Culture medium for pancreatic cancer cells

Abstract

The present invention relates to a culture medium comprising low glucose DMEM, L- glutamine, foetal bovine serum (FBS), antimicrobials and a trypsin inhibitor; the use of said culture medium for culturing mammalian cells and / or maintaining the viability of live tissue or tumour slices, optionally in a perfusion model; a method for 5 identifying a treatment for cancer utilising said culture medium; and a method for perfusion cell culture utilising said culture medium.

Description

[0001] CULTURE MEDIUM FOR PANCREATIC CANCER CELLS

[0002] Field of the Invention

[0003] The invention relates to a culture media composition and culture methods for culturing tumour slices in vitro, and also to the use of the media in live tumour slice imaging and in methods for identifying a treatment for cancer.

[0004] Background to the Invention

[0005] Pancreatic ductal adenocarcinoma (PDAC) is the most common form of pancreatic cancer and remains one of the most fatal gastrointestinal tract malignancies. Overall prognosis is poor primarily due to the prevalence of late-stage diagnosis, with 50% of patients presenting with metastatic disease at the time of diagnosis (Adamska et al., 2017). Even among those with localised disease at the time of diagnosis, survival rates are still low at 42% (Society, 2022), dropping to 9% overall (Siegel et al., 2021). The standard of care for resectable PDAC arising in the head of the pancreas (70% of PDAC cases) involves pancreaticoduodenectomy followed by systemic chemotherapy. The chemotherapeutic regime is dictated by the patient’s overall performance status, whereby patients with a good functional status are commenced on multiagent chemotherapy (FOLFIRINOX), combination gemcitabine and capecitabine or, if unfit, monotherapy with gemcitabine (Regel et al., 2020). While neoadjuvant therapy is currently utilised in the context of locally advanced pancreatic cancer (LAPC), its role in upfront resectable disease has yet to be determined.

[0006] Disease occurs through inflammatory signalling leading to acinar cell remodelling and acinar to ductal metaplasia (ADM). During this increased plasticity, the cells are increasingly susceptible to mutation and form precursor lesions known as pancreatic intraepithelial neoplasms (PanINs) (Orth et al., 2019). While PDAC is considered a relatively immunologically ‘cold’ tumour type, the immune microenvironment is dynamic during lesion development - moving from an initial pro-inflammatory phenotype to becoming increasingly immunosuppressive (Bhatia et al., 2022). Along with the shift towards immune suppression, PDAC lesions build up a characteristic desmoplastic stroma through the secretion of collagen by activated cancer- associated fibroblasts (CAFs) (Rebelo et al., 2023). The fibrous network is formed via tumour signalling through IL-6 and TGF-B, forming a physical barrier thought to hinder the distribution of chemotherapies and prevent immune infiltration (Hartmann et al., 2014).

[0007] Currently, most in vitro therapeutic testing is carried out using patient-derived cell lines, xenografts (PDX) and genetically engineered mouse models (GEM Ms). While murine models provide valuable information about the systemic effects of therapeutics, GEMMs fail to capture the genomic heterogeneity of native tumours and PDX models are challenging to establish orthotopically and do not allow study of the interaction of a tumour with a functional immune system (Kim et al., 2009). Introduction of patient-derived organoids has allowed in vitro analysis of treatment interactions with three-dimensional tumour structures (Ooft et al., 2019) but, similarly to xenograft models, does not capture the complexity of the TME (Larsen et al., 2021). Recent efforts move in the direction of incorporating multiple different cell types and vascular mimics to reconstruct tumour complexities (Neal et al., 2018). Patient-derived tumour slices provide a platform through which tumour, stroma and immune infiltrate can be studied in their native architecture (Ghaderi et al., 2020;

[0008] Kokkinos et al., 2021). Through this system, therapeutics can be investigated for their impact throughout the tumour, allowing analysis of intra-patient variation in a clinically relevant timeframe.

[0009] We have investigated the use of live patient-derived tumour slices for dissection of the PDAC microenvironment and investigation of therapy response. We have established the use of perfusion culture, which maintains superior cellular fitness and preservation of tumour microenvironment compared to standard static culture. Our analysis of transcriptomic changes induced by a novel treatment combination shows the potential of the platform to interrogate treatment responses across cellular compartments, while use of gemcitabine treatment demonstrates the utility of the platform in clinical settings. We demonstrate that organotypic tumour slices can maintain viability and provide novel insights enhancing both novel therapeutic discovery and precision medicine to improve current standard of care.

[0010] Summary of the Invention

[0011] In accordance with a first aspect of the invention there is provided a culture medium comprising low glucose DMEM, L-glutamine, foetal bovine serum (FBS), antimicrobials and a trypsin inhibitor. In a second aspect, the invention relates to the use of the culture medium of the invention for culturing mammalian cells.

[0012] In a third aspect, the present invention relates to the use of the culture medium of the invention for maintaining the viability of live tissue and / or live tumour slices, optionally in a perfusion model.

[0013] In a fourth aspect, the invention relates to the use of the culture medium of the invention for tissue imaging and / or live tumour slice imaging.

[0014] In a fifth aspect, the invention provides a method for identifying a treatment for cancer comprising the steps of: i) providing a cancer tumour slice to an apparatus, ii) providing the culture medium of the invention to the tumour slice, iii) adding a treatment agent, iv) receiving a visual image of the tumour slice, v) analysing the data and vi) identifying the optimal treatment regimen based on the data for the cancer.

[0015] In a sixth aspect, the invention provides a method for perfusion cell culture comprising the step of passing the culture medium according to the invention over a cancer tissue culture in an apparatus.

[0016] Brief Description of the Drawings

[0017] Exemplary arrangements of the disclosure shall now be described with reference to the drawings in which:

[0018] Figure 1

[0019] (A) 5x magnification and (B) 10x magnification. Shows the histology of a pancreatic tumour slice cultured with antimicrobials and soybean trypsin inhibitor to prevent degradation.

[0020] Figure 2

[0021] (A) The right-hand top image (10x magnification, scale bar 50 urn) demonstrates the detrimental effect of contamination on the tumour slice i.e. acellular region, necrotic with no preservation of native tissue architecture; the right-hand bottom image (10x magnification, scale bar 50 urn) is representative of an area of the slice where PDAC histology is preserved. (B) This graph shows quantification of the extent of necrosis (as a percentage of the total surface area) of the tumour slice at 24 hours ex vivo. Data presented as mean ± SEM, statistical analysis performed by unpaired Student T test. Data information: *** P < 0.001.

[0022] Figure 3

[0023] (A) Schematic demonstrating the workflow to establish pancreatic tumour avatar maintenance.

[0024] (B) We assessed the viability of tumour tissue maintained over a 12-day period by determining a proliferation index for the tumour cells using Ki67 staining.

[0025] (C) Baseline values which were obtained for slices from different parts of the same tissue were shown to give similar proliferation indices.

[0026] (D) Spatial transcriptomics of avatar slices. Images show (far left) staining used to gate the samples and (left) improved maintenance of structure over time course in perfusion culture. Heatmaps show the clustering of gene expression data with time (left) and boxplot showing the spread of data (right).

[0027] Figure 4

[0028] (A) Schematic demonstrating the heterogeneity and cell populations present in a pancreatic tumour.

[0029] (B) Representative images of Masson’s Trichrome staining for collagen deposition within the extracellular matrix of 3 independent PDAC avatars at baseline and following 12 days of ex vivo culture, (left) Images taken at 5x magnification, scale bar 100 pm, (right) quantification of percentage positive area (blue stain) per high power field. Dashed horizontal line represents baseline value (58.6%) (n=3) error bars ± S.E.M..

[0030] (C) Immunohistochemical images of tumour avatar after culture in perfusion conditions, stained for a-SMA (left) and quantification (right) from randomly generated sections, (n = 3).

[0031] (D) Heatmaps showing stromal gene expression in perfused (top) and static (bottom) conditions.

[0032] (E) Multiplex immunofluorescence was used to analyse the effect of culture on the immune cells in avatars of 3 patients (PT10, PT12 and PT13) using a panel which detects CD4+ T cells, CD8+ T cells, regulatory T cells, macrophages and B cells. No significant difference is seen for any of the populations in the perfused cultures throughout the 12-day time course. Figure 5

[0033] Summary of immune infiltrate cell numbers and proximity measurements for PDCA10, 12 and 13.

