METHOD AND KIT FOR CELL GROWTH

Abstract

The present invention is related to a method to be performed with one tissue type, wherein a specific combination of hydrogel features has been pre-selected for the said one tissue type to be tested. The present invention is also related to a kit of parts to perform said method.

Claims

1. A method to be performed with one tissue type, optionally, in combination with other cells including stromal cells or immune cells, comprising the steps of: a) providing a fully defined hydrogel matrix array with discrete volumes by crosslinking, onto a substrate or into discrete volumes of a substrate, different combinations of one or more different hydrogel precursor molecules, optionally in the presence of one or more biologically active molecules, optionally at least one crosslinking agent and cells of the tissue type to be tested, so as to create fully defined three-dimensional extracellular matrix conditions that differ from each other in their biological, biophysical and/or biochemical characteristics; b) allowing said cells to grow and expand in said discrete volumes of said hydrogel matrix array in the presence of one or more different culture media; c) performing an operation with the cells grown in said discrete volumes of said hydrogel matrix array; and wherein a specific combination of hydrogel features has been pre-selected for the said one tissue type to be tested.

2. The method according to claim 1, wherein the preselection of at least one of said hydrogel precursor molecules, or of said hydrogel features, and said culture media is made on the basis of selecting suitable extracellular matrix conditions from a method using random extracellular matrix conditions.

3. The method according to claim 1, wherein the tissue type is selected from the group consisting of cancer cells and normal/healthy cells.

4. The method according to claim 1, wherein freshly isolated or frozen cells from a biopsy or tissue resection of a human or from patient-derived xenograft (PDX) tissue are used.

5. The method according to claim 1, wherein in step c) one or more drugs are added to said discrete volumes of said hydrogel matrix.

6. The method according to claim 1, wherein the tissue type is lung cancer cells, overexpressing c-Met, and the hydrogel matrix is preselected as being a non self-degradable PEG hydrogel, wherein the crosslinking agent and said optional bioactive agent do not comprise any RGD motif.

7. The method according to claim 6, wherein said culture medium comprises FBS (serum) or Wnt agonist including R-spondin.

8. The method according to claim 1, wherein the tissue type is pancreatic ductal adenocarcinoma (PDAC) cells, and the hydrogel matrix is preselected as being a non self-degradable PEG hydrogel having a stiffness in the range of 50 to 3000 Pa, wherein at least one of the crosslinking agent and/or said optional bioactive agent comprise a RGD motif.

9. The method according to claim 8, wherein said culture medium comprises Wnt agonists including R-spondin and Wnt 3a.

10. The method according to claim 1, wherein the tissue type is colorectal cancer (CRC) cells, and the hydrogel matrix is preselected as being a PEG hydrogel having at least an initial stiffness in the range of 50 to 2000 Pa, and optionally furthermore comprising one or more biologically active molecules comprising laminin wherein at least one of the crosslinking agent and/or said optional bioactive agent comprise a RGD motif.

11. The method according to claim 10, wherein said culture medium comprises Wnt agonists including R-spondin and Wnt 3a.

12. The method according to claim 1, wherein the tissue type is breast cancer cells, and the hydrogel matrix is preselected as being an enzymatic-degradable PEG hydrogel, wherein at least one of the crosslinking agent comprises an enzymatically degradable motif, and said hydrogel optionally furthermore comprises one or more biologically active molecules comprising laminin.

13. The method according to claim 12, wherein said culture medium comprises FBS (serum) or Wnt agonist including R-spondin.

14. The method according to claim 1, wherein the tissue type is cancer cells that grow ex vivo more slowly than their normal counterparts, and the hydrogel matrix is preselected as being a PEG hydrogel, having a stiffness in the range of 50 to 2000 Pa, wherein said crosslinking agent and said optional bioactive agent do not comprise any RGD motif.

15. Kit of parts for performing an operation on or with one or more tissue type, comprising: a) components for preparing a fully defined hydrogel matrix array, so as to create fully defined three-dimensional extracellular matrix conditions that differ from each other in their biological, biophysical and/or biochemical characteristics, b) said components comprising one or more different hydrogel precursor molecules, optionally at least one crosslinking agent, optionally one or more biologically active molecules, c) one or more different culture media, wherein a specific combination of hydrogel features has been pre-selected for the tissue type to be tested.

