(vii):

[0157] (i) an inducer of the WNT / beta catenin signaling pathway, such as R-spondin and / or WNT, like R-spondin 1. R-spondin, such as R-spondin-1, can be present, for example, in an effective amount in the typical range of 1-10000 ng / ml. Preferably, it can be present in the range of 10-5000 ng / ml, more preferably in the range of 100-2000 ng / ml, and even more preferably in the range of 250-1000 ng / ml. As shown in the experimental section, it can be present in an amount of about 500 ng / ml.

[0158] (ii) FGF, such as FGF-7 and / or FGF-10, preferably both FGF-7 and FGF-10. Effective amounts of FGF-7 can be readily determined and are typically in the range of 0.1-1000 ng / ml, in particular in the range of 1-500 ng / ml, more in particular in the range of 5-200 ng / ml, even more in particular in the range of 10-100 ng / ml, such as in the range of 15-50 ng / ml. As shown in the experimental section, FGF-7 can be present, for example, in an amount of about 25 ng / ml. Similarly, typical effective amounts of FGF-10 are in the range of 1-10000 ng / ml, in particular in the range of 10-5000 ng / ml, more in particular in the range of 25-1000 ng / ml, such as in the range of 50-500 ng / ml. As shown in the experimental section, FGF-10 can be present, for example, in an amount of about 100 ng / ml.

[0159] (iii) a bone morphogenetic protein (BMP) antagonist. An example of such an antagonist is noggin, and effective amounts of noggin are typically in the range of 10-1000 ng / ml, in particular in the range of 25-500 ng / ml, more in particular in the range of 50-250 ng / ml. As shown in the experimental section, effective amounts can be, for example, about 100 ng / ml.

[0160] (iv) an ALK5 inhibitor. An example of an ALK5 inhibitor is A83-01, which is typically effective in the range of 1-10000 nM, in particular in the range of 10-5000 nM, more in particular in the range of 50-2500 nM, for example in the range of 100-1000 nM. As shown in the experimental section, effective amounts can be, for example, about 500 nM.

[0161] (v) Rho kinase inhibitors. One example of a suitable Rho kinase inhibitor is Y-27632. Typically, Y-27632 is applied effectively in the range of 0.01-50 µM, in particular in the range of 0.1-25 µM, more particularly in the range of 1-10 µM. As shown in the experimental section, an effective amount can be, for example, about 5 μΜ.

[0162] (vi) p38 MAPK inhibitors. One example of a suitable p38 MAPK inhibitor is SB202190, which can be applied effectively in the range of 1-10000 nM, in particular in the range of 10-5000 nM, more particularly in the range of 50-2500 nM, for example in the range of 100-1000 nM. As shown in the experimental section, an effective amount can be, for example, about 500 nM.

[0163] (vii) nicotinamide, in particular in the range of 0.1-100 mM, preferably in the range of 0.5 - 50 mM, more preferably in the range of 1-25 mM, even more preferably in the range of 2-10 mM. As shown in the experimental section, a suitable amount can be, for example, about 5 mM.

[0164] Optionally, the culture medium can also contain B27 supplement and N-acetyl cysteine, such as about 1.25 mM N-acetyl cysteine. Typically, the tumor organoids are incorporated in step (ii) using an extracellular matrix gel comprising extracellular matrix proteins, such as laminin and collagen. Typically, the tumor organoids are incorporated in step (ii) by seeding tumor cells at a density of 0.01- 50 x 10 3 cells / µl, preferably in the range of 1-15 x 10 3 cells / µl, more preferably in the range of 1-10 x 10 3 cells / µl.

[0165] In an advantageous embodiment, step (ii) further comprises incorporating cancer associated fibroblasts (CAFs) obtained from said subject. For example, the fibroblasts can be seeded at a ratio of tumor cells: CAFs of about 1-8:1, preferably about 1-4:1. For example, they can be seeded at a density of 0.1-10 x 10 3 cells / µl, for example at a density of about 0.5-2.5 x 10 3 cells / µl.

[0166] In one embodiment, following step (ii), autologous or non-autologous endothelial cells are incorporated onto the tumour organoid, e.g. to mimic an endothelial vascular barrier. Suitable autologous endothelial cells are derived from tumour, artery or vein of the matching patient, and suitable non-autologous endothelial cells are human umbilical vein endothelial cells (HUVEC), human lung microvascular endothelial cells (HLMVEC), human pulmonary microvascular endothelial cells (HPMEC), human intestinal microvascular cells (HIMEC). For example, the endothelial cells can be seeded at a density of 1-50 x 10 3 cells / μl, e.g. at a density of about 7.5-10 x 10 3 cells / μl.

[0167] The immune cells are incorporated in step (iii) by seeding immune cells at a density of 0.01- 50 x 10 3 cells / μl, preferably in the range of 0.75-2 x 10 3 cells / μl.

[0168] Also provided as a result of the above method is an in vitro organ culture system for mimicking the interaction of a tumour with the immune system of a subject, comprising:

[0169] (i) a microfluidic device having a microfluidic channel with at least a first compartment and a second compartment separated by a physical boundary, which physical boundary allows cells to move from the first compartment to the second compartment;

[0170] (ii) a three-dimensional tumour organoid established from a cancer tissue sample obtained from a subject and a suitable tumour cell growth medium in the first compartment; and

[0171] (iii) autologous immune cells from the subject and a suitable immune cell medium in the second compartment, which autologous immune cells have been activated by co-culturing with said established tumour organoid.

[0172] The physical boundary can for example be selected from a membrane, a mechanical barrier, a membrane-free phase guiding boundary, a gel-based boundary, a basement membrane-based boundary or a mesh-based boundary. In preferred embodiments, the tumour organoid is established from a cancer tissue sample obtained from a subject according to the method as specifically described above. Likewise, in preferred embodiments, said autologous immune cells from said subject have been activated by co-culturing with said established tumour organoid according to the method as described above.

[0173] The suitable tumour cell growth medium can be any suitable tumour cell growth medium. However, in preferred embodiments, it is the medium as described above suitable for culturing tumour organoids. Thus, said medium comprises at least two or more, preferably all, of (i)-(vii) below:

[0174] (i) WNT / beta catenin signaling pathway inducer, such as R-spondin and / or WNT, e.g. R-spondin 1. R-spondin, such as R-spondin-1, can be present, for example, in an effective amount in the typical range of 1-10000 ng / ml. Preferably, it can be present in the range of 10-5000 ng / ml, more preferably in the range of 100-2000 ng / ml, and even more preferably in the range of 250-1000 ng / ml. As shown in the experimental section, it can be present in an amount of about 500 ng / ml.

[0175] (ii) FGF, such as FGF-7 and / or FGF-10, preferably both FGF-7 and FGF-10. An effective amount of FGF-7 can be readily determined and is typically in the range of 0.1-1000 ng / ml, in particular in the range of 1-500 ng / ml, more in particular in the range of 5-200 ng / ml, even more in particular in the range of 10-100 ng / ml, such as in the range of 15-50 ng / ml. As shown in the experimental section, FGF-7 can be present, for example, in an amount of about 25 ng / ml. Similarly, a typical effective amount of FGF-10 is in the range of 1-10000 ng / ml, in particular in the range of 10-5000 ng / ml, more in particular in the range of 25-1000 ng / ml, such as in the range of 50-500 ng / ml. As shown in the experimental section, FGF-10 can be present, for example, in an amount of about 100 ng / ml.

[0176] (iii) Bone morphogenetic protein (BMP) antagonist. An example of such an antagonist is noggin, and an effective amount of noggin is typically in the range of 10-1000 ng / ml, in particular in the range of 25-500 ng / ml, more in particular in the range of 50-250 ng / ml. As shown in the experimental section, an effective amount can be, for example, about 100 ng / ml.

[0177] (iv) ALK5 inhibitor. An example of an ALK5 inhibitor is A83-01, which is typically effective in the range of 1-10000 nM, in particular in the range of 10-5000 nM, more in particular in the range of 50-2500 nM, for example in the range of 100-1000 nM. As shown in the experimental section, an effective amount can be, for example, about 500 nM.

[0178] (v) Rho kinase inhibitors. One example of a suitable Rho kinase inhibitor is Y-27632. Typically, Y-27632 is applied effectively in the range of 0.01-50 µM, in particular in the range of 0.1-25 µM, more particularly in the range of 1-10 µM. As shown in the experimental section, an effective amount can be, for example, about 5 μΜ.

[0179] (vi) p38 MAPK inhibitors. One example of a suitable p38 MAPK inhibitor is SB202190, which can be applied effectively in the range of 1-10000 nM, in particular in the range of 10-5000 nM, more particularly in the range of 50-2500 nM, such as in the range of 100-1000 nM. As shown in the experimental section, an effective amount can be, for example, about 500 nM.

[0180] (vii) Nicotinamide, in particular in the range of 0.1-100 mM, preferably in the range of 0.5 - 50 mM, more preferably in the range of 1-25 mM, even more preferably in the range of 2-10 mM. As shown in the experimental section, a suitable amount can be, for example, about 5 mM.

[0181] Optionally, the culture medium can further contain B27 supplement and N-acetyl cysteine, for example about 1.25 mM N-acetyl cysteine. Typically, the tumor organoid is incorporated in step (ii) using an extracellular matrix gel comprising extracellular matrix proteins, such as laminin and collagen.

[0182] In embodiments, the first compartment further comprises cancer associated fibroblasts (CAFs) obtained from said subject. In further embodiments, the first compartment further comprises autologous or non-autologous endothelial cells, preferably wherein the autologous endothelial cells are derived from a tumor, an artery or a vein of the matching patient, and preferably wherein the non-autologous endothelial cells are human umbilical vein endothelial cells (HUVECs), human lung microvascular endothelial cells (HLMVECs), human pulmonary microvascular endothelial cells (HPMECs), human intestinal microvascular cells (HIMECs).

[0183] In embodiments, the organ culture system comprises a microfluidic device having at least two microfluidic channels, wherein each microfluidic channel comprises a different tumor organoid derived from a different tumor of the same subject, and / or wherein at least one microfluidic channel comprises a second compartment free of activated immune cells as a control. Typically, the volume of the first compartment is 0.7 to 100 pi, preferably 0.7 to 80 pi, more preferably 0.7 to 50 pi, and even more preferably 0.7 to 10 pi, and most preferably 0.7 to 2 pi. Typically, the volume of the second compartment is 20 to 200 pi, preferably 20 to 100 pi, more preferably 20 to 50 pi, and most preferably 20 to 40 pi. The microfluidic channels can further comprise a media reservoir.

[0184] In the course of its application, the organ culture system can additionally comprise one or more immunooncology drugs (preferably a combination of at least two) in the second compartment.

[0185] In embodiments, the at least one drug is an immune checkpoint inhibitor, preferably selected from the group consisting of anti-PD-1 / PD-L1 antibodies and anti-CTLA4 antibodies, more preferably an immune checkpoint inhibitor that is an anti-PD-1 / PD-L1 antibody. For example, the anti-PD-1 / PD-L1 antibody can be selected from the group consisting of nivolumab, pembrolizumab, cemiplimab, dostarlimab, retifanlimab, vopratelimab, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, INCMGA00012, AMP-224, AMP-514, and avelumab, atezolizumab, durvalumab, and avelumab. For example, the anti-PD-1 antibody can be nivolumab, as shown in the experimental section. The antibody can be added in a typical amount in the range of 0.1-1000 ng / ml, in particular in the range of 1-500 pg / ml, more in particular in the range of 5-250 pg / ml, even more in particular in the range of 10-150 pg / ml, such as about 100 pg / ml.

[0186] In further embodiments, the at least one drug is a chemotherapeutic agent and / or a small molecule. For example, the chemotherapeutic agent and / or small molecule can be selected from the group consisting of EGFR tyrosine kinase inhibitors, RAS GTPase inhibitors, lymphocyte activation gene-3 inhibitors, folate antimetabolites, TIM3 inhibitors, and STING agonists.

[0187] The EGFR tyrosine kinase inhibitor can be selected from the group consisting of osimertinib, gefitinib, erlotinib, afatinib, and EAI045, more in particular wherein the EGFR tyrosine kinase inhibitor is osimertinib. Typically, the amount of the inhibitor is 1-1000 nM, in particular 10-500 nM, more in particular 50-250 nM, such as about 100 nM.

[0188] The RAS GTPase inhibitor can for example be selected from the group consisting of Sotorasib, Adagrasib, MRTX1133, MRTX849, RSC-1255, BI-1701963. For example, the RAS GTPase inhibitor can be Sotorasib. The inhibitor is typically applied in an amount of 1-1000 nM, in particular in an amount in the range of 100-500 nM, for example about 250 nM.

[0189] An example of a lymphocyte activation gene-3 inhibitor is Relatlimab. It can be added in an amount of 1-1000 ng / ml, in particular in the range of 5-500 µg / ml, more in particular in the range of 10-100 µg / ml, such as about 30 µg / ml.

[0190] The folate antimetabolite can be selected from the group consisting of Pemetrexed and Methotrexate. For example, the folate antimetabolite can be Pemetrexed. It can be added in a typical amount of 0.1-100 µM, in particular in an amount of 0.5-50 µM, more in particular in an amount of 1-25 µM, such as about 10 µM. As shown in the experimental section, it can be added in an amount of for example about 2.5 µM.

[0191] The chemotherapeutic agent can also be selected from the group consisting of Carboplatin, Cisplatin, Gemcitabine, Nab-paclitaxel, Paclitaxel, FOLFOX and FOLFIRI. In some embodiments, the chemotherapeutic agent is Carboplatin. The chemotherapeutic agent (e.g. Carboplatin) can be added in a typical amount of 10-10000 µM, in particular in an amount of 50-5000 µM, for example in an amount of 100-500 µM. As shown in the experimental section, a suitable amount can be about 375 µM.

[0192] The TIM3 inhibitor can for example be selected from the group consisting of Sabatolimab (MBG453), TSR022, Sym023, BGB-A425, AZD7789, RO7121661; and the STING agonist can for example be selected from the group consisting of DMXAA (Vadimezan), ADU-S100 (MIW815), Ulevostinag (MK-1454), BMS-986301, E7766, GSK3745417 and SB11285.

[0193] Since the methods of establishing tumor organoids and autologous tumor organoid-activated immune cells allow for mass production of cells and organoids, these methods allow for advantageously providing a larger number of the same organ culture system. Thus, the present disclosure provides for the first time a plurality of organ culture systems as described above, wherein the plurality comprises at least 5, more preferably at least 10, even more preferably at least 20 organ culture systems. The plurality of organ culture systems allows for performing large-scale or larger-scale screening assays.

[0194] According to the above, there is also provided a method for determining the responsiveness of a tumor to treatment with at least one immuno-oncology drug, comprising the steps of:

[0195] (a) preparing an in vitro organ culture system according to the method as disclosed above, or providing an in vitro organ culture system as disclosed above;

[0196] (b) adding one or more, preferably at least two, immuno-oncology drugs to the second compartment; and

[0197] (c) determining the responsiveness of the in vitro organ culture system to the at least two immuno-oncology drugs.