[0034] Figure 6

[0035] (A)-(F) Proximity of immune cells to tumour cells or proximity of immune cells to other immune cells in patients PDCA10, 12 and 13. Nearest neighbour analysis was performed on cells that were within 50 mm of tumour cells. Box and whisker plots display the median, quartiles and range of the data. Significance is inferred from a Cohen d statistic for the Welch test of > 0.8.

[0036] (G) Immune cells residing within delineated tumour cell areas as a fraction of the total population of that cell type within the avatar.

[0037] (H) Clinical outcome for patients PDCA10.12 and 13.

[0038] Figure 7

[0039] (A) Schematic depicting the spatial transcriptomic analysis of avatars. For each condition 2 sections were analysed using > 6 regions.

[0040] (B) GSVA analysis on tumour component using 3 published gene sets which define the classical and squamous-like phenotypes, with two-way ANOVA multiple comparisons for the tumour types and one-way ANOVA for the stroma, and GSVA analysis on stromal component of avatar using the published gene set for the classical and activated phenotypes.

[0041] (C) We observed a significant increase in the numbers of CD8 T cells after treatment with metformin and ascorbic acid. The increase in the numbers of CD8 T cells after treatment with metformin and ascorbic acid was not seen for any other immune cell population.

[0042] (D) Schematic depicting the effect of treatment on each avatar component.

[0043] Figure 8

[0044] (A) (left) immunohistochemical staining of PT14 avatar after 6 days of gemcitabine treatment: top, Ki67; centre, cleaved caspase-3; bottom, g-H2AX activation. Images taken at 10x magnification, scale bar 50 pm. Quantification (right) using randomly generated sections, n = 3 slides, Data shown is mean ± S.E.M.

[0045] (B) Quantification of GATA6 and KRT81 gene expression in baseline samples of tissue from patients PDCA14, PDCA16 and PDCA17 using qPCR. (C) Schematic of PDCA14’s clinical journey from diagnosis showing tumour marker, CA19-9, trends over time presented as a line graph and times at which treatment was given along with cross-sectional imaging of pre- and post-gemcitabine treatment.

[0046] (D) The patient PT16 responded well to neoadjuvant FOLFIRINOX treatment and did not receive any further chemotherapy during the follow up period. Patient PT17 also did not receive adjuvant therapy. In both cases, on the basis of our results, gemcitabine would not have been expected to be effective.

[0047] Specific Description of the Invention

[0048] Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of them mean “including but not limited to”, and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps. Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

[0049] Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and / or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive. The invention is not restricted to the details of any foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

[0050] For the avoidance of doubt, the information disclosed earlier in this specification under the heading “Background to the Invention” is relevant to the invention and is to be read as part of the disclosure of the invention.

[0051] Culture medium In a first aspect of the invention, there is provided a culture medium comprising low glucose DMEM, L-glutamine, foetal bovine serum (FBS), antimicrobials and a trypsin inhibitor.

[0052] In some embodiments, the culture medium comprises low glucose DMEM which contains between 20-100 mg / dl, 20-200 mg / dl or 20-300 mg / dl glucose. In particular, in some embodiments the low glucose DMEM contains between 50-200 mg / dl glucose. Preferably, in some embodiments the low glucose DMEM contains 100 mg / dl glucose.

[0053] Glucose concentrations in cell culture media range far above those in human blood even in diabetic patients. The culture medium of the invention comprises low glucose DMEM to try to reduce this artificial supplementation which could impact cellular metabolism. In some embodiments the low glucose DMEM contains 100 mg / dl glucose. The normal blood glucose concentration range for an adult is 80-180mg / dL. Even in diabetes (around 68% of PDAC patients are diabetic), sugar levels are seldom above 300mg / dL at their peak. However, standard high glucose DMEM has 450mg / dL glucose, providing nutrients for cell growth but failing to represent bodily conditions.

[0054] In some embodiments, the culture medium comprises antimicrobials selected from the group comprising of amphotericin B, penicillin, streptomycin and neomycin. In some embodiments, the antimicrobials are selected from the group comprising of penicillin and streptomycin. In certain embodiments, the culture medium comprises penicillin. In certain embodiments, the concentration of penicillin is between 10 - 100 ll / rnl, preferably wherein the concentration is 50 ll / rnl. In certain embodiments, the culture medium comprises streptomycin. In certain embodiments, the concentration of streptomycin is between 0.01 - 1.0 mg / ml, preferably wherein the concentration is 0.05 mg / ml. In certain embodiments, the culture medium comprises neomycin. In certain embodiments, the concentration of neomycin is between 0.01 - 1.0 mg / ml, preferably wherein the concentration is 0.1 mg / ml. In certain embodiments, the culture medium comprises amphotericin B. In certain embodiments, the concentration of amphotericin B is between 1.0 - 5.0 mg / ml, preferably wherein the concentration is 2.5 mg / ml.

[0055] Broad spectrum antibiotics, such as penicillin, streptomycin and neomycin were used to prevent infections. The antifungal agent amphotericin B was particularly added to prevent infections seen in patients who had undergone biliary stenting prior to surgical resection.

[0056] In some embodiments, the concentration of FBS is between 0.1 - 1.0 mg / ml, preferably wherein the concentration is between 0.15 - 0.225 mg / ml.

[0057] In the present invention, the culture medium comprises a reduced concentration of FBS, because, while the growth factors are necessary for viability maintenance, batch variability and lack of fidelity to human serum remains an issue in usage. We carried out testing to ensure that a reduction in FBS did not reduce the viability of the fibroblast population of the tumour slices.

[0058] In some embodiments the concentration of trypsin inhibitor is between 0.1 - 1.0 mg / ml, preferably wherein the concentration is 0.2 mg / ml. Further, in some embodiments, the trypsin inhibitor is soybean trypsin inhibitor.

[0059] A trypsin inhibitor is a beneficial component in the culture medium of the invention. It is specifically beneficial in pancreatic media due to the high prevalence of proteolytic enzymes in pancreatic tissue. Culturing tissue in media lacking this supplement led to autodigestion 24-48hrs into ex vivo culture. This was observed through loss of surface area and development of necrotic regions visualised by H&E staining (Figures 1 and 2).

[0060] Previous formulations of the culture medium contained dexamethasone, which is a corticosteroid added to reduce the presence of inflammatory mediators and therefore support tissue viability. This was removed from the culture medium to prevent interference with local immune signalling and to prevent inactivation of immune therapies during testing.

[0061] In some embodiments, the culture medium comprises low glucose DMEM, FBS, soybean trypsin inhibitor, amphotericin B, penicillin, streptomycin, neomycin and L- glutamine. In some embodiments, the culture medium comprises low glucose DMEM, 0.15 - 0.225 mg / ml of FBS, 0.2 mg / ml soybean trypsin inhibitor, 2.5 mg / ml amphotericin B, 50 ll / rnl penicillin, 0.05 mg / ml streptomycin, 0.1 mg / ml neomycin and 0.3 mg / ml L-glutamine. In certain embodiments, the low glucose DMEM contains 100mg / dl glucose. Other components of the culture medium

[0062] The culture medium may also further contain at least one type of amino acid. Examples of amino acids contained in the culture medium may include L-alanine, L- arginine, L-asparagine, L-aspartic acid, L-cysteine, L-cystine, L-glutamic acid, L- glutamine, L-glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L- methionine, L- phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine and combinations thereof.

[0063] L-glutamine is a standard supplement required for tissue maintenance. Glutamine is an essential amino acid for protein and nucleic acid synthesis and for energy production, therefore including it in the culture medium increases the function of many cell types.

[0064] The culture medium may further contain at least one type of vitamin. Examples of vitamins contained in the culture medium may include thiamine (vitamin B1), riboflavin (vitamin B2), niacin (vitamin B3), calcium D-pantothenate (vitamin B5), pyridoxal / pyridoxamine / pyridoxine (vitamin B6), folic acid (vitamin B9), cyanocobalamin (vitamin B12), ascorbic acid (vitamin C), calciferol (vitamin D2), DL- a-tocopherol (vitamin E), biotin (vitamin H) and menadione (vitamin K).