16. Kit according to claim 15, for testing the influence of drugs on lung cancer cells, overexpressing c-Met, the hydrogel matrix is preselected as being a non self-degradable PEG hydrogel, wherein the crosslinking agent and said optional bioactive agent do not comprise any RGD motif, and said culture medium comprises FBS (serum) or Wnt agonist including R-spondin.

17. Kit according to claim 15, for testing the influence of drugs on pancreatic ductal adenocarcinoma (PDAC) cells, the hydrogel matrix is preselected as being a non self-degradable PEG hydrogel having a stiffness in the range of 50 to 3000 Pa, wherein at least one of the crosslinking agent and/or said optional bioactive agent comprise a RGD motif, and said culture medium comprises Wnt agonists including R-spondin and Wnt 3a.

18. Kit according to claim 15, for testing the influence of drugs on colorectal cancer (CRC) cells, and the hydrogel matrix is preselected as being a PEG hydrogel having at least an initial stiffness in the range of 50 to 2000 Pa, and optionally furthermore comprising one or more biologically active molecules comprising laminin, wherein at least one of the crosslinking agent and/or said optional bioactive agent comprise a RGD motif, and wherein said culture medium comprises Wnt agonists including R-spondin and Wnt 3a.

19. Kit according to claim 15, for testing the influence of drugs on breast cancer cells, and the hydrogel matrix is preselected as being an enzymatic-degradable PEG hydrogel, wherein at least one of the crosslinking agent preferably comprises an enzymatically degradable motif, preferably a MMP sensitive motif, and said hydrogel optionally furthermore comprises one or more biologically active molecules comprising laminin, and wherein said culture medium comprises FBS (serum) or Wnt agonist including R-spondin.

20. Kit according to claim 15, for testing the influence of drugs on cancer cells that grow ex vivo more slowly than their normal counterparts, and the hydrogel matrix is preselected as being a PEG hydrogel, having a stiffness in the range of 50 to 2000 Pa, wherein said crosslinking agent and said optional bioactive agent do not comprise any RGD motif.

Description

EXEMPLARY EMBODIMENTS

[0169] The present invention will now be described below with reference to non-limiting exemplary embodiments and drawings.

[0170] FIG. 1a shows the results of c-met expression in different ex vivo examples and drug testing experiments with non-small cell lung cancer cells overexpressing c-met grown in different gels.

[0171] FIG. 1b shows the effect of a SoC treatment and treatment with a c-met inhibitor in an example according to the present invention.

[0172] FIG. 1c shows the effect of a SoC treatment and treatment with a c-met inhibitor in a comparative example (Matrigel®).

[0173] FIG. 1d shows the results of c-met and EGFR expression in different ex vivo examples.

[0174] FIG. 1e shows the effect of a SoC treatment and treatment with EGFR inhibitors in an example according to the present invention.

[0175] FIG. 2a shows the growth of PDX pancreatic ductal adenocarcinoma (PDAC) cells in different gels.

[0176] FIG. 2b shows the drug sensitivity of PDX pancreatic ductal adenocarcinoma (PDAC) cells in different gels.

[0177] FIG. 2c shows the growth of PDX pancreatic ductal adenocarcinoma (PDAC) cells in soft and medium gels.

[0178] FIG. 3 shows Brightfield images of the results of co-culturing 33% PDAC cells with 67% fibroblasts in different gels.

[0179] FIG. 4 shows Brightfield images of the results of human colon cancer organoids grown for 0 and 11 days.

[0180] FIG. 5 shows Brightfield images of the results of growth of human primary or metastatic (Mets) breast cancer cells from four patients of either HER2+ or Triple Negative Breast Cancer (TNBC) (from patient-derived xenograft models).

[0181] FIG. 6a shows Brightfield images of the results of human healthy prostate cells grown for 1 and 14 days.

[0182] FIG. 6b shows Brightfield images of the results of human prostate cancer cells grown for 1, 13 and 20 days.

LUNG CANCER THERAPY

[0183] Well characterized and patient-cell derived preclinical models are essential components to perform reliable translational cancer research, including identifying molecular pathways of oncogenesis and evaluating potential therapeutics.