[0198] For example, step c) can comprise determining tumor cell growth and / or determining the viability of the tumor organoid cells in the first compartment, wherein a decrease in tumor cell growth and / or a decrease in viability indicates that the tumor is effectively responding to treatment with said one or more immuno-oncology drugs. In embodiments, the tumor cell growth and / or the viability of the tumor organoid cells in the first compartment is compared to the tumor cell growth and / or the viability of the tumor organoid cells prior to step (b). Alternatively, the tumor cell growth and / or the viability of the tumor organoid cells in the first compartment can be compared to a control in which the immune cells are replaced by culture medium. Furthermore, step (c) can also comprise single cell sequencing to reveal which tumor cells escape T cell-mediated killing with the aid of the immunotherapy. This is expected to accelerate the opening of IO drugs and guide the selection of clinical treatment regimens. Due to the microfluidic system, the analysis of the effects can easily be automated.

[0199] Accordingly, in an advantageous application, the method is repeated with a plurality of identical in vitro organ culture systems as disclosed above for different immuno-oncology drugs or combinations of different immuno-oncology drugs, thereby identifying a personalized effective immuno-oncology therapy for the patient. Additionally or alternatively, the method is repeated with a plurality of identical in vitro organ culture systems as disclosed above for different treatment regimens of an immuno-oncology drug or combination of drugs, thereby identifying an effective treatment regimen.

[0200] In another advantageous application, the method is repeated with a plurality of different in vitro organ culture systems as disclosed further above for the same immuno-oncology drug or the same combination of immuno-oncology drugs, thereby identifying patient candidates that can benefit from treatment with said immuno-oncology drug or said combination of immuno-oncology drugs.

[0201] In embodiments, the at least one drug in step (b) is an immune checkpoint inhibitor, preferably selected from the group consisting of anti-PD-1 / PD-L1 antibodies and anti- CTLA4 antibodies, more preferably an immune checkpoint inhibitor that is an anti-PD-1 / PD-L1 antibody. For example, the anti-PD-1 / PD-L1 antibody can be selected from the group consisting of nivolumab, pembrolizumab, cemiplimab, dostarlimab, retifanlimab, vopratelimab, spartalizumab, camrelizumab, sindiizumab, tislelizumab, toripalimab, INCMGA00012, AMP-224, AMP-514, and avelumab, atezolizumab, durvalumab, and avelumab. For example, the anti-PD-1 antibody can be nivolumab, as indicated in the experimental section. The antibody can be added in a typical amount in the range of 0.1-1000 ng / ml, in particular in the range of 1-500 µg / ml, more in particular in the range of 5-250 µg / ml, even more in particular in the range of 10-150 µg / ml, such as about 100 µg / ml.

[0202] In further embodiments, the at least one drug in step (b) is a chemotherapeutic agent and / or a small molecule. For example, the chemotherapeutic agent and / or small molecule can be selected from the group consisting of EGFR tyrosine kinase inhibitors, RAS GTPase inhibitors, lymphocyte activation gene-3 inhibitors, folate antimetabolites, TIM3 inhibitors, and STING agonists.

[0203] The EGFR tyrosine kinase inhibitor can be selected from the group consisting of osimertinib, gefitinib, erlotinib, afatinib, and EAI045, more in particular wherein the EGFR tyrosine kinase inhibitor is osimertinib. Typically, the inhibitor is applied in an amount of 1-1000 nM, in particular 10-500 nM, more in particular 50-250 nM, such as about 100 nM.

[0204] The RAS GTPase inhibitor can for example be selected from the group consisting of sotorasib, adagrasib, MRTX1133, MRTX849, RSC-1255, BI-1701963. For example, the RAS GTPase inhibitor can be sotorasib. The inhibitor is typically applied in an amount of 1-1000 nM, in particular in an amount in the range of 100-500 nM, such as about 250 nM.

[0205] An example of a lymphocyte activation gene-3 inhibitor is relatlimab. It can be added in an amount of 1-1000 ng / ml, in particular in the range of 5-500 µg / ml, more in particular in the range of 10-100 µg / ml, such as about 30 µg / ml.

[0206] The folate antimetabolite can be selected from the group consisting of pemetrexed and methotrexate. For example, the folate antimetabolite can be pemetrexed. It can be added in a typical amount of 0.1-100 µM, in particular in an amount of 0.5-50 µM, more in particular in an amount of 1-25 µM, such as in an amount of 2-10 µM. As shown in the experimental section, it can be added in an amount of e.g. about 2.5 µM.

[0207] The chemotherapeutic agent can also be selected from the group consisting of carboplatin, cisplatin, gemcitabine, nab-paclitaxel, paclitaxel, FOLFOX and FOLFIRI. In some embodiments, the chemotherapeutic agent is carboplatin. The chemotherapeutic agent, e.g. carboplatin, can be added in a typical amount of 10-10000 µM, in particular in an amount of 50-5000 µM, such as in an amount of 100-500 µM. As shown in the experimental section, a suitable amount can be about 375 µM.

[0208] The TIM3 inhibitor can for example be selected from the group consisting of Sabatolizumab (MBG453), TSR022, Sym023, BGB-A425, AZD7789, RO7121661; and the STING agonist can for example be selected from the group consisting of DMXAA (Vadimezan), ADU-S100 (MIW815), Ulevostinag (MK-1454), BMS-986301, E7766, GSK3745417 and SB11285.

[0209] The Solid-IO platform also allows to detect biomarkers, e.g. cytokines, that promote or inhibit the response to the drug combination. Thus, in embodiments, the method further comprises the step of comparing the tumor organoids and / or the culture medium in the first compartment treated with response to the tumor organoids and / or the culture medium in the first compartment treated without response, thereby identifying biomarkers of treatment with response or without response. For example, the comparison of the phenotypic changes can comprise comparing the levels of cytokines in the culture medium between the treatment with response and the treatment without response, in particular wherein the biomarker is indicative of (i) a personalized effective or ineffective immuno-oncology therapy for the patient, (ii) an effective or ineffective treatment regimen, and / or (iii) a patient candidate that can or cannot benefit from treatment with the immuno-oncology drug or the combination of immuno-oncology drugs, as outlined above.

[0210] Finally, the present disclosure also provides a kit suitable for use in the above disclosed methods, e.g. a kit for preparing an in vitro organ culture system that mimics the interaction of a tumor with the immune system of a subject. In embodiments, the kit comprises at least two, in particular at least three, more in particular at least four, even more in particular at least five, such as all of the following (a)-(f):

[0211] (a) a microfluidic device having a microfluidic channel with at least a first compartment and a second compartment separated by a physical boundary that allows cells to move from the first compartment to the second compartment, as further disclosed above. In particular embodiments, the physical boundary is selected from a membrane, a mechanical barrier, a membraneless phase-directed boundary, a gel-based boundary, a basement membrane-based boundary, or a mesh-based boundary.

[0212] (b) a tumor cell growth medium comprising at least two, preferably all of:

[0213] (i) a WNT / beta catenin signaling pathway inducer, such as R-spondin and / or WNT, e.g., R-spondin 1. R-spondin, such as R-spondin-1, can be present, for example, in an effective amount in the typical range of 1-10000 ng / ml. Preferably, it can be present in the range of 10-5000 ng / ml, more preferably in the range of 100-2000 ng / ml, and even more preferably in the range of 250-1000 ng / ml. As shown in the experimental section, it can be present in an amount of about 500 ng / ml.

[0214] (ii) FGF, such as FGF-7 and / or FGF-10, preferably both FGF-7 and FGF-10. Effective amounts of FGF-7 can be readily determined and are typically in the range of 0.1-1000 ng / ml, specifically in the range of 1-500 ng / ml, more specifically in the range of 5-200 ng / ml, even more specifically in the range of 10-100 ng / ml, such as in the range of 15-50 ng / ml. As shown in the experimental section, FGF-7 can be present, for example, in an amount of about 25 ng / ml. Similarly, typical effective amounts of FGF-10 are in the range of 1-10000 ng / ml, specifically in the range of 10-5000 ng / ml, more specifically in the range of 25-1000 ng / ml, such as in the range of 50-500 ng / ml. As shown in the experimental section, FGF-10 can be present, for example, in an amount of about 100 ng / ml.

[0215] (iii) a bone morphogenetic protein (BMP) antagonist. An example of such an antagonist is noggin, and effective amounts of noggin are typically in the range of 10-1000 ng / ml, specifically in the range of 25-500 ng / ml, more specifically in the range of 50-250 ng / ml. As shown in the experimental section, an effective amount can be, for example, about 100 ng / ml.

[0216] (iv) ALK5 inhibitor. One example of an ALK5 inhibitor is A83-01, which is typically effective in the range of 1-10000 nM, specifically in the range of 10-5000 nM, more specifically in the range of 50-2500 nM, for example in the range of 100-1000 nM. As shown in the experimental section, an effective amount can be, for example, about 500 nM.

[0217] (v) Rho kinase inhibitor. One example of a suitable Rho kinase inhibitor is Y-27632. Typically, Y-27632 is effective for use in the range of 0.01-50 µM, specifically in the range of 0.1-25 µM, more specifically in the range of 1-10 µM. As shown in the experimental section, an effective amount can be, for example, about 5 μΜ.

[0218] (vi) p38 MAPK inhibitor. One example of a suitable p38 MAPK inhibitor is SB202190, which can be effective for use in the range of 1-10000 nM, specifically in the range of 10-5000 nM, more specifically in the range of 50-2500 nM, for example in the range of 100-1000 nM. As shown in the experimental section, an effective amount can be, for example, about 500 nM.

[0219] (c) immune cell culture medium, a medium comprising

[0220] (i) T cell growth factor, preferably interleukin 2 (IL-2). A T cell growth factor, such as IL-2, is typically added in the range of 1-100000 U / ml, specifically in the range of 10-50000 U / ml, more specifically in the range of 50-10000 U / ml, even more specifically in the range of 100-1000 U / ml, for example in the range of 200-500 U / ml. As shown in the experimental section, a suitable amount can be, for example, about 300 U / ml.

[0221] (ii) an immune checkpoint inhibitor, preferably selected from the group consisting of an anti-PD-1 / PD-L1 antibody and an anti-CTLA4 antibody, more preferably an anti-PD-1 antibody. In embodiments, the anti-PD-1 / PD-L1 antibody is selected from the group consisting of nivolumab, pembrolizumab, cemiplimab, dostarlimab, retifanlimab, vopratelimab, spartalizumab, camrelizumab, sindiizumab, tislelizumab, toripalimab, INCMGA00012, AMP-224, AMP-514, akliesimusab, atezolizumab, durvalumab, and avelumab. In specific embodiments shown below, the anti-PD-1 antibody is nivolumab. Typically, the effective amount of such antibody can be in the range of 0.1-1000 ng / ml, in particular in the range of 1-500 pg / ml, more in particular in the range of 5-250 pg / ml, even more in particular in the range of 10-100 pg / ml, such as in the range of 20-50 pg / ml. As shown in the experimental section, a suitable amount can be for example about 40 pg / ml.

[0222] (iii) nicotinamide, in particular in the range of 0.1-100 mM, preferably in the range of 0.5 - 50 mM, more preferably in the range of 1-25 mM, even more preferably in the range of 2-10 mM. As shown in the experimental section, a suitable amount can be for example about 5 mM.

[0223] (d) a cell culture dish or cell culture vessel pre-coated with an anti-CD28 antibody;

[0224] (e) a container with IFN-gamma; and / or

[0225] (f) a container with an extracellular matrix gel comprising extracellular matrix proteins, such as laminin and collagen.

[0226] Further disclosed are the following embodiments:

[0227] 1. An in vitro method of establishing a tumor organoid from a cancer tissue sample obtained from a subject, the method comprising the following steps:

[0228] (a) dissociating the cancer tissue sample into cells and fragments;

[0229] (b) propagating a first portion of the cells and / or fragments obtained in step (a) in a first 3D culture step;

[0230] (c) propagating a second portion of the cells and / or fragments obtained in step (a) in a first 2D culture step, for example to enrich for epithelial tumor cells and optionally fibroblasts;

[0231] (d) combining the proliferated cells and debris obtained in step (b) with the proliferated cells and debris obtained in step (c) and proliferating the combined cells and debris in a second 3D culture step;

[0232] thereby establishing a tumor organoid from the cancer tissue sample.

[0233] 2. The method according to embodiment 1, wherein the cancer tissue sample is a tissue sample obtained from a tissue selected from the group consisting of lung, bronchus, colon, rectum, prostate, breast, urinary bladder, thyroid, kidney, renal pelvis, uterine corpus, oral cavity or ovarian tissue.

[0234] 3. The method according to embodiment 1 or 2, wherein the cancer tissue sample is a cancer tissue sample from a benign tumor.

[0235] 4. The method according to embodiment 1 or 2, wherein the cancer tissue sample is a cancer tissue sample from a malignant cancer tumor, in particular wherein the cancer is selected from the group consisting of lung cancer, colon cancer, rectal cancer, prostate cancer, breast cancer, bladder cancer, thyroid cancer, kidney cancer, renal cancer, epithelial cancer or ovarian cancer.

[0236] 5. The method according to any one of the preceding embodiments, wherein the cancer tissue sample is a cancer tissue sample from an adenocarcinoma, an epidermoid carcinoma and / or a colorectal cancer.

[0237] 6. The method according to any one of the preceding embodiments, wherein the cancer tissue sample is a cancer tissue sample from a cancer comprising at least one mutated gene, optionally a gene associated with tumor drug resistance, in particular wherein the at least one mutation is a mutation in a gene selected from the group consisting of KRAS, EGFR, KEAP1, ALK, STK11, MET, TP53, ROS1, RB1, NOTCH1, NOTCH2, BRAF, NTRK1 / 2 / 3, RET, ERBB2, HRAS, NRAS, ASCL1, NF1, PI3CA, FGFR1 / 2, MAP2K1, in particular a mutation in a gene selected from the group consisting of KRAS, EGFR, KEAP1, ALK and STK11.

[0238] 7. The method according to embodiment 6, wherein the at least one mutation is a mutation in KRAS, in particular wherein the subject is a human and the at least one mutation in KRAS is a mutation in position Gly 12 and / or Gln 61, more particularly wherein the at least one mutation is a KRAS mutation selected from the group consisting of GL12Ala, Gly 12ASP, Gly 12Val, Gly 12Cys and Gln 61 His.

[0239] 8. The method according to any one of the preceding embodiments, wherein the subject is a mammal; preferably a primate, such as a human; or a rodent, such as a mouse, rat, hamster or guinea pig; or a cat, dog, sheep, horse or cow.

[0240] 9. The method according to any one of the preceding embodiments, wherein step (a) comprises dissociating the cancer tissue sample using mechanical and / or enzymatic methods, in particular wherein the cancer tissue sample is chopped, preferably into 1-2 mm 3 pieces, and / or treated with an enzyme, preferably with an enzyme selected from the group consisting of collagenase, thermolysin, trypsin, hyaluronidase and papain, most preferably wherein the enzyme is collagenase.

[0241] 10. The method according to any one of the preceding embodiments, wherein step (b) is performed in an extracellular matrix gel comprising extracellular matrix proteins, such as laminin and collagen, preferably wherein (b) is performed in Matrigel or Cultrex or Collagen.