[0065] The culture medium may further also contain at least one type of inorganic salt. The inorganic salt contained in the culture medium is for assisting in maintaining the osmotic equilibrium of cells as well as assisting in regulating the membrane potential thereof. Specific examples of inorganic salts include salts of calcium, copper, lead, magnesium, potassium, sodium and zinc. Salts are normally used in the form of chlorides, phosphates, sulfates, nitrates and carbonates.

[0066] The culture medium may further also contain at least one type of sugar capable of serving as a carbon energy source. Examples of sugars contained in the culture medium may include glucose, galactose, maltose and fructose. Among these, glucose is preferable and D-glucose (dextrose) is particularly preferable for the sugar. The concentration of sugar contained in the culture medium is preferably 1 g / L to 10 g / L.

[0067] The culture medium may also further contain at least one type of trace element.

[0068] Examples of trace elements contained in the culture medium may include barium, bromium, cobalt, iodine, manganese, chromium, copper, nickel, selenium, vanadium, titanium, germanium, molybdenum, silicon, iron, fluorine, silver, rubidium, tin, zirconium, cadmium, zinc, aluminium and ions thereof.

[0069] The culture medium may also further contain at least one type of supplementary chemical agent. Examples of such supplementary chemical agents include nutrients or growth factors that have been reported to improve tissue culturing, such as cholesterol, transferrin, albumin, insulin, progesterone, putrescine, selenite or other factors.

[0070] Preparation of the culture medium

[0071] The starting material for the culture medium are obtained and are used to prepare the culture medium as follows.

[0072] - Dehydrated media - dissolved in distilled water using aseptic techniques, pH adjusted, and sterilized prior to use; or

[0073] - Liquid concentrates are usually diluted in distilled water using aseptic techniques, adjusted for pH and stored in sterile containers for further use, or

[0074] - Culture media prepared as working solutions are obtained ready to use, following the addition of additions serum or supplements (e.g. antimicrobials) under sterile filtration, and aseptic conditions, and then pre-warmed to 37°C prior to addition to cells, tissues or tissue / tumour slices.

[0075] Culture media is usually sterilized via filtration, boiling or autoclaving. Ready to use, working media solutions are usually sold as sterile, although sterile filtration and aseptic techniques are recommended when handling. In all cases, aseptic techniques are used to avoid cross contamination, and growth of unwanted microorganisms.

[0076] Serum can be added to cell culture media to facilitate the growth of certain cell lines. Fetal bovine serum (FBS) is among the most common serum added to cell cultures. Hormones and other growth factors can also be added to help the growth of specific types of cells. Additionally, additives such as IPTG can be added to encourage expression of proteins. Furthermore, antimicrobials are routinely added to inhibit the growth of bacteria and fungi. Once prepared cell culture media, in liquid form or as agar are stored at 4°C in the dark, to avoid growth of opportunistic organisms and light degradation of media components. When in use, the media is pre-warmed to 37°C prior to addition of cells, tissues or tissue / tumour slices.

[0077] Storage of the culture medium

[0078] The culture medium of the invention may be stable indefinitely when frozen at -20°C or lyophilised.

[0079] Tumour slices

[0080] The tumour slices can be derived from patient-derived tumour tissues. For example, the collection of tissue samples can be carried out within half an hour after surgical resection or biopsy from the patient. More specifically, in a sterile environment, the tissue sample from non-necrotic sites is cut and then is placed in a pre-cooled medium and transported to a laboratory on ice. The culture medium of the present invention can be used for the storage and / or transport of cells, tissues and / or tumour slices.

[0081] Culture of tumour slices

[0082] The methods of the present invention may be carried out using any culture device suitable for the culture of human tissue, tissue slices or tumour slices. The skilled person will be aware of suitable culture devices.

[0083] A culture vessel used can include, but is particularly not limited to: flask, flask for tissue culture, dish, petri dish, dish for tissue culture, multi dish, micro plate, microwell plate, multi plate, multi-well plate, micro slide, chamber slide, schale, tube, tray, culture bag, and roller bottle, as long as it is capable of culturing the tissue and / or tumour slice therein. The culture vessel can be cellular adhesive or non-adhesive and selected depending on the purpose. The cellular adhesive culture vessel can be coated with any of substrates for cell adhesion such as extracellular matrix (ECM) to improve the adhesiveness of the vessel surface to the cells or tissues.

[0084] Other culturing conditions can be appropriately defined. For example, the culturing temperature can be about 30 to 40°C and preferably about 37°C but particularly not limited to them. The CO2 concentration can be about 1 to 10% and preferably about 2 to 5%. The oxygen tension can be 1-10%.

[0085] Uses of the culture medium

[0086] In a further aspect, the invention relates to the use of the culture medium of the invention for culturing mammalian cells. In some embodiments, the use relates to culturing mammalian cells in a perfusion culture.

[0087] In another aspect, the invention relates to the use of the culture medium of the invention for maintaining the viability of live tissue slices. In some embodiments, the use relates to maintaining the viability of live tissue slices in a perfusion model. In some embodiments, the use relates to maintaining the viability of live tumour slices. In some embodiments, the use relates to maintaining the viability of live tumour slices in a perfusion model. In some embodiments, the tumour slices are pancreatic tumour slices. In some embodiments, the viability of tumour slices is maintained for at least 5 days. In some embodiments, the viability of tumour slices is maintained for at least 10 days. In some embodiments, the viability of tumour slices is maintained for at least 12 days.

[0088] A further aspect of the invention relates to the use of the culture medium of the invention for tissue imaging. In some embodiments, the use relates to live tumour slice imaging.

[0089] In one embodiment, the tumour slices, cultured in the culture medium of the invention, may be used for drug response screening, toxicity assays or regenerative medicine.

[0090] In the case drug response screening, the tissue or tumour slice is cultured in, for example, a multi-well plate such as a 96-well plate or 384-well plate. Molecules are then identified that have an effect on the tumour slice using a molecule library. Examples of molecule libraries include an antibody fragment library, peptide phage display library, peptide library, lipid library, synthetic compound library or natural compound library. Moreover, a gene library may also be used. Examples of gene libraries include a cDNA library, antisense library, and siRNA or other non-coding RNA library. An example of a specific method consists of exposing cells of the tumour slice to multiple concentrations of a test chemical agent over a certain period of time followed by evaluating the culture at completion of exposure.

[0091] Methods for identifying a treatment for cancer

[0092] Another aspect of the invention provides a method for identifying a treatment for cancer comprising the steps of: i) providing a cancer tumour slice to an apparatus, ii) providing the culture medium of the invention to the tumour slice, iii) adding a treatment agent, iv) receiving a visual image of the tumour slice, v) analysing the data and vi) identifying the optimal treatment regimen based on the data for the cancer. In some embodiments, the analysis is performed by a human viewing the displayed image.

[0093] In some embodiments, the cancer is pancreatic cancer. In some embodiments, the cancer is pancreatic ductal adenocarcinoma (PDAC). In other embodiments, the cancer is pancreatic neuroendocrine tumour (PNET).

[0094] In some embodiments, the cancer is a human cancer. In other embodiments, the cancer is a murine cancer.

[0095] In another aspect, the invention provides a method for perfusion cell culture comprising the step of passing the culture medium of the invention over a cancer tissue culture in an apparatus.

[0096] The invention contemplates combinations of any of the foregoing aspects and embodiments of the invention. Each and every embodiment described throughout the application can be combined, and can be applied to each and every aspect of the invention described herein. The present invention is further illustrated by the figures and examples from which further features, embodiments and advantages may be taken.

[0097] Kits

[0098] The present invention can also be a kit. The kits of the present invention can comprise the components of the culture medium separately (i.e. a non-mixed manner). For example, the kit of the present invention can be provided in the form of the each component being packaged in a container individually. The other components which can be contained in the kit of the present invention include, for example, the other components mentioned above, which can be included in the composition of the present invention.

[0099] Definitions

[0100] The term ’’culture medium” refers to any solutions used for growing, storing, handling and maintaining cells, tissues and cell lines. Such solutions generally include various factors necessary for cell attachment, growth and maintenance of the cellular environment. For example, a typical solution may include a basal media formulation, various supplements depending on the cell or tissue type, and occasionally antimicrobials. Sometimes, the solution may include one or more of the following components: an energy source, usually in the form of carbohydrate e.g. glucose; essential amino acids; vitamins and / or other organic compounds required at low concentrations; free fatty acids; or trace elements such as inorganic compounds or naturally occurring elements at very low concentrations.