[0184] Tumor cell lines have long existed as a convenient platform for investigation, and numerous cell lines have been well characterized and used for establishing tumors in animal models (xenograft tumors). However, cell line-derived xenograft tumors suffer a lack of predictable relationship between therapeutic responses in preclinical models when compared to responses in human trials and do not accurately recapitulate the tumor microenvironment in a human (Johnson et al., British Journal of Cancer (2001) 84(10), 1424-1431).

[0185] Patient-derived tumor xenograft models (PDX) are frequently used for translational cancer research and are assumed to behave consistently over serial passaging. Correlations between histopathological and genotypic characteristics of the original patient samples and PDX models have been well documented (Rubio-Viqueira et al., Clin. Cancer Res. 2006, 12(15), 4652). In addition, PDX models grown over multiple passages maintain a correlation between original human tumor therapeutic responses and the responses in PDX derived from these same patients. However, the throughput of PDX-based screening models is low, and furthermore such screening tests are expensive.

[0186] The present invention provides an improved method for cancer research. The present invention provides an improved alternative to PDX models that enables high-throughput screening in a very cost-effective manner.

[0187] According to a preferred embodiment, a pre-selection of suitable extracellular matrix conditions for cancer cell growth can be performed by using cells from a PDX and assaying them as described above in the section “preselection”. The histopathological and genotypic characteristics of cells grown ex vivo under such conditions can be correlated with the ones of in vivo established PDX models, and PDX tumor therapeutic responses derived from those PDX models can be used as an in vivo benchmark to evaluate which extracellular matrix conditions can recapitulate in vivo cancer cell behaviour.

[0188] As shown in the literature above, the microenvironment (i.e. the extracellular matrix conditions) may influence how cancer cells respond to drug treatments, both in vivo and ex vivo. With the method of the present invention, it is possible to establish ex vivo cell culture conditions for drug screening/testing that are capable to capture the different patient tumor characteristics (e.g. different cancer subtypes), in order to more accurately predict drug treatment outcomes for patients.

[0189] This consists in growing cells and testing possible drug treatments on the grown cells ex vivo, using the patient's own cells cultured in a pre-selected range of microenvironments. With the method of the present invention, it is possible to capture the intra- and inter-tumor patient heterogeneity of drug responses (incl. resistance to targeted-therapies).

[0190] Currently, the established method in the prior art is still that cells extracted from patient tissues are grown using a single culture condition composed by e.g. the gold-standard Matrigel®. This single condition does not always allow growing patient cells in a way that all features and the possible heterogeneity of the original patient tumors are captured. Also, sometimes some components of the undefined matrix may interfere with the drug response on tested cells.

[0191] With the present invention, it is possible to culture patient cells that are then exposed to different drug treatments in order to uncover sensitivities and potential resistance to drug treatments (and underlying mechanisms) that better reflect what is happening in the original patient tumors (e.g. tumor heterogeneity, drug response). With the present invention, it is possible to help selecting or excluding drug treatment for cancer patient and/or to help selecting second line treatments to overcome the resistance to previous treatment(s).

[0192] Following this approach, it could be shown that preselected conditions that are suitable for testing the effect of c-Met inhibitors on non-small cell lung cancer (NSCLC) cells overexpressing c-Met are characterized by the absence of any RGD adhesion motif in the hydrogel (see example 1 below).

[0193] This is particularly surprising since from the prior art the opposite result (necessity of presence of RGD adhesion motif in the extracellular matrix conditions) would have been expected. In Mitra et al. (Oncogene 2011, March 31; 30(13): 1566-1576) it was shown that c-Met can be activated independently of its ligand (HGF) via the fibronectin-mediated activation of α5β1-integrin leading to its interaction with c-Met receptor. Inhibition of α5β1-integrin decreased the phosphorylation of c-Met, both in vitro and in vivo (ovarian cancer lines). The crosstalk between integrin β1 with c-Met was also explored for NSCLC in Ju et al. (Cancer Cell International 2013, 13:15). This article showed that interaction of integrin β1 with c-Met induces c-Met activation (i.e. phosphorylation) and permits cancer cells sensitive to inhibition of EGFR receptor to become resistant to EGFR targeted drugs by bypassing the EGF pathway. Both articles show that c-Met can interact with integrin β1, which is a known RGD linker. They also demonstrate that this interaction resulted in c-Met phosphorylation and activation of its downstream pathway (FAK, AKT), inducing cell proliferation and increased survival. In summary, both articles clearly highlight the relationship between c-Met receptor and fibronectin.