[0242] 11. The method according to any one of the preceding embodiments, wherein step (b) comprises performing the proliferation in a medium comprising at least two or more, preferably all, of:

[0243] (i) a WNT / beta catenin signaling pathway inducer, such as R-spondin and / or WNT, preferably R-spondin 1, preferably R-spondin 1, in an amount in particular in the range of 1-10000 ng / ml, more preferably in the range of 10-5000 ng / ml, even more preferably in the range of 100-2000 ng / ml, yet more preferably in the range of 250-1000 ng / ml, and most preferably about 500 ng / ml;

[0244] (ii) FGF, preferably FGF-7 and / or FGF-10, more preferably FGF-7 and FGF-10, in particular wherein FGF-7 is in the range of 0.1-1000 ng / ml, more preferably in the range of 1-500 ng / ml, even more preferably in the range of 5-200 ng / ml, yet more preferably in the range of 10-100 ng / ml, yet more preferably in the range of 15-50 ng / ml, and most preferably about 25 ng / ml, and / or in particular wherein FGF-10 is in the range of 1-10000 ng / ml, preferably in the range of 10-5000 ng / ml, even more preferably in the range of 25-1000 ng / ml, yet more preferably in the range of 50-500 ng / ml, and most preferably about 100 ng / ml;

[0245] (iii) a bone morphogenic protein (BMP) antagonist; preferably wherein the BMP antagonist is noggin, in particular wherein the noggin is in the range of 10-1000 ng / ml, preferably in the range of 25-500 ng / ml, more preferably in the range of 50-250 ng / ml, such as about 100 ng / ml;

[0246] (iv) an ALK5 inhibitor; preferably wherein the ALK5 inhibitor is A83-01, in particular wherein the A83-01 is in the range of 1-10000 nM, preferably in the range of 10-5000 nM, more preferably in the range of 50-2500 nM, even more preferably in the range of 100-1000 nM, such as about 500 nM;

[0247] (v) a Rho kinase inhibitor; preferably wherein the Rho kinase inhibitor is Y-27632, in particular wherein the Y-27632 is in the range of 0.01-50 µM, preferably in the range of 0.1-25 µM, more preferably in the range of 1-10 µM, such as about 5 µM;

[0248] (vi) a p38 MAPK inhibitor; preferably wherein the p38 MAPK inhibitor is SB202190, in particular wherein the SB202190 is in the range of 1-10000 nM, preferably in the range of 10-5000 nM, more preferably in the range of 50-2500 nM, even more preferably in the range of 100-1000 nM, such as about 500 nM;

[0249] (vii) nicotinamide, in particular in the range of 0.1-100 mM, preferably in the range of 0.5 - 50 mM, more preferably in the range of 1-25 mM, even more preferably in the range of 2-10 mM, such as about 5 mM;

[0250] and optionally B27 supplement and N-acetyl cysteine, preferably about 1.25 mM N-acetyl cysteine.

[0251] 12. The method according to any one of the preceding embodiments, wherein step (c) comprises propagating in a culture medium comprising at least two or more, preferably all, of:

[0252] (i) FGF, preferably FGF-basic, in particular wherein the FGF-basic is in the range of 0.1 - 10000 ng / ml, preferably in the range of 0.5-5000 ng / ml, even more preferably in the range of 1-1000 ng / ml, more preferably in the range of 5-500 ng / ml, even more preferably in the range of 10-50 ng / ml, and most preferably is about 20 ng / ml;

[0253] (ii) EGF, in particular wherein the EGF is in the range of 0.1 - 10000 ng / ml, preferably in the range of 0.5-5000 ng / ml, even more preferably in the range of 1-2000 ng / ml, even more preferably in the range of 2-1000 ng / ml, even more preferably in the range of 5-500 ng / ml, even more preferably in the range of 10-100 mg / ml, such as about 50 ng / ml;

[0254] (iii) a Rho kinase inhibitor; preferably wherein the Rho kinase inhibitor is Y-27632, in particular wherein the Y-27632 is in the range of 0.01-50 µM, preferably in the range of 0.1-25 µM, more preferably in the range of 1-10 µM, such as about 5 µM.

[0255] 13. The method according to any one of the preceding embodiments, wherein step (c) is performed for at least 2-6 days, in particular at least 4-6 days, and / or wherein step (d) is performed for no more than passage 3 of the epithelial tumor cells, more in particular no more than passage 2, in particular no more than passage 1, such as at passage 0.

[0256] 14. The method according to any one of the preceding embodiments, wherein step (c) further comprises collecting and propagating cancer-associated fibroblasts, preferably in the same medium as used in step (c).

[0257] 15. The method according to any one of the preceding embodiments, wherein step (d) is performed in an extracellular matrix gel comprising extracellular matrix proteins, such as laminin and collagen, preferably wherein (d) is performed in Matrigel or Cultrex.

[0258] 16. The method according to any one of the preceding embodiments, wherein step (d) comprises propagating in a medium comprising at least two or more, preferably all of:

[0259] (i) a WNT / beta catenin signaling pathway inducer, such as R-spondin and / or WNT, preferably R-spondin 1, preferably R-spondin 1, in particular in an amount in the range of 1-10000 ng / ml, more preferably in the range of 10-5000 ng / ml, even more preferably in the range of 100-2000 ng / ml, yet more preferably in the range of 250-1000 ng / ml, most preferably about 500 ng / ml;

[0260] (ii) FGF, preferably FGF-7 and / or FGF-10, more preferably FGF-7 and FGF-10, in particular wherein FGF-7 is in the range of 0.1-1000 ng / ml, more preferably in the range of 1-500 ng / ml, even more preferably in the range of 5-200 ng / ml, yet more preferably in the range of 10-100 ng / ml, yet more preferably in the range of 15-50 ng / ml, and most preferably about 25 ng / ml, and / or in particular wherein FGF-10 is in the range of 1-10000 ng / ml, preferably in the range of 10-5000 ng / ml, even more preferably in the range of 25-1000 ng / ml, yet more preferably in the range of 50-500 ng / ml, and most preferably about 100 ng / ml;

[0261] (iii) a bone morphogenetic protein (BMP) antagonist; preferably wherein the BMP antagonist is noggin, in particular wherein noggin is in the range of 10-1000 ng / ml, preferably in the range of 25-500 ng / ml, yet more preferably in the range of 50-250 ng / ml, such as about 100 ng / ml;

[0262] (iv) an ALK5 inhibitor; preferably wherein the ALK5 inhibitor is A83-01, in particular wherein A83-01 is in the range of 1-10000 nM, preferably in the range of 10-5000 nM, more preferably in the range of 50-2500 nM, yet more preferably in the range of 100-1000 nM, such as about 500 nM;

[0263] (v) a Rho kinase inhibitor; preferably wherein the Rho kinase inhibitor is Y-27632, in particular wherein Y-27632 is in the range of 0.01-50 µM, preferably in the range of 0.1-25 µM, more preferably in the range of 1-10 µM, such as about 5 µM;

[0264] (vi) a p38 MAPK inhibitor; preferably wherein the p38 MAPK inhibitor is SB202190, in particular wherein SB202190 is in the range of 1- 10000 nM, preferably in the range of 10-5000 nM, more preferably in the range of 50-2500 nM, even more preferably in the range of 100-1000 nM, such as about 500 nM;

[0265] (vii) nicotinamide, in particular in the range of 0.1-100 mM, preferably in the range of 0.5 - 50 mM, more preferably in the range of 1-25 mM, even more preferably in the range of 2-10 mM, such as about 5 mM;

[0266] and optionally B27 supplement and N-acetyl cysteine, preferably about 1.25 mM N-acetyl cysteine.

[0267] 17. The method of any one of the preceding embodiments, wherein step (d) comprises propagating in the same medium as used in step (b).

[0268] 18. The method of any one of the preceding steps, wherein the tumor organoid obtained after step (d) is substantially free of tumor infiltrating leukocytes.

[0269] 19. An in vitro method of providing autologous tumor organoid-activated immune cells, the method comprising:

[0270] (a) establishing a tumor organoid from a cancer tissue sample obtained from a subject according to the method of any one of embodiments 1-19; and

[0271] (b) co-culturing the tumor organoid with immune cells obtained from the subject;

[0272] thereby providing autologous tumor organoid-activated immune cells.

[0273] 20. The method of embodiment 19, wherein step (a) comprises isolating the tumor organoid from an extracellular matrix gel comprising extracellular matrix proteins, such as laminin and collagen.

[0274] 21. The method of embodiment 19 or embodiment 20, wherein step (a) comprises culturing the tumor organoid in a medium comprising at least two or more, preferably all, of:

[0275] (i) a WNT / beta catenin signaling pathway inducer, such as R-spondin and / or WNT, preferably R-spondin 1, preferably R-spondin 1, in particular in an amount in the range of 1-10000 ng / ml, more preferably in the range of 10-5000 ng / ml, even more preferably in the range of 100-2000 ng / ml, yet more preferably in the range of 250-1000 ng / ml, most preferably about 500 ng / ml;

[0276] (ii) FGF, preferably FGF-7 and / or FGF-10, more preferably FGF-7 and FGF-10, in particular wherein FGF-7 is in the range of 0.1-1000 ng / ml, more preferably in the range of 1-500 ng / ml, even more preferably in the range of 5-200 ng / ml, yet more preferably in the range of 10-100 ng / ml, yet more preferably in the range of 15-50 ng / ml, and most preferably about 25 ng / ml, and / or in particular wherein FGF-10 is in the range of 1-10000 ng / ml, preferably in the range of 10-5000 ng / ml, even more preferably in the range of 25-1000 ng / ml, yet more preferably in the range of 50-500 ng / ml, and most preferably about 100 ng / ml;

[0277] (iii) a bone morphogenetic protein (BMP) antagonist; preferably wherein the BMP antagonist is noggin, in particular wherein noggin is in the range of 10-1000 ng / ml, preferably in the range of 25-500 ng / ml, yet more preferably in the range of 50-250 ng / ml, for example about 100 ng / ml;

[0278] (iv) an ALK5 inhibitor; preferably wherein the ALK5 inhibitor is A83-01, in particular wherein A83-01 is in the range of 1-10000 nM, preferably in the range of 10-5000 nM, more preferably in the range of 50-2500 nM, yet more preferably in the range of 100-1000 nM, for example about 500 nM;

[0279] (v) a Rho kinase inhibitor; preferably wherein the Rho kinase inhibitor is Y-27632, in particular wherein Y-27632 is in the range of 0.01-50 µM, preferably in the range of 0.1-25 µM, more preferably in the range of 1-10 µM, for example about 5 µM;

[0280] (vi) a p38 MAPK inhibitor; preferably wherein the p38 MAPK inhibitor is SB202190, in particular wherein SB202190 is in the range of 1 - 10000 nM, preferably in the range of 10 - 5000 nM, more preferably in the range of 50 - 2500 nM, even more preferably in the range of 100 - 1000 nM, such as about 500 nM;

[0281] (vii) nicotinamide, in particular in the range of 0.1 - 100 mM, preferably in the range of 0.5 - 50 mM, more preferably in the range of 1 - 25 mM, even more preferably in the range of 2 - 10 mM, such as about 5 mM;

[0282] and optionally B27 supplement and N-acetyl cysteine, preferably about 1.25 mM N-acetyl cysteine.

[0283] 22. The method of any one of embodiments 19-21, wherein the immune cells are immune cells isolated from whole blood, peripheral blood mononuclear cells (PBMCs), spleen, lymph node, buffy coat, pleural effusion, bone marrow aspirate, tumor, and / or from induced pluripotent stem cells, preferably wherein the immune cells are peripheral immune cells, more preferably wherein the immune cells are PBMCs.

[0284] 23. The method of any one of embodiments 19-22, wherein the tumor organoid of step (a) is stimulated prior to step (b), for example to enhance antigen presentation, in particular by pre-culturing in a culture medium comprising a type II interferon, preferably IFN-g, in an amount in the range of 1 - 100000 ng / ml, more preferably in the range of 10 - 10000 ng / ml, even more preferably in the range of 50 - 5000 ng / ml, even more preferably in the range of 100 - 1000 ng / ml, even more preferably in the range of 150 - 500 ng / ml, such as about 200 ng / ml.

[0285] 24. The method of any one of embodiments 19-23, wherein step (b) comprises co-culturing in a culture medium comprising:

[0286] (i) a T cell growth factor, preferably interleukin 2 (IL-2), preferably in an additive amount in the range of 1-100000 U / ml, more preferably in the range of 10-50000 U / ml, even more preferably in the range of 50-10000 U / ml, yet more preferably in the range of 100-1000 U / ml, even yet more preferably in the range of 200-500 U / ml, such as about 300 U / ml; and / or

[0287] (ii) an immune checkpoint inhibitor, preferably selected from the group consisting of an anti-PD-1 antibody, an anti-CTLA4 antibody, more preferably an anti-PD-1 antibody, in particular an anti-PD-1 antibody, selected from the group consisting of nivolumab, pembrolizumab, cemiplimab, dostarlimab, retifanlimab, vorolanib, spartalizumab, camrelizumab, sintyrozumab, tislelizumab, toripalimab, INCMGA00012, AMP-224, AMP-514, akliesimusab, atezolizumab, durvalumab, and avelumab, more preferably wherein the anti-PD-1 antibody is nivolumab, in a specific additive amount in the range of 0.1 - 1000 pg / ml, preferably in the range of 1-500 pg / ml, more preferably in the range of 5-250 pg / ml, even more preferably in the range of 10-100 pg / ml, even yet more preferably in the range of 20-50 pg / ml, such as about 40 pg / ml; and / or

[0288] (iii) a T cell activator binding to CD28, preferably an anti-CD28 antibody,

[0289] or wherein the anti-CD28 antibody has been pre-coated onto the surface of a cell culture vessel receiving the culture medium.

[0290] 25. The method of any one of embodiments 19-24, wherein step (b) combines the dissociated tumor organoids and immune cells at a ratio of tumor organoids:immune cells of 20:1, preferably added in equal volumes.

[0291] 26. The method of any one of embodiments 19-25, wherein step (b) is performed for at least 5 days, preferably at least 7 days, more preferably at least 10 days, and in particular 14 days or more.

[0292] 27. The method of any one of embodiments 19-26, wherein after co-culturing for 5-7 days, the tumor organoids are replaced with fresh tumor organoids.

[0293] 28. A method of preparing an in vitro organ culture system to mimic the interaction of a tumor with the immune system of a subject, the method comprising:

[0294] (i) providing a microfluidic device having a microfluidic channel with at least a first compartment and a second compartment separated by a physical boundary, the physical boundary allowing cells to move from the first compartment to the second compartment,

[0295] in particular wherein the physical boundary is selected from a membrane, a mechanical barrier, a membraneless phase-directed boundary, a gel-based boundary, a basement membrane-based boundary, or a mesh-based boundary;

[0296] (ii) incorporating a three-dimensional tumor organoid established from a cancer tissue sample obtained from a subject and a suitable tumor cell growth medium into the first compartment; and

[0297] (iii) incorporating autologous immune cells from the subject that have been activated by co-culturing with the established tumor organoid and a suitable immune cell medium into the second compartment;

[0298] thereby providing the in vitro organ culture system.

[0299] 29. The method of embodiment 28, wherein the tumor organoid is established from a cancer tissue sample obtained from a subject according to the method of any one of embodiments 1-19.