[0101] “Dulbecco's Modified Eagle Medium (DM EM)” is a form of cell culture medium produced by Gibco used for the long term culture of mammalian cells.

[0102] “DMEM" or "Dulbecco's Modified Eagle Medium" is a modification of Basal Medium Eagle which contains a four-fold higher concentration of amino acids and vitamins, together with additional components. “Dulbecco's Modified Eagles Medium (DMEM)” contains calcium chloride, ferric nitrate, magnesium sulfate, potassium chloride, sodium bicarbonate, sodium chloride, sodium phosphate moonbasic (anhydrous), L- arginine HCI, L-cysteine HCI, glycine, L-histidine HClTW, L-isoleucine, L-leucine, L- lysine HCI, L-methionine, L-tryptophan, L-tyrosine 2Na H2O, L-valine, choline chloride, folic acid, myo-inositol, niacinamide, D-pantothenic acid, pyridoxal- HCI, pyridoxine, riboflavin, thiamine-HCI, D-glucose, phenol red sodium, and pyruvic acid.

[0103] The term “low glucose DMEM” refers to DMEM which contains between 20-100 mg / dl, 20-200 mg / dl or 20-300 mg / dl glucose. In particular, low glucose DMEM contains 100 mg / dl glucose.

[0104] “Glucose” is a 6-carbon structure with the chemical formula CeH^Oe. It is a ubiquitous source of energy for every organism in the world and is essential to fuel both aerobic and anaerobic cellular respiration. “Foetal bovine serum (FBS)” is a supplement added to mammalian cell culture media which provides cells with nutrients and factors promoting growth. It is derived from clotted blood and both promotes growth and enhances the survival of cells.

[0105] “L-glutamine” is a standard supplement required for tissue maintenance. L-glutamine is an L-a-amino acid containing five carbons; its molecular weight is 146.15 kDa and its elemental composition comprises carbon (41.09%), hydrogen (6.90%), oxygen (32.84%), and nitrogen (19.17%). With respect to its physiological pH, L-glutamine is classified as a neutral amino acid, whereas it is nutritionally classified as a non- essential amino acid. Glutamine has two amino groups, namely the a-amino group and the easily-hydrolysable side-chain amide group, and these features enable the role played by glutamine as a nitrogen transporter and NH3 carrier. Glutamine is also a proteinogenic amino acid, i.e. , amino acids that are incorporated into proteins, and accounts for 5 to 6% of bound amino acids.

[0106] By “trypsin inhibitor” is meant a type of serine protease inhibitor that reduces the biological activity of trypsin by controlling the activation and catalytic reactions of proteins. This enzyme inhibitor is useful specifically in pancreatic media due to the high prevalence of proteolytic enzymes in pancreatic tissue. Culturing tissue in media lacking this supplement led to autodigestion 24-48hrs into ex vivo culture. This was observed through loss of surface area and development of necrotic regions visualised by H&E staining.

[0107] By “soybean trypsin inhibitor” is meant a trypsin inhibitor made from soybeans.

[0108] The term “antibiotics” refers to agents that kill bacteria. Penicillin, streptomycin and neomycin are examples of antibiotics which act against bacteria. “Antimycotics” are agents that kill funguses. Amphotericin B is an antimycotic agent. The term “antimicrobials” encompass all of these agents i.e. antibiotics and antimycotics are subsets of antimicrobials.

[0109] “Contamination” means infection with bacteria or fungus and can lead to degradation of tissue.

[0110] “mg / dl” stands for milligrams (mg) per decilitre (dL) and is a unit of measure that shows the concentration of a substance in a specific amount of fluid. “mg / ml” stands for milligrams (mg) per millilitre (dL) and is a unit of measure that shows the concentration of a substance in a specific amount of fluid.

[0111] “ll / ml” stands for units per millilitre . A unit is an amount agreed upon by scientists and doctors. A millilitre is a unit of fluid volume.

[0112] The term “culturing”, “cell culture” or “tissue culture” refers to cells grown in suspension or grown adhered to a variety of surfaces or substrates in vessels.

[0113] “Mammalian cells” are cells that are derived or isolated from the tissue of a mammal. “Mammalian cell culture” is the technique of growing animal cells outside of their original tissue but in a growth medium instead.

[0114] The term "tissue" is used to refer to an aggregation of similarly specialized cells united in the performance of a particular function. Tissue is intended to encompass all types of biological tissue including both hard and soft tissue. A "tissue" is a collection or aggregation of particular cells embedded within its natural matrix, wherein the natural matrix is produced by the particular living cells. The term may also refer to ex vivo aggregations of similarly specialized cells which are expanded in vitro such as in artificial organs.

[0115] The term “(live) tissue slice” refers to three-dimensional (3D) sections of tissue cut from larger pieces of tissue or extracted organs that can be cultured ex vivo for an extended period of time.

[0116] The term “(live) tumour slice” (referred to also as “organotypic tumour slices”, “ex vivo tumour slices”, “cancer tumour slice”, “tumour avatars” and “patient derived explants”) refers to sections of tumour tissue derived from patients. The sections can be taken following surgery or biopsy.

[0117] The term “pancreatic tumour slices” refers to sections of tumour tissue derived from pancreatic tumours.

[0118] By “tissue imaging” is meant the study of living tissues and cells using time lapse microscopy. This allows the study of cell movement and function while cells are still alive. “(Live) tumour slice imaging” refers to the study of living tumour tissues and cells in tumour slices using time lapse microscopy.

[0119] By “perfusion culture” or a “perfusion model” means a form of cell culture utilising a pump system to provide a continual flow of media. This is differentiated from ‘static’ culture by the constant supply and removal of fluid.

[0120] In “static culture”, the culture medium is supplied in a batch-wise manner, and is replaced by fresh medium manually in regular intervals.

[0121] The term “tissue culture chamber” refers to a device in which tissues or cells are grown or preserved using an artificial medium outside the patent organism.

[0122] The term “cancer tissue culture” refers to the process by which cancer cells are grown under controlled conditions, generally outside of their natural environment.

[0123] “Pancreatic ductal adenocarcinoma (PDAC)” is the most common form of pancreatic cancer.

[0124] The term "therapeutic agent" as used herein refers to any of a variety of agents that exhibit one or more beneficial therapeutic effects when used in conjunction with methods, matrices and / or skin tissues of the present disclosure. Examples of therapeutic agents that may be used include, without limitation, proteins, peptides, drugs, cytokines, extracellular matrix molecules, and / or growth factors. One of skill in the art will be aware of other suitable and / or advantageous therapeutic agents that may be used in accordance with the present disclosure.

[0125] “A treatment for cancer” (also referred to as a “treatment agent” or an “optimal treatment regimen”) refers to medications or substances intended for medical treatment, in particular the treatment of cancer.

[0126] The term “apparatus” refers to a structure, device or receptacle in which tissue culture can take place e.g. a perfusion culture apparatus.

[0127] By “immunotherapy” is meant a form of treatment which uses stimulation of the immune response to combat disease. “Cell viability” or “viability” refers to relative amounts of living and dead cells, present within a population of cells at any given time. Cell viability may be determined by measuring the relative numbers of living and dead cells in any given sample of the population. Cell viability may also be estimated by measuring the rate of cell proliferation of the entire population which represents the overall balance of the rates of cell growth and cell death. Rates of cell growth may also be directly measured, by counting the number of cells and by using any number of commercially available cell proliferation assays which directly score the rate of cell growth.

[0128] “A visual image of the tumour slice” refers to an image of the cancer tissue or tumour slice observed with the naked eye, or with a microscope. This image may be captured i.e. in a micrograph or photomicrograph (which is a photograph or digital image taken through a microscope or similar device to show a magnified image of an object).

[0129] “Analysing data” is the practice of inspecting and working with data to glean useful information, which can then be used to make informed decisions. Data analysis of the data obtained from the visual image of a cancer tumour slice can be done by a human viewing the displayed image, by histology assessments, immunohistochemistry and / or immunofluorescence or using a software.

[0130] Examples

[0131] Reference is now made to the following examples, which together with the above description illustrate some embodiments of the invention in a non-limiting fashion.