[0194] According to the present invention, however, it could be shown that the presence of a RGD motif in the hydrogel matrix led to a downregulation of the c-Met receptor and the absence of activated c-Met receptor (i.e. phosphorylated receptor) in NSCLC cells, resulting in their resistance against treatment with a c-Met inhibitor. It was an important finding of the present invention that the absence of any RGD adhesion motif in the hydrogel provided the correct preselected conditions for identifying suitable drug candidates for NSCLC cancer cells exhibiting an activated c-met receptor. On the other hand, NSCLC cancer cells growing in the presence of a RGD motif do not possess and do not rely on an activated c-met receptor, and this indicates that they may also have to be treated with other drug candidates than a c-met inhibitor (possibly along with a c-met inhibitor). Without the use of the “preselected growth conditions”, it would not have been possible to understand that these cells may rely on other mechanisms of growth than c-Met. One of the major added value of using said “preselected growth conditions” compared to single growth conditions as used in the prior art, is that it enables uncovering the heterogeneity (e.g. genetic, phenotypic) of the specific cancer tissue and cancer type, as well as a better possible range of treatments that are needed to cure said cancer. This has relevance in personalize medicine as well as drug development applications.

[0195] Thus, according to this embodiment, the present invention is related to a method of testing the influence of c-Met inhibitors on lung cancer cells, preferably non-small cell lung cancer cells, overexpressing c-Met, comprising the steps of: [0196] a) providing preselected extracellular matrix conditions comprising a fully defined non self-degradable hydrogel matrix array with discrete volumes prepared by crosslinking, onto a substrate or into discrete volumes of a substrate, preferably a multi-well plate, different combinations of one or more different PEG hydrogel precursor molecules in the presence of optionally one or more biologically active molecules, at least one crosslinking agent, and said lung cancer cells, preferably non-small cell lung cancer cells, so as to create fully defined three-dimensional extracellular matrix conditions that differ from each other in their biological, biophysical and/or biochemical characteristics; [0197] b) allowing said lung cancer cells, preferably non-small cell lung cancer cells, to grow in said discrete volumes of said hydrogel matrix in the presence of one or more different culture media, preferably comprising FBS (serum) or Wnt agonist such as R-spondin, especially preferred also comprising FGF-7, FGF-10, HGF, and a TGF-β inhibitor; [0198] c) adding a drug targeting c-Met receptor or c-Met pathway to the cells grown in said discrete volumes of said hydrogel matrix;

[0199] wherein said crosslinking agent and said optional bioactive agent do not comprise any RGD motif.

[0200] According to a very preferred embodiment, the present invention is related to a method of testing the influence of c-Met inhibitors and other drugs on lung cancer cells, preferably non-small cell lung cancer cells, comprising the steps of: [0201] a) providing, in a first array of a substrate, preselected extracellular matrix conditions comprising a fully defined non self-degradable hydrogel matrix array with discrete volumes prepared by crosslinking, onto a substrate or into discrete volumes of a substrate, preferably a multi-well plate, different combinations of one or more different PEG hydrogel precursor molecules in the presence of optionally one or more biologically active molecules, at least one crosslinking agent, and said lung cancer cells, preferably non-small cell lung cancer cells, so as to create fully defined three-dimensional extracellular matrix conditions that differ from each other in their biological, biophysical and/or biochemical characteristics, wherein said crosslinking agent and said optional bioactive agent do not comprise any RGD motif, and providing, in a second array of the substrate, preselected extracellular matrix conditions that differ from the preselected extracellular matrix conditions in the first array by the presence of an RGD motif in said crosslinking agent and/or said optional bioactive agent; [0202] b) allowing said lung cancer cells, preferably non-small cell lung cancer cells, to grow in said discrete volumes of said hydrogel matrix in the first array and second array in the presence of one or more different culture media, preferably comprising FBS (serum) or Wnt agonist such as R-spondin, especially preferred also comprising FGF-7, FGF-10, and a TGF-β inhibitor; [0203] c) adding a drug targeting c-Met receptor or c-Met pathway to the cells grown in said discrete volumes of said hydrogel matrix in the first array and second array; [0204] d) adding at least one other drug, preferably a EGFR-receptor inhibitor, to the cells grown in said discrete volumes of said hydrogel matrix in the first array and second array, into wells where no drug targeting c-Met receptor or c-Met pathway has been added.