[0300] 30. The method of embodiment 28 or embodiment 29, wherein the suitable tumor cell growth medium in step (ii) comprises at least two or more, preferably all, of:

[0301] (i) a WNT / beta catenin signaling pathway inducer, such as R-spondin and / or WNT, preferably R-spondin 1, preferably R-spondin 1, in an amount in particular in the range of 1-10000 ng / ml, more preferably in the range of 10-5000 ng / ml, even more preferably in the range of 100-2000 ng / ml, yet more preferably in the range of 250-1000 ng / ml, most preferably about 500 ng / ml;

[0302] (ii) FGF, preferably FGF-7 and / or FGF-10, more preferably FGF-7 and FGF-10, in particular wherein FGF-7 is in the range of 0.1-1000 ng / ml, more preferably in the range of 1-500 ng / ml, even more preferably in the range of 5-200 ng / ml, more preferably in the range of 10-100 ng / ml, even more preferably in the range of 15-50 ng / ml, and most preferably about 25 ng / ml, and / or in particular wherein FGF-10 is in the range of 1-10000 ng / ml, preferably in the range of 10-5000 ng / ml, even more preferably in the range of 25-1000 ng / ml, more preferably in the range of 50-500 ng / ml, and most preferably about 100 ng / ml;

[0303] (iii) a bone morphogenetic protein (BMP) antagonist; preferably wherein the BMP antagonist is noggin, in particular wherein noggin is in the range of 10-1000 ng / ml, preferably in the range of 25-500 ng / ml, more preferably in the range of 50-250 ng / ml, such as about 100 ng / ml;

[0304] (iv) an ALK5 inhibitor; preferably wherein the ALK5 inhibitor is A83-01, in particular wherein A83-01 is in the range of 1-10000 nM, preferably in the range of 10-5000 nM, more preferably in the range of 50-2500 nM, more preferably in the range of 100-1000 nM, such as about 500 nM;

[0305] (v) a Rho kinase inhibitor; preferably wherein the Rho kinase inhibitor is Y-27632, in particular wherein Y-27632 is in the range of 0.01-50 µM, preferably in the range of 0.1-25 µM, more preferably in the range of 1-10 µM, such as about 5 µM;

[0306] (vi) a p38 MAPK inhibitor; preferably wherein the p38 MAPK inhibitor is SB202190, in particular wherein SB202190 is in the range of 1- 10000 nM, preferably in the range of 10-5000 nM, more preferably in the range of 50-2500 nM, more preferably in the range of 100-1000 nM, such as about 500 nM;

[0307] (vii) nicotinamide, in particular in the range of 0.1-100 mM, preferably in the range of 0.5 - 50 mM, more preferably in the range of 1-25 mM, even more preferably in the range of 2-10 mM, such as about 5 mM;

[0308] and optionally B27 supplement and N-acetyl cysteine, preferably about 1.25 mM N-acetyl cysteine.

[0309] 31. The method of any one of embodiments 28-30, wherein the tumor organoid is incorporated in step (ii) using an extracellular matrix gel comprising extracellular matrix proteins, such as laminin and collagen.

[0310] 32. The method of any one of embodiments 28-31, wherein the tumor organoid is incorporated in step (ii) by seeding tumor cells at a density of 0.01- 50 x 10 3 cells per pl, preferably in the range of 1-15 x 10 3 cells per pl, more preferably in the range of 1-10 x 10 3 cells per pl.

[0311] 33. The method of any one of embodiments 28-32, wherein the autologous immune cells from the subject have been activated according to the method of any one of embodiments 20-27, by co-culturing with the established tumor organoid.

[0312] 34. The method of any one of embodiments 28-33, wherein the immune cells are incorporated in step (iii) by seeding immune cells at a density of 0.01- 50 x 10 3 cells per pl, preferably in the range of 0.75-2 x 10 3 cells per pl.

[0313] 35. The method of any one of embodiments 28-34, wherein step (ii) further comprises incorporating cancer-associated fibroblasts (CAFs) obtained from the subject, preferably wherein the fibroblasts are seeded at a tumor cell:CAF ratio of about 1-8:1, preferably a ratio of 1-4:1, more preferably at a density of 0.1-10 x 10 3 cells per pl, such as a density of about 0.5-2.5 x 10 3 cells per pl.

[0314] 36. The method of any one of embodiments 28-35, wherein after step (ii) autologous or non-autologous endothelial cells are incorporated onto the tumor organoid, preferably wherein the autologous endothelial cells are derived from a tumor, artery or vein of a matching patient and the non-autologous endothelial cells are human umbilical vein endothelial cells (HUVEC), human lung microvascular endothelial cells (HLMVEC), human pulmonary microvascular endothelial cells (HPMEC), human intestinal microvascular endothelial cells (HIMEC), more preferably wherein the endothelial cells are seeded at a density of 1-50 x 10 3 cells / µl, such as a density of about 7.5-10 x 10 3 cells / µl.

[0315] 37. An ex vivo tumor organoid obtained by the method according to embodiments 1-18.

[0316] 38. The tumor organoid of embodiment 37, wherein the tumor organoid is established from a cancer tissue sample obtained from a tissue selected from lung, bronchus, colon, rectum, prostate, breast, bladder, thyroid, kidney, renal pelvis, uterine body, oral cavity or ovarian tissue.

[0317] 39. The tumor organoid of embodiment 37 or 38, wherein the tumor organoid is established from a benign tumor.

[0318] 40. The tumor organoid of embodiment 37 or 38, wherein the tumor organoid is established from a malignant cancer tumor, in particular wherein the cancer is selected from lung cancer, colon cancer, rectal cancer, prostate cancer, breast cancer, bladder cancer, thyroid cancer, kidney cancer, renal cancer, epithelial cancer or ovarian cancer.

[0319] 41. The tumor organoid of any one of embodiments 37-40, wherein the tumor organoid is established as an adenocarcinoma, an epidermoid carcinoma and / or a colorectal cancer.

[0320] 42. The tumor organoid of any one of embodiments 37-41, wherein the tumor organoid comprises at least one mutated gene, optionally a gene associated with tumor drug resistance, in particular wherein the at least one mutation is a mutation in a gene selected from KRAS, EGFR, KEAP1, ALK and STK11.

[0321] 43. The tumor organoid of embodiment 42, wherein the at least one mutation is a mutation in KRAS, in particular wherein the subject is a human and the at least one mutation in KRAS is a mutation of position Glyl2 and / or Gln61, more in particular wherein the at least one mutation is a KRAS mutation selected from GL12Ala, Glyl2ASP, Glyl2Val, Glyl2Cys and Gln61His.

[0322] 44. The method of any one of embodiments 37-42, wherein the tumor organoid is established from a subject, wherein the subject is a mammal; preferably a primate, such as a human; or a rodent, such as a mouse, rat, hamster or guinea pig; or a cat, dog, sheep, horse or cow.

[0323] 45. An in vitro cell co-culture composition comprising a tumor organoid established from a cancer tissue sample obtained from a subject as further defined in any one of embodiments 37-44 and immune cells obtained from the subject.

[0324] 46. The cell co-culture composition of embodiment 45, wherein the immune cells are immune cells isolated from peripheral blood mononuclear cells (PBMCs), spleen, buffy coat, pleural effusion, bone marrow aspirate, tumor, and / or immune cells derived from induced pluripotent stem cells, preferably wherein the immune cells are peripheral immune cells, more preferably wherein the immune cells are PBMCs.

[0325] 47. The cell co-culture composition of embodiment 45 or 46, further comprising a culture medium comprising at least two or more, preferably all, of:

[0326] (i) a T cell activating agent that binds to CD28, preferably an anti-CD28 antibody;

[0327] (ii) a T cell growth factor, preferably interleukin 2 (IL-2), preferably in an amount in the range of 1-100000 U / ml, more preferably in the range of 10-50000 U / ml, even more preferably in the range of 50-10000 U / ml, yet more preferably in the range of 100-1000 U / ml, even yet more preferably in the range of 200-500 U / ml, such as about 300 U / ml; and / or

[0328] (iii) an immune checkpoint inhibitor, preferably selected from the group consisting of an anti-PD-1 / PD-L1 antibody and an anti-CTLA4 antibody, more preferably an immune checkpoint inhibitor selected from the group consisting of nivolumab, pembrolizumab, cemiplimab, dostarlimab, retifanlimab, vorsetuzumab, spartalizumab, camrelizumab, sindigimab, tislelizumab, toripalimab, INCMGA00012, AMP-224, AMP-514 and avelumab, more preferably wherein the anti-PD-1 antibody is nivolumab, in a specific added amount in the range of 0.1 - 1000 pg / ml, preferably in the range of 1 - 500 pg / ml, more preferably in the range of 5 - 250 pg / ml, even more preferably in the range of 10 - 100 pg / ml, even more preferably in the range of 20 - 50 pg / ml, such as about 40 pg / ml.

[0329] 48. The cell co-culture composition of any one of embodiments 45-47, comprising dissociated tumor organoids and immune cells, at a tumor organoid:immune cell ratio of 20:1.

[0330] 49. An in vitro organ culture system for modeling the interaction of a tumor with the immune system of a subject, comprising:

[0331] (i) a microfluidic device having a microfluidic channel with at least a first compartment and a second compartment separated by a physical boundary, the physical boundary allowing cells to move from the first compartment to the second compartment, in particular wherein the physical boundary is selected from the group consisting of a membrane, a mechanical barrier, a membraneless phase-directed boundary, a gel-based boundary, a basement membrane-based boundary, or a mesh-based boundary;

[0332] (ii) a three-dimensional tumor organoid established from a cancer tissue sample obtained from the subject in the first compartment and a suitable tumor cell growth medium; and

[0333] (iii) autologous immune cells from the subject in the second compartment that have been activated by co-culturing with the established tumor organoid and a suitable immune cell medium.

[0334] 50. The organ culture system of embodiment 49, wherein the tumor organoid is a tumor organoid according to embodiments 37-44.

[0335] 51. The organ culture system of embodiment 49 or 50, wherein the suitable tumor cell growth medium in step (ii) comprises at least two or more, preferably all, of:

[0336] (i) a WNT / beta catenin signaling pathway inducer, such as R-spondin and / or WNT, preferably R-spondin 1, preferably R-spondin 1, in particular in an amount in the range of 1-10000 ng / ml, more preferably in the range of 10-5000 ng / ml, even more preferably in the range of 100-2000 ng / ml, yet more preferably in the range of 250-1000 ng / ml, most preferably about 500 ng / ml;

[0337] (ii) FGF, preferably FGF-7 and / or FGF-10, more preferably FGF-7 and FGF-10, in particular wherein FGF-7 is in the range of 0.1-1000 ng / ml, more preferably in the range of 1-500 ng / ml, even more preferably in the range of 5-200 ng / ml, yet more preferably in the range of 10-100 ng / ml, yet more preferably in the range of 15-50 ng / ml, and most preferably about 25 ng / ml, and / or in particular wherein FGF-10 is in the range of 1-10000 ng / ml, preferably in the range of 10-5000 ng / ml, even more preferably in the range of 25-1000 ng / ml, yet more preferably in the range of 50-500 ng / ml, and most preferably about 100 ng / ml;

[0338] (iii) a bone morphogenetic protein (BMP) antagonist; preferably wherein the BMP antagonist is noggin, in particular wherein noggin is in the range of 10-1000 ng / ml, preferably in the range of 25-500 ng / ml, yet more preferably in the range of 50-250 ng / ml, for example about 100 ng / ml;

[0339] (iv) an ALK5 inhibitor; preferably wherein the ALK5 inhibitor is A83-01, in particular wherein A83-01 is in the range of 1-10000 nM, preferably in the range of 10-5000 nM, more preferably in the range of 50-2500 nM, yet more preferably in the range of 100-1000 nM, for example about 500 nM;

[0340] (v) a Rho kinase inhibitor; preferably wherein the Rho kinase inhibitor is Y-27632, in particular wherein Y-27632 is in the range of 0.01-50 µM, preferably in the range of 0.1-25 µM, more preferably in the range of 1-10 µM, for example about 5 µM;

[0341] (vi) a p38 MAPK inhibitor; preferably wherein the p38 MAPK inhibitor is SB202190, in particular wherein SB202190 is in the range of 1- 10000 nM, preferably in the range of 10-5000 nM, more preferably in the range of 50-2500 nM, even more preferably in the range of 100-1000 nM, such as about 500 nM;

[0342] (vii) nicotinamide, in particular in the range of 0.1-100 mM, preferably in the range of 0.5 - 50 mM, more preferably in the range of 1-25 mM, even more preferably in the range of 2-10 mM, such as about 5 mM;

[0343] and optionally B27 supplement and N-acetyl cysteine, preferably about 1.25 mM N-acetyl cysteine.

[0344] 52. The organ culture system of any one of embodiments 49-51, wherein the tumor organoid is incorporated into an extracellular matrix gel comprising extracellular matrix proteins, such as laminin and collagen.

[0345] 53. The organ culture system of any one of embodiments 49-52, wherein the first compartment further comprises cancer-associated fibroblasts (CAFs) obtained from the subject.

[0346] 54. The organ culture system of any one of embodiments 49-53, wherein the first compartment further comprises an endothelial tube formed by autologous or non-autologous endothelial cells, preferably wherein the autologous endothelial cells are derived from a tumor, an artery or a vein of the matched patient, and preferably wherein the non-autologous endothelial cells are human umbilical vein endothelial cells (HUVECs), human lung microvascular endothelial cells (HLMVECs), human pulmonary microvascular endothelial cells (HPMECs), human intestinal microvascular endothelial cells (HIMECs).

[0347] 55. The organ culture system of any one of embodiments 49-54, comprising a microfluidic device having at least two microfluidic channels, wherein each microfluidic channel comprises a different tumor organoid derived from a different tumor of the same subject, and / or wherein at least one microfluidic channel comprises a second compartment without activated immune cells as a control.

[0348] 56. The organ culture system of any one of embodiments 49-55, wherein the volume of the first compartment is 0.7 to 100 pi, preferably 0.7 to 80 pi, more preferably 0.7 to 50 pi, and even more preferably 0.7 to 10 pi, and most preferably 0.7 to 2 pi.

[0349] 57. The organ culture system of any one of embodiments 49-56, wherein the volume of the second compartment is 20 to 200 pi, preferably 20 to 100 pi, more preferably 20 to 50 pi, and most preferably 20 to 40 pi.

[0350] 58. The organ culture system of any one of embodiments 49-57, wherein the microfluidic channel further comprises a media reservoir.

[0351] 59. The organ culture system of any one of embodiments 49-58, wherein the second compartment additionally comprises one or more, preferably at least two, immunooncology drugs.

[0352] 60. The organ culture system of embodiment 59, wherein at least one of the drugs is an immune checkpoint inhibitor, preferably an immune checkpoint inhibitor selected from the group consisting of anti-PD-1 / PD-L1 antibodies and anti-CTLA4 antibodies, more preferably an immune checkpoint inhibitor that is an anti-PD-1 / PD-L1 antibody, in particular an immune checkpoint inhibitor selected from the group consisting of nivolumab, pembrolizumab, cemiplimab, dostarlimab, retifanlimab, vopratelimab, spartalizumab, camrelizumab, sindigimab, tislelizumab, toripalimab, INCMGA00012, AMP-224, AMP-514, and avelumab, atezolizumab, durvalumab, and avelumab, more preferably wherein the anti-PD-1 antibody is nivolumab, in particular in an amount in the range of 0.1 - 1000 pg / ml, preferably in the range of 1-500 pg / ml, more preferably in the range of 5-250 pg / ml, even more preferably in the range of 10-150 pg / ml, such as about 100 pg / ml.