[0132] Example 1

[0133] Trypsin inhibitor preserves the integrity of human tumour avatars

[0134] We cultured pancreatic tumour slices in a perfusion system using the media of the invention.

[0135] Figure 1 shows the histology of a pancreatic tumour slice cultured with antimicrobials and soybean trypsin inhibitor, i.e. the culture medium of the invention, to prevent degradation. The two top images ‘A’ (left hand side is 5x and right hand side is 10x magnification) show the normal morphology of the tissue at the baseline timepoint. In the top right hand image, the arrow identifies a pancreatic islet, the white arrowhead identifies a duct and the black arrowhead identifies acinar cells. The bottom two images ‘B’ (taken at 5x magnification) show that before and after culture perfused with the media of the invention, the morphology of the pancreatic tumour slice remains the same.

[0136] Figure 2 (10x magnification, scale bar 50 urn) shows the staining of a pancreatic tumour slice that had become contaminated (identified within 24hrs of culture) and degraded when cultured in baseline media without antimicrobials or trypsin inhibitor. H&E staining shows that the degraded area (top right hand image) is acellular and is necrotic with no preservation of native tissue architecture. The bottom right hand image is representative of an area of the slice where PDAC histology is preserved.

[0137] ‘Contamination’ means infection with bacteria or fungus. Tumour slices are susceptible both to degradation due to enzymatic digestion and also degradation due to infection, when cultured in media without antimicrobials or trypsin inhibitor.

[0138] Figure 2B shows quantification of the extent of necrosis (as a percentage of the total surface area) of the tumour slice at 24 hours ex vivo, under contamination and no contamination conditions. The data is presented as mean ± SEM and statistical analysis was performed by unpaired Student T test (Data information: *** P < 0.001).

[0139] Example 2

[0140] Perfusion culture preserves the integrity of human tumour slices

[0141] Having previously established that perfusion conditions are preferable for cell culture, we investigated the effect of perfusion on maintenance of ex vivo tumour avatars. These were obtained and processed as described in the methods section, cultured under perfused or static conditions in the culture medium of the invention and then analysed using a variety of techniques (Fig 3A). Initially, we assessed the viability of tumour tissue maintained over a 12-day period by determining a proliferation index for the tumour cells using Ki67 staining (Fig 3B). Relative to the baseline value (dashed line), a proliferative population persists to some extent throughout but is maintained to a greater degree when slices are cultured under perfusion conditions. To confirm that this effect is not due to the position in the original biopsy material from which the slices originated, baseline values which were obtained for slices from different parts of the same tissue were shown to give similar proliferation indices (Fig 3C). Since much of the tumour cell population is not proliferative, we also used spatial transcriptomic analysis to interrogate whether there was a change in gene expression in the tumour cells during the course of culture. A clustered heatmap of the expression data demonstrates that, while the static culture shows a progressive change in gene expression with time, gene expression in the perfused avatars clusters randomly over the time course (Fig 3D). This strongly suggests that avatars maintained under perfusion culture retain their integrity. In addition, the gene expression data has a greater range for the static culture, indicative of less stable culture conditions (Fig 3D, right).

[0142] The tumour microenvironment (TME) plays a crucial role in the survival of the cancer cells and is a particular challenge in the treatment of PDAC as it forms the greater part of the tumour mass and is very heterogenous, both within a tumour and between patients (Fig 4A) (Weniger et al., 2018). The use of patient avatars allows us to study the role of the TME in maintaining the integrity of the tumour and its impact on treatment response. We therefore first needed to assess whether our perfusion culture conditions were sufficient to also maintain the integrity of the other main components of the TME; the stromal and the immune components. We chose a window of 12 days for the time course of the assay, since for the patient the average length of stay following resection is 7 -12 days, enabling us to allow selection of a drug regime for the next stage of treatment by the time the patient has recovered post-surgery.

[0143] The stromal compartment was analysed initially using Masson’s trichrome stain and proved to be stable under both perfusion and static conditions (Fig 4B). Fibroblasts are a major component of the stroma and therefore maintenance of the fibroblast population was also assessed through aSMA staining. This similarly showed consistency throughout the treatment time course (Fig 4C) in the perfused culture. A heatmap of spatial transcriptomic gene expression data on the stromal compartment of our avatars largely confirmed this observation, although subtle differences could be observed between the two conditions (Fig 4D). Notably, we saw no timedependent clustering for either set of data, indicating a lack of progressive alterations during the assay, although the static culture did result in a greater range of gene expression values indicating a greater stability of stromal structure integrity in the perfused condition. Multiplex immunofluorescence was used to analyse the effect of culture on the immune cells in avatars of 3 patients (PT10, PT12 and PT13) using a panel which detects CD4+ T cells, CD8+ T cells, regulatory T cells, macrophages and B cells. No significant difference is seen for any of the populations in the perfused cultures throughout the 12-day time course (Fig 4E). In the case of the static culture, most populations are also constant, although the CD4 cell population is increased. This could be due to a local proliferation facilitated by the conditions of this particular microenvironment. As a result of these investigations, we were confident that our perfused system maintains the tumour avatars in a state which recapitulates that of the original tumour and we could therefore go on to assess its robustness in interrogating tumour biology. Our main areas of interest in the present study were (i) spatial characterisation of the immune filtrate, (ii) the effect of the TME on drugs that are known to affect tumour cells and (iii) whether the system has potential to enable selection of personalised chemotherapy regimens.

[0144] Example 3

[0145] Tumour slice culture gives valuable insight into immune infiltrate interactions

[0146] As part of an ongoing study on the immunobiology of PDAC we wished to analyse the location of immune cells within a tumour. Therefore, in this work we extended our multiplex IF analysis of the three patient avatars discussed in the previous section to include the distances between immune cells and the tumour cells along with inter- immune cell distances. We reasoned that the functional state of the local immune environment could be inferred from measurements of proximity indicating cellular interactions. To do this, we used the ‘proximity’ calculations in the HALO software, including cells that were within 50 mm of the target cell. Due to the large numbers of cells involved (in all cases n > 200 cells and in most cases n > 1000 cells. Table S2) all p values from ANOVA analyses were very low and therefore misleading. We therefore used size effect as a measure of significance, using Cohen’s d statistic for the Welch test, adjusted for the different cell population sizes and variances. Values > 0.8 were taken as significant (Cohen, 1998). We first tested whether culture conditions affected the location of cells in the avatars and noted that day 12 of the perfused culture gave very similar results to the baseline (Fig 5).

[0147] In Figure 6, baseline data is shown and boxplots represent distances between B cells, Tregs and CD4+ cells and the remaining immune cells and tumour cells (Fig 6). The plots show effect sizes between patients PT10 and PT12, PT10 and PT13 and PT12 and PT13 and the dashed line gives the Cohen d statistic of 0.8. It is notable that results for patients PT10 and PT12 tended to give similar results whereas patient PT13 differed in many respects. Taking instances in which PT13 shows a large size effect difference from both PT10 and PT12, B cells and Treg cells were more closely

[0148] 5 associated with tumour cells in PT13 (Fig 6A). In addition, B cells, Tregs and CD4+ cells resided further from CD8+ cells in the PT13 sample (Fig 6E, 6D and 6C respectively), whereas B cells were proximal to Treg cells (Figure 6E). Macrophages and CD8+ cells showed no differences across the patients (Fig 6A). 0 We also used the IF images to investigate the numbers of cells residing within the tumour cell areas (Fig 6G and Table 1). In this analysis the peritumoural area was defined as up to 15 pm outside the interface, while the intratumour area was the entire area of a cluster of tumour cells. Across the three patient avatars at baseline, we observed variation with the intratumoural immune cell infiltrate. The PT13 avatar 5 had the greatest proportion of intratumoural B cells, Tregs and, to a lesser extent, CD4+ cells in comparison to PT10 and PT12, which is in agreement with our proximity measurements.

[0149] Table 1 0

[0150] We note that patient survival also differed along the same lines as observed with the IF results, with patient PT13 surviving for considerably longer than the other two patients (Fig 6H). Thus, such analyses of patient avatars may give valuable insight into the role of the immune infiltrate in patient survival.