[0205] Preferably, said hydrogel matrix array has a soft or medium stiffness in the range of 50-2000 Pa.

[0206] Preferably, said PEG hydrogel precursor molecule is PEG-VS (Polyethylene glycol with terminal vinylsulfone moieties), especially preferable 4-arm or 8-arm PEG-VS.

[0207] More preferably, said fully defined non self-degradable hydrogel matrix array is prepared by crosslinking PEG-VS, especially preferable 4-arm or 8-arm PEG-VS, with a peptide comprising at least two, preferably two cysteine moieties as a crosslinking agent, wherein said crosslinking agent does not comprise any RGD motif. Especially preferred, no bioactive ligand is attached to the hydrogel matrix.

[0208] As an optional bioactive ligand, a ligand comprising a bioactive motif except any RGD adhesion motif may be used.

[0209] Preferably, said optional bioactive ligand is selected from the group consisting of natural laminins, for example laminin-111, in particular mouse laminin-111, recombinant laminin isoforms, and biofunctional fragments thereof. Examples of suitable recombinant laminin isoforms are laminin-111, laminin-211, laminin-332, laminin-411, laminin-511, or laminin-521.

[0210] As an optional bioactive ligand, a ligand comprising glycosaminoglycans such as hyaluronic acid and hyaluronan may be used. Examples of hyaluronic acid are hyaluronic acid 50k, hyaluronic acid 1000k, hyaluronate thiol 50k or hyaluronate thiol 1000k.

[0211] Preferably, said culture medium is characterized by the presence of FBS (serum) or Wnt agonists such as R-spondin. According to a preferred embodiment, a culture medium may be used that is adapted from the medium described in Sachs et al. (The EMBO Journal e 100300|2019). The preferred culture medium comprises AdDMEM/F12 medium supplemented with glutamine, Noggin, EGF, fibroblast growth factor 7 and 10 [FGF7 and FGF10], HGF, R-spondin-conditioned medium, Primocin, penicillin/streptomycin, N-acetyl-L-cysteine, Nicotinamide, A83-01, SB202190 (p38-inhibitor), Y-27632 (rock inhibitor), B27 supplement and HEPES. Other media like the ones described in Lancaster et al. (Nat Bi otechnol 2017 35(7): 659-666), or the commercially available culture media from PromoCell (Small Airway Epithelial Cell Growth Medium (C-39175)) or from Invitrogen (StemPro™ hESC SFM) may be used.

[0212] Especially preferable, said lung cancer cells, preferably nonsmall cell lung cancer cells, overexpressing c-Met are from freshly isolated or frozen human cells from a biopsy or tissue resection of a human or from patient-derived xenograft (PDX) tissue.

[0213] With said method, it is possible to grow, expand and subsequently test said lung cancer cells, preferably non-small cell lung cancer cells, in a selected medium under extracellular matrix conditions that recapitulate drug results observed in vivo.

[0214] According to an especially preferred embodiment, the extracellular matrix conditions are chosen such that the use of a naturally-derived matrix such as Matrigel® can be completely avoided.

[0215] The present invention provides a method with preselected extracellular matrix conditions that sustain the growth as well as the expansion of lung cancer cells, preferably NSCLC cells, using a fully defined or preferably fully synthetic hydrogel matrix. The method allows the reproduction of target expression and drug responses observed in vivo in PDX lung models and not achieved with Matrigel®. Preselection is important, since different ex vivo conditions can promote different drug responses confirming that using a single culture condition may not reflect what is happening in the original patient tumor.