[0353] 61. The organ culture system of embodiment 59 or 60, wherein at least one of the drugs is a chemotherapeutic agent and / or a small molecule,

[0354] preferably wherein the chemotherapeutic agent and / or small molecule is selected from the group consisting of EGFR tyrosine kinase inhibitors, RAS GTPase inhibitors, lymphocyte activation gene-3 inhibitors, folate antimetabolites, TIM3 inhibitors, and STING agonists;

[0355] in particular wherein the EGFR tyrosine kinase inhibitor is selected from the group consisting of osimertinib, gefitinib, erlotinib, afatinib, and EAI045, more in particular wherein the EGFR tyrosine kinase inhibitor is osimertinib, even more in particular in an amount in the range of 1-1000 nM, preferably 10-500 nM, more preferably 50-250 nM, such as about 100 nM; and / or

[0356] In particular wherein the RAS GTPase inhibitor is selected from the group consisting of Sotorasib, Adagrasib, MRTX1133, MRTX849, RSC-1255, BI-1701963, more in particular wherein the RAS GTPase inhibitor is Sotorasib, even more in particular in an amount of 1-1000 nM, preferably 100-500 nM, such as about 250 nM; and / or

[0357] In particular wherein the Lymphocyte-activation gene-3 inhibitor is Relatlimab, even more in particular in an amount of 1-1000 pg / ml, preferably in the range of 5-500 pg / ml, more preferably in the range of 10-100 pg / ml, such as about 30 pg / ml; and / or

[0358] In particular wherein the folate ant metabolite is selected from the group consisting of Pemetrexed and Methotrexate, more in particular wherein the folate ant metabolite is Pemetrexed, even more in particular in an amount of 0.1-100 pM, preferably 0.5-50 pM, more preferably 1-25 pM, even more preferably 2-10 pM, such as about 2.5 pM; and / or in particular wherein the chemotherapeutic agent is selected from the group consisting of Carboplatin, Cisplatin, Gemcitabine, Nab-paclitaxel, Paclitaxel, FOLFOX and FOLFIRI, more in particular wherein the chemotherapeutic agent is Carboplatin, even more in particular in an amount of 10-10000 pM, preferably 50-5000 pM, more preferably 100-500 pM, such as about 375 pM;

[0359] In particular wherein the TIM3 inhibitor is selected from the group consisting of Sabatolimab (MBG453), TSR022, Sym023, BGB-A425, AZD7789, RO7121661; and

[0360] In particular wherein the STING agonist is selected from the group consisting of DMXAA (Vadimezan), ADU-S100 (MIW815), Ulevostinag (MK-1454), BMS-986301, E7766, GSK3745417 and SB11285.

[0361] 62. A plurality of organ culture systems according to any one of embodiments 49-61, preferably according to any one of embodiments 49-58, wherein the plurality comprises at least 5, more preferably at least 10, even more preferably at least 20 organ culture systems according to any one of embodiments 49-61, preferably according to any one of embodiments 49-58.

[0362] 63. A method for determining the responsiveness of a tumor to treatment with at least one immuno-oncology drug comprising the steps of:

[0363] (a) preparing an in vitro organ culture system according to the method of any one of embodiments 28-36, or providing an in vitro organ culture system according to any one of embodiments 49-58;

[0364] (b) adding one or more, preferably at least two, immuno-oncology drugs to the second compartment; and

[0365] (c) determining the responsiveness of the in vitro organ culture system to the at least two immuno-oncology drugs.

[0366] 64. The method of embodiment 63, wherein step c) comprises determining tumor cell growth and / or determining the viability of the tumor organoid cells in the first compartment, wherein a decrease in tumor cell growth and / or a decrease in viability is indicative of an effective response of the tumor to treatment with the one or more immuno-oncology drugs.

[0367] 65. The method of embodiment 64, wherein the tumor cell growth and / or the viability of the tumor organoid cells in the first compartment is compared to the tumor cell growth and / or viability of the tumor organoid cells prior to step (b); or

[0368] wherein the tumor cell growth and / or the viability of the tumor organoid cells in the first compartment is compared to a control, wherein the immune cells are replaced by culture medium.

[0369] 66. The method of any one of embodiments 63-65, wherein the method is repeated with a plurality of identical in vitro organ culture systems according to any one of embodiments 49-58 for different immuno-oncology drugs or combinations of different immuno-oncology drugs, thereby identifying an individualized effective immuno-oncology therapy for a patient.

[0370] 67. The method of any one of embodiments 63-65, wherein the method is repeated with a plurality of identical in vitro organ culture systems according to any one of embodiments 49-58 for different treatment regimens of an immuno-oncology drug or combination of drugs, thereby identifying an effective treatment regimen.

[0371] 68. The method of any one of embodiments 63-65, wherein the method is repeated with a plurality of different in vitro organ culture systems according to any one of embodiments 49-58 for the same immuno-oncology drug or the same combination of immuno-oncology drugs, thereby identifying patient candidates who can benefit from treatment with the immuno-oncology drug or the combination of immuno-oncology drugs.

[0372] 69. The method of any one of embodiments 63-68, wherein at least one of the drugs of step (b) is an immune checkpoint inhibitor, preferably an immune checkpoint inhibitor selected from the group consisting of anti-PD-1 / PD-L1 antibodies and anti-CTLA4 antibodies, more preferably an immune checkpoint inhibitor which is an anti-PD-1 / PD-L1 antibody, in particular an immune checkpoint inhibitor which is an anti-PD-1 / PD-L1 antibody selected from the group consisting of nivolumab, pembrolizumab, cemiplimab, dostarlimab, retifanlimab, vorolanib, spartalizumab, camrelizumab, sintyrozumab, tislelizumab, toripalimab, INCMGA00012, AMP-224, AMP-514 and avelumab, atezolizumab, durvalumab and avelumab, more preferably wherein the anti-PD-1 antibody is nivolumab, in particular in an amount in the range of 0.1 - 1000 pg / ml, preferably in the range of 1-500 pg / ml, more preferably in the range of 5-250 pg / ml, even more preferably in the range of 10-150 pg / ml, such as about 100 pg / ml.

[0373] 70. The method of any one of embodiments 63-69, wherein at least one of the drugs is a chemotherapeutic agent, a small molecule and / or an anti-cancer agent, other than an immune checkpoint inhibitor,

[0374] preferably wherein the chemotherapeutic agent and / or small molecule is selected from the group consisting of an EGFR tyrosine kinase inhibitor, a RAS GTPase inhibitor, a lymphocyte activation gene-3 inhibitor, a folate antimetabolite, a TIM3 inhibitor and a STING agonist;

[0375] in particular wherein the EGFR tyrosine kinase inhibitor is selected from the group consisting of osimertinib, gefitinib, erlotinib, afatinib and EAI045, more in particular wherein the EGFR tyrosine kinase inhibitor is osimertinib, even more in particular in an amount in the range of 1-1000 nM, preferably 10-500 nM, more preferably 50-250 nM, such as about 100 nM; and / or

[0376] In particular wherein the RAS GTPase inhibitor is selected from the group consisting of Sotorasib, Adagrasib, MRTX1133, MRTX849, RSC-1255, BI-1701963, more in particular wherein the RAS GTPase inhibitor is Sotorasib, even more in particular in an amount of 1-1000 nM, preferably 100-500 nM, such as about 250 nM; and / or

[0377] In particular wherein the Lymphocyte-activation gene-3 inhibitor is selected from the group consisting of Relatlimab, [if necessary, expand], more in particular wherein the Lymphocyte-activation gene-3 inhibitor is Relatlimab, even more in particular in an amount in the range of 1-1000 µg / ml, such as about 30 µg / ml; and / or

[0378] In particular wherein the folate antimetabolite is selected from the group consisting of Pemetrexed and Methotrexate, more in particular wherein the folate antimetabolite is Pemetrexed, even more in particular in an amount of 0.1-100 µM, preferably 0.5-50 µM, more preferably 1-25 µM, even more preferably 2-10 µM, such as about 2.5 µM; and / or

[0379] In particular wherein the chemotherapeutic agent is selected from the group consisting of Carboplatin, Cisplatin, Gemcitabine, Nab-paclitaxel, Paclitaxel, FOLFOX and FOLFIRI, more in particular wherein the chemotherapeutic agent is Carboplatin, even more in particular in an amount of 10-10000 µM, preferably 50-5000 µM, more preferably in an amount of 100-500 µM, such as about 375 µM;

[0380] In particular wherein the TIM3 inhibitor is selected from the group consisting of Sabatolimab (MBG453), TSR022, Sym023, BGB-A425, AZD7789, RO7121661; and

[0381] In particular wherein the STING agonist is selected from the group consisting of DMXAA (Vadimezan), ADU-S100 (MIW815), Ulevostinag (MK-1454), BMS-986301, E7766, GSK3745417 and SB11285.

[0382] 71. The method of any one of embodiments 63-70, further comprising the step of comparing the tumor organoids and / or culture medium in the first compartment with the reactive treatment, to the tumor organoids and / or culture medium in the first compartment without the reactive treatment, thereby identifying biomarkers of a reactive or non-reactive treatment.

[0383] 72. The method of embodiment 71, wherein the comparison of the phenotypic changes comprises comparing cytokine levels in the culture medium between the responsive and non-responsive treatment, in particular wherein the biomarker is indicative of (i) an individual effective or ineffective immunooncology therapy for the patient, (ii) an effective or ineffective treatment regimen, and / or (iii) a patient candidate capable or not capable of benefiting from treatment with the immunooncology drug or the combination of immunooncology drugs.

[0384] 73. A kit for preparing an in vitro organ culture system mimicking the interaction of a tumor with the immune system of a subject, the kit comprising at least two, preferably at least three, more preferably at least four, even more preferably at least five, most preferably all of:

[0385] (a) a microfluidic device having a microfluidic channel with at least a first compartment and a second compartment separated by a physical boundary allowing cells to move from the first compartment to the second compartment, in particular wherein the physical boundary is selected from a membrane, a mechanical barrier, a membraneless phase-directed boundary, a gel-based boundary, a basement membrane-based boundary, or a mesh-based boundary;

[0386] (b) a tumor cell growth medium comprising at least two, preferably all of:

[0387] (i) a WNT / beta catenin signaling pathway inducer, such as R-spondin and / or WNT, preferably R-spondin 1, preferably R-spondin 1, in an amount in particular in the range of 1-10000 ng / ml, more preferably in the range of 10-5000 ng / ml, even more preferably in the range of 100-2000 ng / ml, even more preferably in the range of 250-1000 ng / ml, most preferably about 500 ng / ml;

[0388] (ii) FGF, preferably FGF-7 and / or FGF-10, more preferably FGF-7 and FGF-10, in particular wherein FGF-7 is in the range of 0.1-1000 ng / ml, more preferably in the range of 1-500 ng / ml, even more preferably in the range of 5-200 ng / ml, more preferably in the range of 10-100 ng / ml, even more preferably in the range of 15-50 ng / ml, and most preferably about 25 ng / ml, and / or in particular wherein FGF-10 is in the range of 1-10000 ng / ml, preferably in the range of 10-5000 ng / ml, even more preferably in the range of 25-1000 ng / ml, more preferably in the range of 50-500 ng / ml, and most preferably about 100 ng / ml;

[0389] (iii) a bone morphogenic protein (BMP) antagonist; preferably wherein the BMP antagonist is noggin, in particular wherein noggin is in the range of 10-1000 ng / ml, preferably in the range of 25-500 ng / ml, more preferably in the range of 50-250 ng / ml, even more preferably about 100 ng / ml;

[0390] (iv) an ALK5 inhibitor; preferably wherein the ALK5 inhibitor is A83-01, in particular wherein A83-01 is in the range of 1-10000 nM, preferably in the range of 10-5000 nM, more preferably in the range of 50-2500 nM, even more preferably in the range of 100-1000 nM, such as about 500 nM;

[0391] (v) a Rho kinase inhibitor; preferably wherein the Rho kinase inhibitor is Y-27632, in particular wherein Y-27632 is in the range of 0.01-50 µM, preferably in the range of 0.1-25 µM, more preferably in the range of 1-10 µM, such as about 5 µM;

[0392] (vi) a p38 MAPK inhibitor; preferably wherein the p38 MAPK inhibitor is SB202190, in particular wherein SB202190 is in the range of 1- 10000 nM, preferably in the range of 10-5000 nM, more preferably in the range of 50-2500 nM, even more preferably in the range of 100-1000 nM, such as about 500 nM;

[0393] (c) an immune cell culture medium, a medium comprising

[0394] (i) a T cell growth factor, preferably interleukin 2 (IL-2), preferably in an amount in the range of 1-100000 U / ml, more preferably in the range of 10-50000 U / ml, even more preferably in the range of 50-10000 U / ml, even more preferably in the range of 100-1000 U / ml, even more preferably in the range of 200-500 U / ml, such as about 300 U / ml;

[0395] (ii) an immune checkpoint inhibitor, preferably selected from the group consisting of an anti-PD-1 / PD-L1 antibody and an anti-CTLA4 antibody, more preferably an immune checkpoint inhibitor selected from the group consisting of an anti-PD-1 / PD-L1 antibody, in particular an anti-PD-1 / PD-L1 antibody, selected from the group consisting of nivolumab, pembrolizumab, cemiplimab, dostarlimab, retifanlimab, vopratelimab, spartalizumab, camrelizumab, sindigimab acastin, tislelizumab, toripalimab, INCMGA00012, AMP-224, AMP-514, akliesimus, atezolizumab, durvalumab and avelumab, more preferably wherein the anti-PD-1 antibody is nivolumab, in particular in an amount in the range of 0.1 - 1000 pg / ml, preferably in the range of 1 - 500 pg / ml, more preferably in the range of 5 - 250 pg / ml, even more preferably in the range of 10 - 100 pg / ml, even more preferably in the range of 20 - 50 pg / ml, such as about 40 pg / ml;

[0396] (iii) nicotinamide, in particular in the range of 0.1 - 100 mM, preferably in the range of 0.5 - 50 mM, more preferably in the range of 1 - 25 mM, even more preferably in the range of 2 - 10 mM, such as about 5 mM;

[0397] and optionally B27 supplement and N-acetyl cysteine, preferably about 1.25 mM N-acetyl cysteine;

[0398] (d) a cell culture dish or cell culture vessel that has been pre-coated with an anti-CD28 antibody;

[0399] (e) a container with IFN-gamma; and / or

[0400] (f) a container with an extracellular matrix gel comprising extracellular matrix proteins, such as laminin and collagen.

[0401] Examples

[0402] Example 1 - Establishment and culturing of tumor organoids

[0403] To be able to study anti-tumor immunity, the inventors first generated a living biobank of 3D organoids of lung tumors and benign tissue.