[0151] Example 4 Treatment of patient tumour slices with metformin and ascorbic acid modifies stromal and immune cells

[0152] We have previously demonstrated in a preclinical study that treatment of tumours with a combination of metformin and ascorbic acid induces phenotypic change of pancreatic tumour cells from aggressive squamous-like to more classical and therefore renders cells potentially more susceptible to chemotherapy (Eyres et al., 2021). It has been reported that the stromal component of PDAC tumours can also exist in an activated form which is found in, and assists in maintenance of, more aggressive tumours (Moffitt et al., 2015). We therefore decided to study the effect of metformin and ascorbic acid on the TME by perfusing tumour slices with the drug combination and analysing the effect on cell phenotype using spatial transcriptomics (Fig 7A).

[0153] Gene expression data for both the tumour and stroma sections were analysed using GSVA with the published gene sets that describe the squamous-like and classical phenotypes (Moffitt et al., 2015), (Bailey et al., 2016), (Collisson et al., 2011). The means of the GSVA scores for each condition and for each gene set were then further processed by subtracting the day 1 values from those of day 5 and results plotted for control and treated samples (Fig 7B). For each of the three gene sets used to analyse tumour cells, there is a marked increase over time in the representation of the less aggressive classical gene set in the treated samples compared to the control samples and that there is a greater representation of classical over squamous-like genes. This confirms our previous observations on the effect of metformin and ascorbic acid. When we analysed the stromal compartment using the gene set for the stromal subtypes (Moffitt et al., 2015), we observed an increase in the form of stroma associated with less aggressive tumours after the 5 day treatment regime and a decrease in the more aggressive activated stroma (Fig 7B). Thus, treatment alters the phenotype of both tumour cells and associated stroma.

[0154] Finally, we investigated the effect of the drugs on the immune infiltrate of our avatars. In order to do this, we deconvoluted the gene expression data for the immune compartment using the CIBERSORTx software and followed change in numbers of different types of immune cells at day 1 compared with the later timepoints of days 3 and 5. We observed a significant increase in the numbers of CD8 T cells after treatment with metformin and ascorbic acid (Fig 7C), an effect which was not seen for any other immune cell population. This could be indicative of CD8 cell proliferation or competition for energy resources as evidenced by the increased CD8:CD4 ratios observed.

[0155] Together, these data strongly suggest that the metformin and ascorbic acid combination modifies not only the tumour but also the key components of the TME to enhance the therapeutic effect of the combination (Fig 7D). Use of tumour avatars to dissect the effect of potential drugs on all aspects of tumour biology is a powerful addition to currently available techniques.

[0156] Example 5

[0157] Drug testing on avatars can allow rapid, efficient personalised treatment response analysis

[0158] A key aim of the current work was to use avatars as a precision medicine tool to ascertain which adjuvant treatment is likely to be the most appropriate for each patient. The procedure is ideal for this since chemotherapy starts approximately 3 months after surgical resection. Thus, avatars can be tested with potential drugs and results analysed in time to select further treatment.

[0159] To assess whether our system can be used in this way, we tested avatars from three patients by perfusing them with gemcitabine for 24hr and culturing for five subsequent days, followed by fixation and IHC to test for proliferation and cell death (Fig 8). We also isolated RNA from baseline samples of each section and assessed GATA6 and KRT81 gene expression by qPCR (Fig 8B). The low expression of GATA6 and high expression of the KRT81 genes in patient PT14 (Fig 8A) suggest that this tumour is squamous-like and therefore a more aggressive subtype. PT16 and PT17 show the opposite expression profile and therefore are likely to be classical tumours. Squamous-like tumours are reported to have a greater sensitivity to gemcitabine therapy which we observed within our drug-treated avatar cohort;

[0160] PT14 showed decreased cell proliferation and increased cell death (Fig 8A), whereas PT16 and PT17 showed no response to treatment (Fig 8E).

[0161] Significantly, the clinical outcome of the patients (taken at 18 months) correlated with our avatar observations. PT14 was treated with surgery, adjuvant FOLFIRINOX and palliative gemcitabine, PT16 with neo-adjuvant FOLFIRINOX, surgery and no further chemotherapy and PT17 with surgery and no further chemotherapy.

[0162] For PT14, the FOLFIRINOX regime may have initially secured systemic control over the disease. However, over time the therapeutic effect was lost and tumour outgrowth and metastases occurred. A comparison of tumour behaviour after the administration of palliative gemcitabine show that there was not only was a striking reduction in the Ca19-9 tumour marker, but also, there was 8 months of stable metastatic disease with no radiological evidence of progression (Fig 8C). This is very different to the tumour behaviour prior to gemcitabine therapy, where the disease burden had significantly increased. The lack of increase in the metastatic burden since completing palliative therapy implies sensitivity of the tumour to gemcitabine in agreement with our observations with the patient avatar.

[0163] The patient PT16 responded well to neoadjuvant FOLFIRINOX treatment and did not receive any further chemotherapy during the follow up period. Patient PT17 also did not receive adjuvant therapy. In both cases, on the basis of our results, gemcitabine would not have been expected to be effective (Fig 8D).

[0164] The correlation observed between avatar behaviour and patient response makes this a viable option for assessing which treatments would be suitable for each patient and is therefore a rapid, economical method for personalising PDAC treatment.

[0165] Materials and methods

[0166] Acquisition of tissue and blood samples

[0167] The study (REC number 19 / A056) was approved for the collection of tumour and healthy pancreatic tissue by the Oxford Radcliffe BioBank. Collection of the specimens was supported by the Oxford Centre for Histopathology Research. All patients recruited to the study provided written consent confirming voluntary participation and permission for tissue donation for research. Biopsy punch samples (5mm diameter) were obtained by the pathologist at the John Radcliffe hospital following surgery provided that clear surgical margins could be determined.

[0168] Sectioning and culture of live tumour slices Samples were transported on ice in unsupplemented low glucose (LG) DMEM media prior to suspension in agarose scaffolds. Following a manual wash in LG DMEM media, biopsy punches were suspended in 4% low-gelling-temperature agarose and cooled. The agarose scaffold structure was generated by melting the solution and suspending the slice in a small embedding mould. 250 pm sections were generated using the Leica VT1200 vibratome (blade angle +21 °C, speed 1.5 mms-1, amplitude 2 mm) in a bath of ice-cold PBS and transported in LG DMEM media on ice.

[0169] Alvetex perfusion plates (REPROCELL) were used to conduct dynamic perfusion experiments, with syringe pumps maintaining a constant flow of 10 pLmin-1. The apparatus was assembled within a sterile tissue culture hood. To construct the circuit, 60 mL syringes were connected to silicone tubing and flushed with 70% ethanol prior to washing in unsupplemented LG DMEM media. Alvetex 12-well tissue culture inserts were activated in 70% ethanol for 2 minutes prior to washing in LG DMEM media. Long term culture of tissue slices was conducted using LG DMEM complete pancreatic medium at +37°C and 5% CO2.

[0170] Treatment of slices in perfusion culture

[0171] All ex vivo treatment of avatars was commenced on day 0 (day of tumour acquisition). Systemic chemotherapy was prepared in DMSO. Drug delivery commenced after the avatars had been created and subsequently placed in the perfusion plate, via a new inflow circuit comprising a syringe and tubing, attached to the perfusion plate and connected to the perfusion pump. After the intended time period for drug delivery, the infusion was stopped, and the perfusion plate removed from the incubator and placed within a tissue culture hood. The inflow circuit was removed and the inflow channel on the perfusion plate temporarily occluded using a 2ml syringe. A new inflow circuit was then created, and the syringe filled with culturing media. The inflow circuit was primed with the media of the invention (from the attached syringe) to remove any air bubbles present in the tubing prior to attachment to the perfusion plate to ensure that there was a constant column of media from the syringe and the tubing to the perfusion plate.

[0172] Histological analysis - haematoxylin and eosin (H&E)

[0173] Samples were fixed in 4% neutral buffered formalin (NBF) for 12 hours and stored in 70% ethanol prior to processing and embedding in paraffin wax. To prevent loss of the small tissues during processing each slice was placed into a Cellsafe biopsy insert inside the processing / embedding cassette. 4-5pm microtome sections were generated and mounted on negatively charged slides. Slides were kept overnight at +50°C prior to rehydration in Citroclear and decreasing concentrations of ethanol. After rehydration, slides were submerged in Meyer’s haematoxylin for 5mins and washed with tap water. Brief washes in acid alcohol, Scott’s tap water and lithium carbonate followed with distilled water rinse between each step. Dehydration in increasing ethanol (50%, 70%, 90%) concentrations preceded 15sec eosin submersion and final submersion in 100% ethanol. To mount samples, slides were first kept in xylene for 1min intervals before coverslips were mounted with DPX mountant and left to dry overnight.