[0216] According to this embodiment, the present invention is also related to a kit of parts for testing the influence of c-Met inhibitors on lung cancer cells, preferably non-small cell lung cancer cells, overexpressing c-Met, comprising: [0217] a) components for preparing a fully defined non self-degradable hydrogel matrix array so as to create fully defined three-dimensional extracellular matrix conditions that differ from each other in their biological, biophysical and/or biochemical characteristics, said components comprising [0218] one or more different PEG hydrogel precursor molecules, preferably PEG-VS, especially preferable 4-arm or 8-arm PEG-VS, [0219] at least one crosslinking agent, preferably a peptide comprising at least two, preferably two, cysteine moieties, [0220] optionally one or more biologically active molecules, wherein said crosslinking agent and said bioactive agent do not comprise any RGD motif; [0221] b) one or more different culture media, preferably comprising FBS (serum) or Wnt agonist such as R-spondin.

[0222] According to this embodiment, the present invention is also related to a kit of parts for testing the influence of c-Met inhibitors on lung cancer cells, preferably non-small cell lung cancer cells, overexpressing c-Met, comprising: [0223] a) components for preparing a fully defined non self-degradable hydrogel matrix array so as to create fully defined three-dimensional extracellular matrix conditions that differ from each other in their biological, biophysical and/or biochemical characteristics, said components comprising [0224] one or more different PEG hydrogel precursor molecules, preferably PEG-VS, especially preferable 4-arm or 8-arm PEG-VS, [0225] at least one crosslinking agent, preferably a peptide comprising at least two, preferably two, cysteine moieties, [0226] optionally one or more biologically active molecules, wherein said crosslinking agent and said bioactive agent do not comprise any RGD motif; [0227] b) one or more different culture media, preferably comprising FBS (serum) or Wnt agonist such as R-spondin [0228] c) optionally, cells from a cell repository/biobank that have been created using the same extracellular matrix conditions.

[0229] According to another preferred variant of this embodiment, the present invention is also related to a kit of parts for testing the influence of c-Met inhibitors and other drugs on lung cancer cells, preferably non-small cell lung cancer cells, comprising: [0230] a) components for preparing a fully defined non self-degradable hydrogel matrix array so as to create fully defined three-dimensional extracellular matrix conditions that differ from each other in their biological, biophysical and/or biochemical characteristics, said components comprising [0231] one or more different PEG hydrogel precursor molecules, preferably PEG-VS, especially preferable 4-arm or 8-arm PEG-VS, [0232] at least one crosslinking agent, preferably a peptide comprising at least two, preferably two, cysteine moieties, [0233] optionally one or more biologically active molecules, wherein said crosslinking agent and said bioactive agent do not comprise any RGD motif; [0234] b) components for preparing a fully defined non self-degradable hydrogel matrix array so as to create fully defined three-dimensional extracellular matrix conditions that differ from each other in their biological, biophysical and/or biochemical characteristics, said components comprising [0235] one or more different PEG hydrogel precursor molecules, preferably PEG-VS, especially preferable 4-arm or 8-arm PEG-VS, [0236] at least one crosslinking agent, preferably a peptide comprising at least two, preferably two, cysteine moieties, [0237] optionally one or more biologically active molecules, wherein said crosslinking agent and/or said bioactive agent comprise an RGD motif; [0238] c) one or more different culture media, preferably comprising FBS (serum) or Wnt agonist such as R-spondin.

[0239] According to another preferred variant of this embodiment, the present invention is also related to a kit of parts for testing the influence of c-Met inhibitors and other drugs on lung cancer cells, preferably non-small cell lung cancer cells, comprising: [0240] a) components for preparing a fully defined non self-degradable hydrogel matrix array so as to create fully defined three-dimensional extracellular matrix conditions that differ from each other in their biological, biophysical and/or biochemical characteristics, said components comprising [0241] one or more different PEG hydrogel precursor molecules, preferably PEG-VS, especially preferable 4-arm or 8-arm PEG-VS, [0242] at least one crosslinking agent, preferably a peptide comprising at least two, preferably two, cysteine moieties, [0243] optionally one or more biologically active molecules, wherein said crosslinking agent and said bioactive agent do not comprise any RGD motif; [0244] b) components for preparing a fully defined non self-degradable hydrogel matrix array so as to create fully defined three-dimensional extracellular matrix conditions that differ from each other in their biological, biophysical and/or biochemical characteristics, said components comprising [0245] one or more different PEG hydrogel precursor molecules, preferably PEG-VS, especially preferable 4-arm or 8-arm PEG-VS, [0246] at least one crosslinking agent, preferably a peptide comprising at least two, preferably two, cysteine moieties, [0247] optionally one or more biologically active molecules, wherein said crosslinking agent and/or said bioactive agent comprise an RGD motif; [0248] c) one or more different culture media, preferably comprising FBS (serum) or Wnt agonist such as R-spondin; [0249] d) optionally, cells from a cell repository/biobank that have been created using the same extracellular matrix conditions.