[0404] Specifically, patient tumor and blood samples were collected from known consented lung cancer patients according to the Declaration of Helsinki and submitted to the Biobank simultaneously. The study has been approved by the HUS IRB (HUS / 237 / 2021) with the statement of the institutional ethics committee (HUS / 970 / 2021).

[0405] Lung tumor resections and biopsies were kept in wash medium (Advanced DMEM / F12 low serum + 1% Glutamax + 1% Hepes buffer + 1% Penicillin / Streptomycin) and sent to the lab within one hour of tissue collection. Upon arrival, a small piece of tissue was cut and quickly put in a cryotube for storage at -80°C for future sequencing needs. The rest of the tissue was cut into 1-2 mm 3 pieces with a scalpel and further dissociated in a shaking incubator at 37°C with digestion medium (Advanced DMEM / F12 supplemented with 1% Ultra-Glutamine I, 1% Penicillin / Streptomycin and 1.5 mg / ml collagenase type II) for 2 hours for biopsies and overnight for resections. Digestion was stopped by washing once with cold DMEM containing 20% FBS and twice with PBS.

[0406] To improve the chances of successfully establishing tumor organoids from most patients and to be able to isolate tumor cells and fibroblasts, the inventors established a two-step building culture system.

[0407] First, the inventors embedded half of the cells / pieces in a 3D culture using Matrigel. 30 minutes after embedding, the Matrigel solidified and a special organoid culture medium was added (Advanced DMEM / F12 supplemented with 500 ng / ml R-protein 1 (PeproTech), 25 ng / ml FGF-7 (PeproTech), 100 ng / ml FGF-10 (PeproTech), 100 ng / ml Noggin (PeproTech), 500 nM A83-01 (ALK5 inhibitor, Merck), 5 µM Y-27632 (Rho kinase inhibitor, Selleckchem), 500 nM SB202190 (p38 MAPK inhibitor, MedChemExpress), 1x B27 supplement, 1.25 mM N-acetyl cysteine, 5 mM Nicotinamide and 1% Ultra-Glutamine, 10 mM Hepes buffer, 1% Penicillin / Streptomycin and 50 µg / ml Primocin (a broad-spectrum antibiotic preparation).

[0408] Second, the other half of the cells / pieces were resuspended with standard medium (Advanced DMEM / F12 supplemented with 1% Ultra-Glutamine (Lonza), 1% Penicillin / Streptomycin, 20 ng / ml FGF-basic (R&D systems), 50 ng / ml EGF (Invitrogen), 5 mM Y-27632 (Rho kinase inhibitor; Selleckchem)) to establish 2D culture, then plated into two wells of tissue-treated 6-well plates. 2 to 6 days after the start of 2D culture, cells in each well were resuspended according to cell status, and the suspension was transferred to the adjacent well, while more medium was added to the original well. This step was repeated two days later, and finally each sample had six or more wells in new wells. This allowed enrichment of epithelial tumor cells or fibroblasts in different wells. Fibroblasts were then collected and propagated in standard medium (ingredients as described above).

[0409] Meanwhile, confluent epithelial cells at passage 0 to 2 were collected and embedded in Matrigel to allow formation of organoids, or mixed directly with 3D cells. Organoid growth was assessed using a Nikon Eclipse TS100 microscope.

[0410] Depending on the growth of the organoids, passaging was performed every 0.5-2 weeks with a split ratio of 1:2. Organoids obtained from 2D culture steps and 3D culture steps were dissociated with TrypLE Express pre-warmed to 37°C for 5-10 minutes. To terminate the action of TrypLE Express, cold DMEM / F12 containing 20% FBS was added, then the sample was centrifuged (4°C, 300xg, 5 minutes), then washed with cold Advanced DMEM / F12. In this step, organoids were broken into single cells or aggregates of 2-5 or 3-5 cells, which were resuspended with Matrigel and formed domes, then solidified at 37°C for 30 minutes, then covered with special organoid medium (ingredients as described above).

[0411] Using the two-step organoid establishment system disclosed herein, the inventors successfully established 3D tumor tissue organoids from 85% (23 / 27) of patient tumor samples and 92% (26 / 28) of benign tissue samples. Matched fibroblast lines were also established from 48% (13 / 27) of samples.

[0412] The following table shows the characteristics of patients, including mutation profile, mutant allele frequency, and PDL-1 expression of samples.

[0413]

[0414]

[0415] Generation of autoreactive immune cells

[0416] To study the systemic immune response and to achieve a tumor-specific immune response, we isolated circulating peripheral blood mononuclear cells (PBMCs) from patient blood and simulated systemic anti-tumor activation by co-culturing these cells with autologous tumor organoids with functional MHC-I (see also Figure 1 ).

[0417] Patient tumor and blood samples were collected from lung cancer patients with informed consent according to the Declaration of Helsinki, and samples were simultaneously submitted to the Biobank as described in Example 1. Peripheral blood was collected in lithium heparin vacutainers in the patient’s chosen laboratory according to the standard protocol of HUSLAB.

[0418] Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood using SepMate density gradient centrifugation. Ficoll-Paque (density gradient medium at room temperature) was transferred into a 50 ml SepMate tube by a central hole insert according to the manufacturer’s instructions. Peripheral whole blood was diluted with an equal volume of PBS + 2% fetal bovine serum (FBS) and then the diluted solution was gently dropped into the tube wall above the insert. Then centrifugation was performed at room temperature for 10 minutes at 1200 x g with the brake on. Peripheral blood mononuclear cells (PBMCs) and plasma were poured into another 50 ml centrifuge tube and washed with PBS + 2% FBS for 2 times. PBMCs were resuspended in RPMI-1640 medium supplemented with 2 mM L-glutamine, 100 U / ml penicillin, 100 μg / ml streptomycin, and 10% FBS (complete RPMI medium), then the viability was assessed and cultured or cryopreserved.

[0419] Alternatively, PBMCs were isolated by LeucoSep (Greiner bio-one) density gradient centrifugation. Ficoll-Paque (density gradient medium at room temperature) was pipetted into a 50 ml LeucoSep tube, then centrifuged at 1000 x g for 30 seconds according to the manufacturer’s instructions. Peripheral whole blood was diluted with an equal volume of PBS and then pipetted into the LeucoSep tube. Then the sample was centrifuged at 800 x g for 25 minutes at room temperature with the brake off. The topmost plasma was discarded and the remaining plasma containing the PBMC layer was collected into a new 50 ml Falcon tube using a disposable pipette. To wash the plasma, PBS was added and centrifuged at 160 x g for 10 minutes at room temperature with the brake off. Then the sample was washed with PBS and centrifuged at 250 x g for 10 minutes.

[0420] The separated PBMCs were divided into 5-10x10 6 Resuspend the PBMCs at a concentration of [number] cells / mL in cryopreservation medium containing 7-10% DMSO and 90-93% FBS, and place them in a -80°C freezer for 24 hours to allow for uniform cooling. The next day, transfer the samples to -150°C for long-term storage. Carefully thaw the PBMCs by gently rotating them in a 37°C water bath for 30 seconds. Wash the thawed samples twice with RPMI 1640 supplemented with 1% penicillin / streptomycin and 10% FBS (500xg for 5 minutes at room temperature). After thawing, incubate the PBMCs overnight in RPMI 1640 medium supplemented with 2 mM L-glutamine, 1% penicillin / streptomycin, and 10% FBS for recovery.

[0421] In order to generate reactive immune cells, the inventors followed the protocol established by Catteneo et al. in 2020 (incorporated herein by reference), but used tumor organoids prepared according to Example 1.

[0422] Two days before co-culture, tumor organoids (each 2 × 10⁻⁶) were placed... 6 One PBMC, 1-5×10 4 Organoids were separated from Matrigel using mechanical dissociation and then incubated with pre-warmed dispersant (2 mg / ml PBS solution) at 37°C for 15 minutes. The dispersant was inactivated with 0.5 M EDTA (100 µl EDTA per 1 ml dispersant solution), and the organoids were washed with PBS at 300 × g for 5 minutes at room temperature. They were then resuspended in specially prepared organoid culture medium (as described above) and plated into 6-well plates prepared for tissue culture. The organoids were cultured at 37°C for 24 hours.

[0423] The day before co-culturing, organoids were stimulated overnight with 200 ng / ml IFNγ to enhance antigen presentation.

[0424] 96-well U-shaped plates were coated with 50 µl of anti-CD28 antibody (used to provide co-stimulatory signal, mouse anti-human CD28 antibody (eBioscience)) in 5 µg / ml PBS. The plates were sealed with film and incubated at 4°C for 24 hours.

[0425] Freeze-stored PBMC were thawed in 15 mL T-cell thawing medium (RPMI1640 supplemented with 1% Ultra-Glutamine I (Lonza), 1% Penicillin / Streptomycin and 10% FBS) and centrifuged at 200xg for 10 min at room temperature with moderate deceleration. PBMC were then incubated with 1:1000 Benzonase for 15 min at 37°C in 5 mL T-cell thawing medium, then washed with T-cell thawing medium and cells were pelleted at 200xg for 12 min at room temperature. Cells were resuspended at 2x10 6 / mL in T-cell medium (RPMI1640 supplemented with 1% Ultra-Glutamine I (Lonza), 1% Penicillin / Streptomycin and 10% male human serum (Merck)) with 150 U / mL IL-2 (PreproTech) and incubated overnight at 37°C in 15 mL centrifuge tubes.

[0426] The next day, IFNy-stimulated organoids were collected and pelleted by centrifugation at 300xg for 5 min at room temperature. The pellet was resuspended with 1 mL TrypLE Express and combined with the remaining cells adhered to the bottom of the 6-well plate for 5 min at 37°C, then washed with PBS and cells were centrifuged at 300xg for 5 min at room temperature. The isolated organoids were then resuspended at 5 x 10 4 cells / ml T-cell medium (composition as above). The overnight incubated PBMC were washed with PBS and resuspended at 1x10 6 cells / ml T-cell medium and supplemented with 300 U / mL IL-2 (PreproTech) and 40 pg / mL anti-PD-1 (Nivolumab, Selleckchem). Equal volumes of isolated organoids and PBMC were mixed, with a ratio of 20:1 of PBMC to organoids. Anti-CD28 coated 96-well plates were washed twice with PBS. Tumor cells and PBMC were co-cultured by plating 200 mΐ of dissociated organoid-PBMC suspension per well. The co-culture medium was changed three times per week with 300 U / mL IL-2 and 40 pg / mL anti-PD-1.

[0427] After seven days of co-culture, PBMC were collected and counted, then re-stimulated for seven days with freshly isolated organoids (stimulated with IFNy 24 hours prior to co-culture) by seeding 2x10 5 PBMC with 10 4 isolated organoids per well of a pre-coated anti-CD28 plate.

[0428] At the end of co-culture, reactive immune cells were collected and cryopreserved, or used for downstream analysis and tumor killing assays. Reactive immune cells were cryopreserved at 5 x 10 6 cells / mL in cold male human serum containing 10% DMSO, then placed in a cryogenic container at -80°C.

[0429] To investigate potential changes in T cell subsets resulting from co-culture, flow cytometry-based immunological analysis was employed to measure the percentage of helper (CD3+ / CD4+) and cytotoxic (CD3+ / CD8+) T cells in baseline PBMCs and compared to these T cells isolated from two-week co-cultures. At the population level, two-week co-culture of PBMCs with matched organoids did not result in a significant shift in the original proportions of CD3+ cells or CD4+, CD8+ cells Figure 2 ). However, in 4 samples (ORG-29, ORG-36, ORG-43, and ORG-55), CD4 + T cell numbers were slightly reduced. At the same time, CD8 + T cell populations were enriched in 5 samples (ORG-36, ORG-43, ORG-55, lung-4, and lung-35T).

[0430] To further assess changes in T cell activation status following stimulation, we measured IFNy expression in cytotoxic T cells by flow cytometry Figure 3 A and 3B).

[0431] Briefly, 10 5 immune cells isolated from 14-day co-cultures were re-stimulated with 5 x 10 4 tumor cells at a 2: 1 effector to target ratio. Tumor cells and immune cells, including T cells, were co-cultured in coated anti-CD28 96-well plates in T cell media with the addition of 20 ug / mL anti-PD1 antibody (1:150) (Nivolumab, Selleckchem). For positive controls, 10 5 PBMCs were seeded with ImmunoCult (25:1000) (CD3 / CD28 / CD2 T cell activator (StemCell)). Both conditions were co-cultured at 37°C for 1 hour, then Golgi-Stop (1:1500) (BD) and Golgi-Plug (1:1000) (BD) were added and co-culturing was continued at 37°C for an additional 4 hours to inhibit surface protein translocation, thus improving detection of antibodies to them.

[0432] Cells were pelleted at 330xg for 5 min at 4°C and washed twice with FACS buffer (PBS supplemented with 1% EDTA (0.5 M) and 5 mg BSA). After washing, a 30 min cell surface staining was performed in FACS buffer with the addition of 1 :20 mouse anti-CD4-FITC (Stemcell technologies), 1 :20 mouse anti-CD3-PerCP-Cy5.5 (BD), 1 :200 anti-CD8-V450 (BD) antibodies and 1 :1000 near infrared live dye. Cells were washed twice with FACS buffer, then fixed by the addition of 100 μΐ of Fixation / Permeabilization solution (BD Biosciences) and permeabilized for 20 min on ice, washed twice with 1 x Permeabilization / Wash Buffer (with FBS and saponin) in distilled water (BD Biosciences) and intracellular staining was performed with 1 :40 mouse γ-APC (BD) in Permeabilization / Wash Buffer for 30 min at 37°C. Cells were washed twice with Perm / Wash Buffer, resuspended in FACS buffer and acquired by BD FACSVERSE TM Recorded.

[0433] IFNy production was induced in 60% (9 / 15) of samples, with 47% (7 / 15) of samples having no baseline IFNy expression, and 13% (2 / 15) of patients having pre-existing IFNy production, and IFNy production was further increased by co-culture (lung-4, ORG-43) Figure 3 B). It was observed that IFNy production by CD8+ T cells was patient and time point dependent Figure 4 ).

[0434] Finally, the inventors wanted to investigate whether established reactive immune cells, including T cells, could be specifically activated against the matched tumor sample. To investigate this, baseline PBMCs and reactive immune cells compared to them were co-cultured with the matched tumor organoid for 48 hours, after which cell death was assessed.

[0435] Briefly, tumor organoids were isolated using 5 U / mL dispase for 10-15 min, then inactivated with EDTA and washed. Organoids were then resuspended with 2 mL of special organoid medium (as described above) per well and plated in a 6-well plate, then incubated at +37°C, 5% CO2. After 24 hours, organoids were collected, and the suspension aliquot was isolated, then treated with TrypLE to dissociate the organoids into single cells, with the aim of determining the number of tumor single cells in the original organoid suspension. Tumor organoids were plated at 1 x 10 4Cells per well were plated in 100 mI of special medium in 96-well flat bottom plates (Corning) (with or without stimulated immune cells), including T cells plated in T cell medium (target effector ratio of 1 :4) (with or without 200 pg / mL anti-PD-1 (nivolumab), and with or without 200 pg / mL anti-PD-1 (nivolumab) in combination with chemotherapy (450 mI / mL carboplatin + 2.5 mI / mL pemetrexed)). For conditions containing tumor organoids only, 100 mI of T cell medium was added to bring the final volume to 200 mI. For stimulated T cells, 10 ng / mL IL-2 was added. The co-cultures were then incubated at 37°C, 5% CO2 for 48 hours. Subsequently, a caspase 3 / 7 Glo assay (Promega) was performed. Luminescence was measured using a Spark Multifunction Microplate Reader. Alternatively, a CellTox green (Promega) assay was performed and fluorescence was measured by a Cellcyte Live Cell Imager at 48 time points.