[0174] Immunohistochemistry (IHC)

[0175] Slides were treated as in ‘histological analysis’ section for IHC up to the point of rehydration in successively decreasing ethanol concentrations and ddH20 submersion.

[0176] Following rehydration, an antigen retrieval step was performed to improve staining quality. Retrieval was optimised for individual antibodies using a pressure cooker (reaching +125°C for 2mins and +90°C for 1min prior to cooling). Staining bins were kept on ice for 1 hr before a hydrophobic PAP pen was used to circle the specimens. Samples were then blocked for 15mins with dual endogenous enzyme block and 45mins with 5% goat serum in TBS to reduce non-specific binding. Primary antibodies were applied and kept overnight at +4°C in a humid chamber. All antibodies were diluted in antibody diluent. Slides were washed three times prior to 45min incubation with secondary antibody HRP.

[0177] Staining was visualised through short exposure to diaminobenzidine diluted 1:50 before the reaction was ceased by submersion in distilled water. Slides were then counterstained with 20 second immersion in haematoxylin Gill, washed and dehydrated. Following xylene submersion, coverslips were applied using DPX mountant and slides were dried in the fume hood for 48hrs prior to imaging at 20X / 40X magnification using the Aperio Brightfield Slide Scanner.

[0178] Image quantification was carried out using QPath with methods supplied by Dr Hannah Bolland (Department of Oncology). Tissue was identified using stain vector estimation with deprecated tissue detection used to map tissue area prior to analysis. Deprecated cell and membrane detection was subsequently used to identify cells with parameters kept constant between samples. Positive cell detection was used to quantify staining intensity within pre-defined tumour and stroma areas and an average taken across the slice. H-score thresholding was kept constant between samples for each individual marker. Three independent patient samples were used for experiments unless stated otherwise.

[0179] RNA extraction and qPCR

[0180] RNA extraction and isolation from the avatars was performed using Monarch® Total RNA Miniprep Kit. The avatar was initially suspended in 300 uL of DNA / RNA protection reagent from the Monarch® Total RNA Miniprep Kit. This was followed by manual dissociation using a pestle and mortar (TaKaRa BioMasher Standard). Manual dissociation was performed for 5 minutes; the sample was subsequently transferred to a qiashredder spin column and spun for 2 minutes at maximum speed. Enzymatic dissociation was performed with proteinase K as per the Monarch® Total RNA Miniprep. All subsequent steps were performed exactly as specified by the Monarch® Total RNA Miniprep Kit protocol. Residual gDNA was removed using on- column DNase I treatment.

[0181] RNA was converted into cDNA using the LunaScript RT SuperMix kit (by New England Biolabs) as per the manufacturer’s protocol. Luna® Universal qPCR Master Mix was used in order to measure expression of individual genes of interest. Primers were designed online using PrimerBank (Spandidos et al., 2010). The reactions were performed in duplicate and were run for 45 cycles on a StepOnePlus™ Real-Time PCR machine. In order to determine the change in mRNA expression, normalisation to b-2-microglobulin was performed followed by the cycle threshold (CT) calculation method.

[0182] GeoMX spatial transcriptomic analysis

[0183] Spatial transcriptomic analysis was provided by NanoString Technologies, Inc. through their GeoMX® DSP Technology Access Program Grant. Formalin-fixed paraffin-embedded (FFPE) samples were provided cut at 5 pm thickness and mounted on negatively-charged slides. Samples were sent to the NanoString Technology Access Program labs for analysis using the GeoMX Digital Spatial Profiler. Slides were incubated with oligonucleotide-antibody conjugates with photocleavable linkers. UV light was then used to selectively release oligonucleotide barcodes which were read and quantified through sequencing. Four immunofluorescent markers were used for morphology definition, facilitating region of interest (ROI) selection. A total of 68 ROIs were selected across treated and control samples. The Whole Transcriptome Atlas was used for sample profiling, with genes mapped to barcodes using an in-house algorithm produced by NanoString Technologies, Inc., generating spatially resolved transcriptional data. Quality control involved assessing raw read threshold, percent-aligned reads and sequencing saturation. A quantification limit was set based upon negative probe signal (mean + two standard deviations). Reads were filtered based upon their expression in >5% AOIs and counts were normalised to account for differences in AOI size and cellularity.

[0184] Multiplexed immunofluorescence and HALO analysis

[0185] The multiplex IF staining of avatars was performed in collaboration with the Oxford Translational Histopathology Lab. Slides were stained using the Leica BOND RXm autostainer machine (Leica, Microsystems) whilst following the OPAL™ protocol 28 (AKOYA Biosciences). A total of 6 staining cycles were subsequently performed. The following primary antibody-opal fluorophore pairing was used: CD4 - Opal 520, CD8 - Opal 570, CD20 - Opal 480, Foxp3 - Opal 620, CD68 - Opal 690, pan Cytokerratin - Opal 780. In adherence to the manufacturer's instructions, the primary antibodies were incubated for one hour and subsequently detected with the BOND™ 4 Polymer Refine Detection System (DS9800, Leica Biosystems). The DAB step was replaced by the opal fluorophores which consisted of a 10 minute incubation with no haematoxylin step. The antigen retrieval step was performed with Epitope Retrieval (ER) Solution 2 (AR9640, Leica 8 Biosystems) for 20 minutes at 100°C. This was performed prior to the application of each primary antibody. VECTASHIELD® Vibrance™ Antifade Mounting Medium with DAPI 10 (H-1800-10, Vector Laboratories.) was used to mount each slide. The AKOYA Biosciences Vectra® Polaris™ was used in order to obtain whole slide scans and multispectral images (MSI). A batch analysis of every MSIs was performed using the inForm 2.4.8 software. The final step consisted of fusing the multiple batched analysed MSIs on the HALO (Indica Labs) software. This resulted in the creation of a spectrally unmixed reconstructed whole image of the avatar.

[0186] The analysis of the multiplex IF was performed on the Indica Labs HALO® (version 3.0.311.407). This software allows for deconvolution of the image by selecting individual fluorophores for analysis. The initial step was teaching the software a Random Forest Classifier module in which the images were segmented into tumour and stromal regions. Manual annotation of the slides was performed to exclude areas with staining artefacts. Cell detection and subsequent phenotyping was performed using the Indica Labs - HighPlex FL v3.1.0 (fluorescent images). Individual cells were defined by their expression of the specific markers: Tumour (DAPI+ panCytokeratin+), CD4 helper (DAPI+CD4+), CD8 cytotoxic (DAPI+CD8+), regulatory T-cell (DAPI+CD4+Foxp3+), B cells (DAPI+CD20+) and Macrophages (DAPI+CD68+). Proximity and spatial analysis were performed using the relevant software packages within the Indica Labs HALO® software using the “proximity analysis” setting.

[0187] Statistical Analysis

[0188] All statistical analyses were performed using GraphPad Prism v9.3.1 or Rstudio.

[0189] All data are represented as means ± SD unless otherwise specified, with significance determined at P < 0.05. *=p<0.05, **=p<0.01 , ***=p<0.005, ****=p<0.001.

[0190] The Shapiro-Wilk and Kolmogorov-Smirnov Normalcy tests were carried out and data determined to be Normally distributed was analysed using T tests or ANOVA for comparing multiple variables. Multiplex IF proximity measurements were analysed using Cohen’s ds statistic and 95% confidence intervals using the effect size package in RStudio (Ben-Shachar M, 2020).

[0191] Numbered embodiments

[0192] 1. A culture medium comprising low glucose DMEM, L-glutamine, foetal bovine serum (FBS), antimicrobials and a trypsin inhibitor.

[0193] 2. The culture medium according to embodiment 1 , wherein the low glucose DMEM contains between 20-100 mg / dl, 20-200 mg / dl or 20-300 mg / dl glucose.

[0194] 3. The culture medium according to any preceding embodiment, wherein the low glucose DMEM contains between 50-200 mg / dl glucose, preferably wherein the low glucose DMEM contains 100 mg / dl glucose.

[0195] 4. The culture medium according to any preceding embodiment, wherein the antimicrobials are selected from the group comprising of amphotericin B, penicillin, streptomycin and neomycin.

[0196] 5. The culture medium according to any preceding embodiment, wherein the antimicrobials are selected from the group comprising of penicillin and streptomycin.

[0197] 6. The culture medium according to any preceding embodiment, wherein the concentration of penicillin is between 10 - 100 ll / rnl, preferably wherein the concentration is 50 ll / rnl.

[0198] 7. The culture medium according to any preceding embodiment, wherein the concentration of streptomycin is between 0.01 - 1.0 mg / ml, preferably wherein the concentration is 0.05 mg / ml.

[0199] 8. The culture medium according to any preceding embodiment, wherein the concentration of neomycin is between 0.01 - 1.0 mg / ml, preferably wherein the concentration is 0.1 mg / ml.

[0200] 9. The culture medium according to any preceding embodiment, wherein the concentration of amphotericin B is between 1.0 - 5.0 mg / ml, preferably wherein the concentration is 2.5 mg / ml. 10. The culture medium according to any preceding embodiment, wherein the concentration of FBS is between 0.1 - 1.0 mg / ml, preferably wherein the concentration is between 0.15 - 0.225 mg / ml.

[0201] 11. The culture medium according to any preceding embodiment, wherein the concentration of trypsin inhibitor is between 0.1 - 1.0 mg / ml, preferably wherein the concentration is 0.2 mg / ml.

[0202] 12. The culture medium according to any preceding embodiment, wherein the trypsin inhibitor is soybean trypsin inhibitor.

[0203] 13. The culture medium according to any preceding embodiment, comprising low glucose DMEM, FBS, soybean trypsin inhibitor, amphotericin B, penicillin, streptomycin, neomycin and L-glutamine.

[0204] 14. The culture medium according to embodiment 13, comprising low glucose DMEM, 0.15 - 0.225 mg / ml of FBS, 0.2 mg / ml soybean trypsin inhibitor, 2.5 mg / ml amphotericin B, 50 ll / rnl penicillin, 0.05 mg / ml streptomycin, 0.1 mg / ml neomycin and 0.3 mg / ml L-glutamine.

[0205] 15. The culture medium according to embodiment 14, wherein the low glucose DMEM contains 100mg / dl glucose.

[0206] 16. The use of the culture medium according to any preceding embodiment for culturing mammalian cells.

[0207] 17. The use according to embodiment 16 for culturing mammalian cells in a perfusion culture.

[0208] 18. The use of the culture medium according to any preceding embodiment for maintaining the viability of live tissue slices.

[0209] 19. The use of the culture medium according to any preceding embodiment for maintaining the viability of live tissue slices in a perfusion model.

[0210] 20. The use of the culture medium according to any preceding embodiment for maintaining the viability of live tumour slices. 21. The use of the culture medium according to any preceding embodiment for maintaining the viability of live tumour slices in a perfusion model.

[0211] 22. The use according to embodiments 20 or 21, wherein the tumour slices are pancreatic tumour slices.

[0212] 23. The use according to any one of embodiments 20-22, wherein the viability of tumour slices is maintained for at least 5 days.

[0213] 24. The use according to any one of embodiments 20-22, wherein the viability of tumour slices is maintained for at least 10 days.

[0214] 25. The use according to any one of embodiments 20-22, wherein the viability of tumour slices is maintained for at least 12 days.

[0215] 26. The use of the culture medium according to any preceding embodiment for tissue imaging.

[0216] 27. The use of the culture medium according to any preceding embodiment for live tumour slice imaging.

[0217] 28. A method for identifying a treatment for cancer comprising the steps of: i) providing a cancer tumour slice to an apparatus, ii) providing the culture medium according to any preceding embodiment to the tumour slice, iii) adding a treatment agent, iv) receiving a visual image of the tumour slice, v) analysing the data, vi) identifying the optimal treatment regimen based on the data for the cancer.

[0218] 29. The method according to embodiment 28, wherein the cancer is pancreatic ductal adenocarcinoma (PDAC).

[0219] 30. The method according to embodiments 28 or 29, wherein the analysis is performed by a human viewing the displayed image. 31. The method according to any one of embodiments 28-30, wherein the analysis is performed by histology assessments, immunohistochemistry and / or immunofluorescence.

[0220] 32. The method according to embodiments 28 or 29, wherein the analysis is performed using a software.

[0221] 33. A method for perfusion cell culture comprising the step of passing the culture medium according to any preceding embodiment over a cancer tissue culture in an apparatus.

Claims

Claims1. A culture medium comprising low glucose DMEM, L-glutamine, foetal bovine serum (FBS), antimicrobials and a trypsin inhibitor.

2. The culture medium according to claim 1 , wherein the low glucose DMEM contains between 20-100 mg / dl, 20-200 mg / dl or 20-300 mg / dl glucose, optionally wherein the low glucose DMEM contains between 50-200 mg / dl glucose, preferably wherein the low glucose DMEM contains 100 mg / dl glucose.

3. The culture medium according to any preceding claim, wherein the concentration of trypsin inhibitor is between 0.1 - 1.0 mg / ml, preferably wherein the concentration is 0.2 mg / ml.

4. The culture medium according to any preceding claim, wherein the trypsin inhibitor is soybean trypsin inhibitor.

5. The culture medium according to any preceding claim, wherein the antimicrobials are selected from the group comprising of amphotericin B, penicillin, streptomycin and neomycin, optionally wherein the antimicrobials are selected from the group comprising of penicillin and streptomycin, optionally wherein a) the concentration of penicillin is between 10 - 100 ll / rnl, preferably wherein the concentration is 50 ll / rnl; and / or b) the concentration of streptomycin is between 0.01 - 1.0 mg / ml, preferably wherein the concentration is 0.05 mg / ml; and / or c) the concentration of neomycin is between 0.01 - 1.0 mg / ml, preferably wherein the concentration is 0.1 mg / ml; and / or d) the concentration of amphotericin B is between 1.0 - 5.0 mg / ml, preferably wherein the concentration is 2.5 mg / ml.

6. The culture medium according to any preceding claim, wherein the concentration of FBS is between 0.1 - 1.0 mg / ml, preferably wherein the concentration is between 0.15 - 0.225 mg / ml.

7. The culture medium according to any preceding claim, comprising low glucose DMEM, FBS, soybean trypsin inhibitor, amphotericin B, penicillin, streptomycin, neomycin and L-glutamine;optionally comprising low glucose DM EM, 0.15 - 0.225 mg / ml of FBS, 0.2 mg / ml soybean trypsin inhibitor, 2.5 mg / ml amphotericin B, 50 ll / rnl penicillin, 0.05 mg / ml streptomycin, 0.1 mg / ml neomycin and 0.3 mg / ml L-glutamine; optionally wherein the low glucose DM EM contains 100mg / dl glucose.

8. The use of the culture medium according to any preceding claim for: a) culturing mammalian cells, optionally for culturing mammalian cells in a perfusion culture, and / or b) maintaining the viability of live tissue slices, optionally for maintaining the viability of live tissue slices in a perfusion model, and / or c) maintaining the viability of live tumour slices, optionally for maintaining the viability of live tumour slices in a perfusion model, optionally wherein: i) the tumour slices are pancreatic tumour slices, and / or ii) the viability of tumour slices is maintained for at least 5 days, and / or iii) the viability of tumour slices is maintained for at least 10 days, and / or iv) the viability of tumour slices is maintained for at least 12 days.

9. The use of the culture medium according to any preceding claim for tissue imaging, optionally for live tumour slice imaging.

10. A method for identifying a treatment for cancer comprising the steps of: i) providing a cancer tumour slice to an apparatus, ii) providing the culture medium according to any preceding claim to the tumour slice, iii) adding a treatment agent, iv) receiving a visual image of the tumour slice, v) analysing the data, vi) identifying the optimal treatment regimen based on the data for the cancer, optionally wherein the cancer is pancreatic ductal adenocarcinoma (PDAC).

11. The method according to claim 10, wherein the analysis is performed: a) by a human viewing the displayed image; and / or b) by histology assessments, immunohistochemistry and / or immunofluorescence; or c) using a software.

12. A method for perfusion cell culture comprising the step of passing the culture medium according to any preceding claim over a cancer tissue culture in an apparatus.

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