[0436] In the 2D killing validation assay, reactive immune cells, including reactive T cells, resulted in significantly higher tumor killing compared to baseline PBMCs, indicating tumor-specific activation occurred during co-culture Figure 5 ).

[0437] Single cell sequencing (scSeq) was used to more broadly assess the activation state of autologous immune cells when co-cultured with tumor cells, as well as to understand the response of tumor and immune cells in co-culture in the presence or absence of immune cells and immunomodulatory drugs in individual patients. The inventors found that co-culture induced widespread activation of immune cells, including different T cell subtypes and NK cells, but the level of activation varied for each subject patient.

[0438] Tumor organoids were isolated using 5 U / mL dispase for 10-15 minutes, then inactivated with EDTA and washed. The organoids were then resuspended with 2 mL special media per well and plated in a 6-well plate and then incubated at 37 °C, 5% CO2. After 48 hours, the organoids were collected and an aliquot of the suspension was isolated and then treated with TryplE to dissociate the organoids into single cells with the goal of determining the number of tumor single cells in the original organoid suspension. Tumor organoids were plated at 50,000 cells per well in 200 ul special media on a 48-well plate with or without stimulated immune cells, including T cells plated in T cell media (target effector ratio of 1:4) with or without 200 pg / mL nivolumab. For conditions containing tumor organoids only, 100 ul T cell media was added to bring the final volume to 200 ul. For stimulated immune cells, including T cells, 10 ng / mL IL-2 was added. The co-cultures were then incubated at 37 °C, 5% CO2 for 48 hours. Co-cultures were performed in duplicate for each condition. Experiments were completed using tumor organoids, stimulated immune cells, including T cells, and PBMCs from three patients. After 48 hours of co-culture, cells from each well were dissociated into single cells using TrypLE and then washed 2-3 times with 10 mL PBS. Cells were resuspended in 100 ul cold wash buffer 1 or wash buffer 2, 10 ul TruStain FcX blocking reagent (Biolegend) was added, and cells were blocked at +4 °C for 10 minutes. Unique TotalSeq-C Hashed antibodies (Biolegend) were added to each sample (2 ul / 2 pg per sample), and cells were incubated at +4 °C for 30 minutes. Cells were then washed 3-5 times with 3.5 mL wash buffer 1 or wash buffer 2, and samples were then combined in cold PBS + 0.04% bovine serum albumin (BSA) and subjected to scRNA-seq. Single cell gene expression profiles were investigated using the 10x Genomics Chromium Single Cell 5’ Gene Expression platform. Chromium Single Cell 5’ RNAseq runs and library preparation were completed using Chromium Next GEM Single Cell 5’ Immune Profiling Analysis by Feature Barcode Technology version 2 chemistry. Sample libraries were sequenced on an Illumina NovaSeq 6000 system with read lengths of 26 bp (read 1), 10 bp (i7 index), 10 bp (i5 index), and 90 bp (read 2), respectively.

[0439] As Figure 13As shown, it can be demonstrated that the method of providing immune cells activated by autologous tumor organoids has personalized features, with multiple shared and individual genes upregulated in CD4+ (helper) and CD8+ (cytotoxic) T cells after the PBMC / tumor co-culture protocol applied to different patients.

[0440] Example 3 - Ex vivo study of response to anti-PD-1 and chemotherapy using the Solid-IO platform

[0441] To study the systemic cytotoxic effects of immune cells under stimulation with or without IO drugs, we combined tumor organoids prepared as described in Example 1, tumor-reactive immune cells prepared as described in Example 2, and tumor microenvironment components on a high-throughput microfluidic chip organ (Solid-IO platform). To mimic the tumor site, organoids were loaded into the 3D extracellular matrix in the middle channel of the microfluidic chip. To mimic the process of immune cells (including T cells) infiltrating from blood vessels to the tumor site, previously obtained patient-matched reactive immune cells were added to the proximal side channels of the microfluidic chip. To study drug efficacy and perfusion to the tumor site, standard of care drugs or immunotherapy drugs were added to the distal side channels of the chip. Figure 6 ).

[0442] Specifically, the AIM identTX3 chip (AIMbiotech) and the Mimetas OrganoPlate 3-lane 40 / 64 (Mimetas) microfluidic devices were used. Tumor organoids were mechanically dissociated from Matrigel and then incubated with Try-plE for 10-15 min at 37°C to dissociate them into single cells or aggregates of 2-5 cells. Dissociated tumor cells were resuspended in 90% cold Matrigel and seeded at 1 x 10 3 cells / µl (for the AIM device), or at 10 x 10 3 cells / µl (for the Mimetas device). 10 µl and 2 µl of tumor cell Matrigel suspension were loaded into the middle channel of the AIM and Mimetas, respectively. Matrigel was allowed to solidify for 15-30 min at 37°C. In both devices, 50 µl of special organoid culture medium (composition as described above) was added to the upper-lower culture medium channels on both left and right sides. In the AIM chip, an additional 50 µl of special organoid culture medium was added to each of the four culture medium inlets. Tumor cells in the Matrigel matrix within the microfluidic chip were allowed to form organoids at 37°C for 48 h.

[0443] To set up the Solid-IO co-culture and screen for immunotherapy responses, on day 2, 50 pl of special organoid medium was aspirated from the inlet of the medium and 50 pl of T cell medium (composition as described above) with or without 2 x 10 5 or 1 x 10 5 activated immune cells was inoculated into the inlet of the right-top medium in the AIM device and Mimetast device, respectively, and another 50 pl of T cell medium was inoculated into the left-bottom channel.

[0444] Subsequently, 50 pl of special organoid medium with or without selected drugs was added to the inlet of the right-top and bottom medium channels. In the AIM chip, 50 pl of special medium was additionally added to each of the four medium inlets.

[0445] When all components were loaded into the microfluidic chip, the device was placed in an incubator at 37 °C and placed on a shaker at 1 rpm for 48 hours to generate flow. The drugs used were: 100 nM / mL osimertinib, 250 nM / mL sotorasib, 100 pg / mL nivolumab, 30 pg / mL relatlimab, and chemotherapeutic agents were a combination of 450 pM / mL carboplatin + 2.5 pM / mL pemetrexed.

[0446] Live / dead cell fluorescent staining was performed by loading 1 :5 dilution in PBS of Nexcelom ViaStain™ AO / PI stain. Cells were incubated with the dye for 10 minutes in the dark and then washed three times with PBS. After that, cells were fixed by adding 2% PFA for 10 minutes and then washed with PBS. Each condition was imaged by a Nikon Eclipse Ti-E microscope.

[0447] Quantitative analysis of images was performed by ImageJ software. All data were processed blindly to avoid bias before quantification. Detailed quantification steps were as follows: I- Open image and separate color channels (live cells - green and dead cells - red), and each channel was processed separately; II- From Edit, select Transform option and select a- Scale, b- Weighted RGB transformation when transforming; III- From Analyze, select Set Measurements, then select Area, limit to threshold, and set Decimals to 3; IV- Convert each channel to 8-bit by selecting Image, Type, then 8-bit will convert the channel to grayscale; V- Generate measurement mask independently to fit the area of the original channel, open the mask and place it on the area to be measured; VI- From Image, select Adjust, then select Threshold, then select "Add / Subtract" and set, then set lower threshold to 30 and upper threshold to 255, each channel will be converted to blue, while the organoids / cells will remain gray; VII- Measure the coverage area of the organoids / cells in the live and dead cell channels, press Analyze, then measure each channel. Add the values obtained for each channel to obtain the total value. To obtain the ratio of live and dead cells, the inventors used the value of the green channel / total value x 100 = percentage of live organoids, while the value of the red channel / total value x 100 = percentage of dead organoids.

[0448] We first used Solid-IO to screen 8 NSCLC patients carrying different clinically relevant mutations for response to anti-PD-1 (nivolumab) or its combination with chemotherapy (carboplatin / pemetrexed). Except for one patient (ORG-2), all patients had very low clinical baseline PD-L-1 expression (<5%) ( Figure 7 ), which can indicate poor clinical response to anti-PD-1. In the vehicle control treatment group, the addition of tumor reactive T cells led to significant killing of tumor tissue in Solid-IO ( Figure 8 ). As suggested by the clinical PD-L-1 expression data, blockade of PD-1 by nivolumab did not enhance T cell killing of tumor organoids. The same was true for ORG-2, which had 15% of the cells expressing PD-L-1.

[0449] At the population average level, the combination of nivolumab with chemotherapy and stimulated immune cells did not have a significant difference when compared to the same conditions without immune cells ( Figure 8 ).

[0450] However, in 43% (3 / 8) of the patients, we were able to detect individual responses to the combination of nivolumab and chemotherapy when used in combination with reactive immune cells (ORG-2, ORG-55 and Lung-19, with 20%, 10% and 11% increase in tumor killing, respectively) Figure 8 ,Figure 9 Interestingly, all patients that responded to the combination harbored different mutations in the KRAS oncogene, which is known to be an inducer of high tumor mutational burden and thus associated with higher anti-tumor immunity. Surprisingly, one of the combination treatment responders (lung-19) also harbored a STK11 mutation (allele frequency 28%), which is usually associated with a poor response to anti-PD-1. Since the allele frequency of the STK11 mutation was relatively low, the responding tumor cells originated from the unmutated population, however, this was not tested in our study.

[0451] In summary, our study results show that the Solid-IO platform can provide valuable tools to unravel patient individualized IO drug responses. Some NSCLC patients can benefit from the combination of nivolumab and chemotherapy even with low PD-L1 expression levels.

[0452] Example 4 - Advanced Solid-IO with artificial vasculature system

[0453] In the advanced Solid-IO setup, endothelial tubes obtained as described in Example 1 and patient-matched fibroblasts were added to the system to better mimic the patient’s vasculature and immunosuppressive microenvironment Figure 10 ).

[0454] An advanced Solid-IO was established with a 3-channel Mimetas Organo-Plate with different components Figure 10 , Figure 12 ).

[0455] Tumor organoids were mechanically dissociated from Matrigel and incubated with TryplE for 10-15 min at 37°C to dissociate them into single cells or aggregates of 2-5 cells.

[0456] Cancer-associated fibroblasts (CAFs) were dissociated from 2D cultures by incubation with trypsin for 5 min at 37°C. A suspension of 10 x 10 3 cells / µl + 2.5 x 10 3 CAF / µl in ice-cold Matrigel was prepared and 2 µl of the ice-cold tumor fibroblast Matrigel mixture was loaded into the middle channel inlet. The Matrigel was allowed to solidify for 15 min at 37°C.

[0457] After solidification, human umbilical vein endothelial cells (HUVEC) were dissociated from 2D cultures using trypsin and 2 µl of 7.5 x 10 3HUVEC cells were injected into the right-upper media inlet at 50 mΐ, followed by the addition of 50 mΐ endothelial cell media to the inlet. The side of the Mimetas plate was then placed in a custom-made holder at a 75° angle into the incubator to allow the HUVEC cells in the channel to settle and attach to the ECM Matrigel for 4 hours.

[0458] After HUVEC cells were attached to the ECM gel, 50 mΐ endothelial cell media was added to the right-lower media inlet and 50 mΐ special organoid media was added to each of the left-side media channel inlets. The Mimetas plate was then placed in the incubator on a shaker at 1 rpm to generate flow. Cells were cultured in the chip for four days to allow organoid and endothelial tube formation.

[0459] Immune cells and drugs were then added to the Solid-IO as described in Example 3.

[0460] Endothelial tubes were successfully established in the Solid-IO Figure 10 ) and matched fibroblasts were also successfully cultured in the Solid-IO.

[0461] To assess whether the tubes could prevent drug diffusion to the tumor site, we compared the control (organoid + endothelial tubes) to the chemotherapy condition.

[0462] Chemotherapy resulted in higher tumor cell killing, while HUVEC cells remained viable (99%) after 6 days of culture Figure 11 ).

[0463] Example 5 - Cytokine profiling using the Solid-IO platform

[0464] Cytokines are signaling proteins that play a crucial role in shaping the immune response to tumor cells in the tumor microenvironment (TME). To investigate the differences in cytokine secretion by baseline PBMCs and tumor-reactive immune cells in the presence or absence of IO drugs, the inventors collected the media after 48 hours of co-culture from the Solid-IO platform and analyzed the secreted cytokines using Luminex cytokine profiling. Profiling was performed using the Bio-Plex Pro Human Immunotherapy panel utilizing a Luminex 200 instrument.

[0465] It was found that reactive immune cells induced the secretion of all immune-related cytokines compared to baseline PBMCs, indicating that tumor stimulation enhanced the overall immune activity and cytokine secretion in the TME.

[0466] Next, the inventors analyzed cytokines collected from Solid-IO from a responder (Lung-24) and a non-responder (Lung-25) patient. Interestingly, in the responder patient, the inventors detected higher levels of IFNy-induced chemokines IP-10, MIP-la and MIP-lb, which are known to be facilitators of T cell recruitment and activation. In contrast, the non-responder patient had higher levels of STAT6-activated cytokines, which have been shown to have immunosuppressive effects and can inhibit T cell and dendritic cell activity. This therefore provides a potential mechanism for the poor anti-tumour activity in this patient.

[0467] In summary, the different responses of patients to the IO combination were associated with differences in cytokine profiles. Responder patients exhibited higher levels of IFNy-induced chemokines, which are involved in T cell recruitment and activation, while non-responder patients exhibited higher levels of specific immunosuppressive cytokines.

[0468] Developing biomarkers based on cytokine profiles extracted from the Solid-IO platform, by targeting the differential cytokine profiles observed in responders and non-responders, can lead to more personalized and effective immunotherapeutic approaches.

[0469] It will be obvious to a person skilled in the art that, as technology advances, the inventive concept can be implemented in various ways. The application and its embodiments are not limited to the examples described above but can vary within the scope of the claims.

[0470] References

[0471] 1 -Al-Samadi, A., Poor, B., Tuomainen, K., Liu, V., Hyytiainen, A., Suleymanova, I.,... & Salo, T. (2019). In vitro humanized 3D microfluidic chip for testing personalized immunotherapeutics for head and neck cancer patients. Experimental Cell Research, 383(2), 111508.

[0472] 2-Aung, A., Kumar, V., Theprungsirikul, J., Davey, S. K., & Varghese, S. (2020). An engineered tumor-on-a-chip device with breast cancer-immune cell interactions for assessing T-cell recruitment. Cancer research, 80(2), 263-275.

[0473] 3-Ayuso, J. M., Rehman, S., Virumbrales-Munoz, M., McMinn, P. H., Geiger, P., Fitzgerald, C.,... & Beebe, D. J. (2021). Microfluidic tumor-on-a-chip model to evaluate the role of tumor environmental stress on NK cell exhaustion. Science Advances, 7(8), eabc2331.

[0474] 4-Ayuso, J. M., Truttschel, R., Gong, M. M., Humayun, M., Virumbrales-Munoz, M., Vitek, R.,... & Skala, M. C. (2019). Evaluating natural killer cell cytotoxicity against solid tumors using a microfluidic model. Oncoimmunology, 8(3), 1553477.

[0475] 5-Cattaneo, C. M., Dijkstra, K. K., Fanchi, L. F., Kelderman, S., Kaing, S., van Rooij, N.,... & Voest, E. E. (2020). Tumor organoid-T-cell coculture systems. Nature protocols, 15(1), 15-39.

[0476] 6- de Haan, L., Suijker, J., van Roey, R., Berges, N., Petrova, E., Queiroz, K.,... & van den Broek, L. J. (2021). A microfluidic 3D endothelium-on-a-chip model to study transendothelial migration of T cells in health and disease. International Journal of Molecular Sciences, 22(15), 8234.

[0477] 7- Nguyen, M., De Ninno, A., Mencattini, A., Mermet-Meillon, F., Fornabaio, G., Evans, S. S.,... & Parrini, M. C. (2018). Dissecting effects of anti-cancer drugs and cancer-associated fibroblasts by on-chip reconstitution of immunocompetent tumor microenvironments. Cell reports, 25(13), 3884-3893.

[0478] 8- Rosenberg, S. A., & Restifo, N. P. (2015). Adoptive cell transfer as personalized immunotherapy for human cancer. Science, 348(6230), 62-68.

[0479] 9-Simoni, Y., Becht, E., Fehlings, M., Loh, C. Y., Koo, S. L., Teng, K. W. W.,... & Newell, E. W. (2018). Bystander CD8+ T cells are abundant and phenotypically distinct in human tumour infiltrates. Nature, 557(7706), 575-579.

[0480] 10-Schuth, S., Le Blanc, S., Krieger, T.G. et al. Patient-specific modeling of stroma-mediated chemoresistance of pancreatic cancer using a three-dimensional organoid-fibroblast co-culture system. J Exp Clin Cancer Res 41, 312 (2022).

[0481] 11-Maulana et al. Immunocompetent cancer-on-chip models to assess immuno-oncology therapy, Advanced Drug Delivery Reviews, 173: 281-305 (2021).

[0482] 12-Gopal, S., Kwon, SJ., Ku, B. et al. 3D tumor spheroid microarray for high-throughput, high-content natural killer cell-mediated cytotoxicity. Commun Biol 4, 893 (2021).

[0483] 13- Saraiva et al. Establishment of a 3D Co-culture With MDA-MB-231 Breast Cancer Cell Line and Patient-Derived Immune Cells for Application in the Development of Immunotherapies Front. Oncol., Sec. Cancer Immunity and Immunotherapy Volume 10 (2020).

[0484] 14- US Patent No. 10,472,599

Claims

1. An in vitro method for establishing tumor organoids from cancer tissue samples obtained from a subject, the method comprising the following steps: (a) Dissociating the cancer tissue sample into cells and fragments; (b) The first portion of the cells and / or fragments obtained in step (a) during the first 3D culture step; (c) In the 2D culture step, a second portion of the cells and / or debris obtained in step (a) is proliferated, such as to enrich epithelial tumor cells and optional fibroblasts. (d) Combine the proliferating cells and debris obtained in step (b) with the proliferating cells and debris obtained in step (c), and proliferate the combined cells and debris in a second 3D culture step; Thus, tumor organoids are created from the cancer tissue samples.

2. The method as described in claim 1, in, The cancer tissue sample is a cancer tissue sample from a malignant tumor, specifically, the cancer is selected from lung cancer, colon cancer, rectal cancer, prostate cancer, breast cancer, bladder cancer, thyroid cancer, kidney cancer, renal cancer, epithelial cancer, or ovarian cancer; and / or The cancer tissue sample mentioned above is a cancer tissue sample from adenocarcinoma, epidermoid carcinoma, and / or colorectal cancer.

3. The method as claimed in any of the preceding claims, wherein, The cancer tissue sample is a cancer tissue sample from a cancer containing at least one mutation in a gene associated with tumor drug resistance. Specifically, the at least one mutation is a mutation in a gene selected from KRAS, EGFR, KEAP1, ALK, STK11, MET, TP53, ROS1, RB1, NOTCH1, NOTCH2, BRAF, NTRFK1 / 2 / 3, RET, ERBB2, HRAS, NRAS, ASCL1, NF1, PI3CA, FGFR1 / 2, and MAP2K1, specifically a mutation in a gene selected from KRAS, EGFR, KEAP1, ALK, and STK11.

4. The method as described in any of the preceding claims, in, Step (b) includes proliferation in a culture medium containing at least two or more of the following, preferably all of them: (i) WNT / β-catenin signaling pathway inducers, such as R-reactive protein and / or WNT, preferably R-reactive protein 1; (ii) FGF, preferably FGF-7 and / or FGF-10, more preferably FGF-7 and FGF-10; (iii) Bone morphogenetic protein (BMP) antagonist; preferably, the BMP antagonist is head protein; (iv) ALK5 inhibitor; preferably, the ALK5 inhibitor is A83-01; (v) Rho kinase inhibitor; preferably, the Rho kinase inhibitor is Y-27632; (vi) p38 MAPK inhibitor; preferably, the p38 MAPK inhibitor is SB202190; (vii) Nicotinamide; And optional B27 supplements and N-acetylcysteine; and / or Step (c) includes propagation in a culture medium containing at least two or more, preferably all of, the following: (i) FGF, preferably FGF-basic; (ii) EGF; (iii) Rho kinase inhibitor; preferably, the Rho kinase inhibitor is Y-27632.

5. The method as claimed in any of the preceding claims, wherein, Step (c) also includes collecting and proliferating cancer-associated fibroblasts, preferably in the same culture medium used in step (c).

6. The method as described in any of the preceding claims, in, Step (d) is performed in an extracellular matrix gel containing extracellular matrix proteins, such as laminin and collagen, and / or Step (d) includes propagation in a culture medium containing at least two or more, preferably all of, the following: (i) WNT / β-catenin signaling pathway inducers, such as R-reactive protein and / or WNT, preferably R-reactive protein 1; (ii) FGF, preferably FGF-7 and / or FGF-10, more preferably FGF-7 and FGF-10; (iii) Bone morphogenetic protein (BMP) antagonist; preferably, the BMP antagonist is head protein; (iv) ALK5 inhibitor; preferably, the ALK5 inhibitor is A83-01; (v) Rho kinase inhibitor; preferably, the Rho kinase inhibitor is Y-27632; (vi) p38 MAPK inhibitor; preferably, the p38 MAPK inhibitor is SB202190; (vii) Nicotinamide; And optional B27 supplements and N-acetylcysteine.

7. An in vitro method for providing autologous tumor organoid-activated immune cells, wherein the method comprises: (a) The method of any one of claims 1-6 to establish tumor organoids from cancer tissue samples obtained from a subject; as well as (b) Co-culturing the tumor organoids with immune cells obtained from the subject; This provides immune cells activated by autologous tumor organoids.

8. The method of claim 7, wherein, The immune cells mentioned are immune cells isolated from whole blood, peripheral blood mononuclear cells (PBMCs), spleen, lymph nodes, leukocyte layer, pleural effusion, bone marrow aspiration fluid, tumors, and / or immune cells derived from induced pluripotent stem cells. Preferably, the immune cells are peripheral immune cells. More preferably, the immune cells mentioned above are PBMCs.

9. The method according to any one of claims 7-8, wherein, Prior to step (b), the tumor organoids of step (a) are stimulated, such as to enhance antigen presentation, specifically by pre-culturing in a medium containing type II interferon, preferably IFN-γ.

10. The method according to any one of claims 7-9, wherein, Step (b) includes co-culturing in a culture medium containing: (i) T cell growth factor, preferably interleukin-2 (IL-2); and / or (ii) An immune checkpoint inhibitor, preferably selected from anti-PD-1 / PD-L1 antibodies and anti-CTLA4 antibodies, more preferably an anti-PD-1 / PD-L1 antibody, specifically selected from nivolumab, pembrolizumab, cimipril, dostalimumab, retivalimumab, vorapramilab, spartalizumab, camrelizumab, sintilimab, tislelizumab, torepalimumab, INCMGA00012, AMP-224, AMP-514, aclalimumab, atezolizumab, durvalumab, and avelumab, more preferably wherein the anti-PD-1 antibody is nivolumab; and / or (iii) T-cell activators that bind to CD28, preferably anti-CD28 antibodies. Alternatively, the anti-CD28 antibody may have been pre-coated onto the surface of the cell culture vessel receiving the culture medium.

11. A method for preparing an in vitro organ culture system to simulate the interaction between a tumor and a subject's immune system, the method comprising: (i) Providing a microfluidic device having a microfluidic channel having at least a first compartment and a second compartment separated by a physical boundary, the physical boundary allowing cells to move from the first compartment to the second compartment. Specifically, the physical boundary is selected from membrane, mechanical barrier, membrane-free phase guided boundary, gel-based boundary, basement membrane-based boundary, or network-based boundary. (ii) A three-dimensional tumor organoid constructed from a cancer tissue sample obtained from the subject and a suitable tumor cell growth culture medium are incorporated into the first compartment; as well as (iii) Autologous immune cells from the subject and a suitable immune cell culture medium are introduced into the second compartment, the autologous immune cells having been activated by co-culturing with the established tumor organoids; This provides the in vitro organ culture system.

12. The method of claim 11, wherein, The tumor organoids are established from cancer tissue samples obtained from the subject using the method according to any one of claims 1-6; and / or The autologous immune cells from the subject have been activated by co-culturing with the established tumor organoids according to any one of claims 7-10.

13. The method as described in any one of claims 11-12, in, Step (ii) further includes incorporating cancer-associated fibroblasts (CAFs) obtained from the subject; and / or In step (ii), autologous or non-autologous endothelial cells are incorporated into the tumor organoid, wherein the autologous endothelial cells are derived from the tumor, artery or vein of the matched patient, and wherein the non-autologous endothelial cells are preferably human umbilical vein endothelial cells (HUVEC), human pulmonary microvascular endothelial cells (HLMVEC), human pulmonary microvascular endothelial cells (HPMEC), or human intestinal microvascular cells (HIMEC).

14. An in vitro organ culture system for simulating the interaction between a tumor and a subject's immune system, comprising: (i) A microfluidic device having a microfluidic channel having at least a first compartment and a second compartment separated by a physical boundary that allows cells to move from the first compartment to the second compartment, wherein the physical boundary is selected from a membrane, a mechanical barrier, a membrane-free phase-guided boundary, a gel-based boundary, a basement membrane-based boundary, or a network-based boundary; (ii) a three-dimensional tumor organoid constructed from a cancer tissue sample obtained from the subject and a suitable tumor cell growth culture medium in the first compartment; and (iii) Autologous immune cells from the subject and a suitable culture medium for the immune cells in the second compartment, the autologous immune cells having been activated by co-culturing with the established tumor organoids.

15. A method for determining the responsiveness of a tumor to treatment with at least one immuno-oncology drug, comprising the following steps: (a) The method of any one of claims 11-12 is used to prepare an in vitro organ culture system, or to provide an in vitro organ culture system according to claim 14; (b) Adding one or more, preferably at least two, immuno-oncology drugs to the second compartment; and (c) Determine the responsiveness of the in vitro organ culture system to the at least two immuno-oncology drugs; Specifically, step c) includes determining tumor cell growth and / or determining the viability of the tumor organoid cells in the first compartment, wherein reduced tumor cell growth and / or decreased viability indicate that the tumor has responded effectively to treatment with one or more of the immuno-oncology drugs.

16. The method as described in any one of claims 15, in, The method is repeated using multiple identical in vitro organ culture systems for different immuno-oncology drugs or combinations of different immuno-oncology drugs, thereby identifying personalized and effective immuno-oncology therapies for patients; or In this method, for different treatment regimens of immuno-oncology drugs or drug combinations, the same in vitro organ culture system is used to repeatedly perform the above method to identify effective treatment regimens; or Specifically, the method is repeated using multiple different in vitro organ culture systems for the same immuno-oncology drug or the same combination of immuno-oncology drugs, thereby identifying patient candidates who may benefit from treatment with the immuno-oncology drug or the combination of immuno-oncology drugs.

17. The method of any one of claims 15-16, further comprising the step of: The tumor organoids and / or culture medium in the first compartment that responded to treatment were compared with those in the first compartment that did not respond to treatment, thereby identifying biomarkers of response or non-response to treatment. Specifically, the comparison of phenotypic changes includes a comparison of cytokine levels in the culture medium between responsive and non-responsive treatments. Specifically, the biomarker indicates (i) a personalized, effective or ineffective immuno-oncology therapy for the patient, (ii) an effective or ineffective treatment regimen, and / or (iii) a patient candidate who may or may not benefit from treatment with the immuno-oncology drug or a combination of the immuno-oncology drugs.

18. A kit for preparing an in vitro organ culture system, said in vitro organ culture system simulating the interaction between a tumor and a subject's immune system, said kit comprising at least two of the following, preferably at least three, more preferably at least four, even more preferably at least five, and most preferably all of the following: (a) A microfluidic device having a microfluidic channel having at least a first compartment and a second compartment separated by a physical boundary that allows cells to move from the first compartment to the second compartment, wherein the physical boundary is selected from a membrane, a mechanical barrier, a membrane-free phase-guided boundary, a gel-based boundary, a basement membrane-based boundary, or a network-based boundary; (b) A tumor cell growth medium comprising at least two of the following, preferably all of them: (i) WNT / β-catenin signaling pathway inducers, such as R-reactive protein and / or WNT, preferably R-reactive protein 1; (ii) FGF, preferably FGF-7 and / or FGF-10, more preferably FGF-7 and FGF-10; (iii) Bone morphogenetic protein (BMP) antagonist; preferably, the BMP antagonist is head protein; (iv) ALK5 inhibitor; preferably, the ALK5 inhibitor is A83-01; (v) Rho kinase inhibitor; preferably, the Rho kinase inhibitor is Y-27632; (vi) p38 MAPK inhibitor; preferably, the p38 MAPK inhibitor is SB202190; (c) An immune cell culture medium, a culture medium containing... (i) T cell growth factor, preferably interleukin-2 (IL-2); (ii) Immune checkpoint inhibitors, preferably selected from anti-PD-1 / PD-L1 antibodies and anti-CTLA4 antibodies, more preferably anti-PD-1 / PD-L1 antibodies, specifically selected from nivolumab, pembrolizumab, simiprelimab, dostalimab, retivalimab, vorapram, spartalizumab, camrelizumab, sintilimab, tislelizumab, torepalimab, INCMGA00012, AMP-224, AMP-514, aclalimab, atezolizumab, durvalumab, and avelumab, more preferably wherein the anti-PD-1 antibody is nivolumab; (iii) Nicotinamide; And optional B27 supplement and N-acetylcysteine, preferably about 1.25 mM N-acetylcysteine; (d) Cell culture dishes or cell culture containers pre-coated with anti-CD28 antibody; (e) Containers with IFN-γ; and / or (f) A container having an extracellular matrix gel containing extracellular matrix proteins such as laminin and collagen.

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