[0250] With the method and kit of the present invention, it is also possible to culture and test drugs on additional lung cancer cells that bear different disease features (e.g. different lung cancer subtypes, mutations such as EGFR, KRAS or PI3 kinase mutations). By the same manner as described above, extracellular matrix conditions can be preselected for those other cells.

[0251] According to a particularly preferred embodiment, the hydrogel does not comprise any RGD binding site, and especially preferred no integrin binding site at all.

[0252] As can be seen from example 1 and related FIG. 1 discussed below, when testing drugs on their activity against non-small cell lung cancer cells overexpressing c-Met, preselection of the correct conditions is of paramount importance. With Matrigel®, i.e. the standard matrix in the prior art, it is not possible to identify a drug candidate targeting c-Met. As has been found in comparative example 1, this is probably because under conditions of employing Matrigel® the drug target c-Met is not sufficiently expressed and is not activated. Accordingly, under conditions of employing Matrigel® it is not possible to identify suitable drug candidates that act against the most important target in those lung cancer cells, i.e. the overexpressed c-Met.

[0253] Also, in example 1 and related FIG. 1 discussed below it was shown that targeting c-Met is actually a key feature for inhibiting growth of non-small cell lung cancer cells overexpressing c-Met.

[0254] In addition, with the preselected conditions of this embodiment of the present invention, it is also possible to better identify an optimal treatment regime for a specific patient. It was found that certain patients did not respond to drug treatment with a c-met inhibitor alone, presumably because such patients had a cell phenotype that could compensate for c-met inhibition. With the preselected conditions of this embodiment of the present invention, it is possible to screen for drug combination treatment using a c-met inhibitor together with another drug type. As discussed above, this is not possible with prior art conditions using Matrigel®. The present invention provides a better predictability for patient response.

[0255] The preselected extracellular matrix conditions of this embodiment can also be used in a method for testing the efficiency of therapies for a cancer patient, comprising the steps: [0256] a) providing freshly isolated or frozen lung cancer cells, preferably non-small cell lung cancer cells, from a biopsy or a tissue resection of a cancer patient; [0257] b) establishing and expanding organoids from said cells, and applying one or more drugs to said organoids by the method described above; [0258] c) comparing the activity of the one or more drugs applied in step b) with the result of the treatment of said patient with one of said drugs applied in step b); [0259] d) and/or providing drug activity results on patient organoids and corresponding genetic and phenotypic data of the disease to support physician in making decisions on how to treat the patient.

[0260] Patient biopsies or resections dedicated for the isolation of lung cancer cells, preferably non-small cell lung cancer (NSCLC) cells, to establish organoids in step b), can be collected during a standard diagnostic procedure and subsequently transported to the site where step b) is to be conducted.

[0261] Step b) of this method is conducted as described above, i.e. by providing preselected extracellular matrix conditions comprising a fully defined non self-degradable hydrogel matrix array with discrete volumes having a stiffness in the range of 50 to 2000 Pa and prepared by crosslinking, onto a substrate or into discrete volumes of a substrate, preferably a multi-well plate, different combinations of one or more different PEG hydrogel precursor molecules in the presence of optionally one or more biologically active molecules, at least one crosslinking agent, and said lung cancer cells, preferably non-small cell lung cancer cells, so as to create fully defined three-dimensional extracellular matrix conditions that differ from each other in their biological, biophysical and/or biochemical characteristics;

[0262] allowing said lung cancer cells, preferably non-small cell lung cancer cells, to grow in said discrete volumes of said hydrogel matrix in the presence of one or more different culture media preferably comprising FBS (serum) or Wnt agonist such as R-spondin, especially preferred also comprising FGF-7, FGF-10, and a TGF-β inhibitor;

[0263] and adding one or more drugs to the cells grown in said discrete volumes of said hydrogel matrix;

[0264] wherein said crosslinking agent and said optional bioactive agent do not comprise a RGD motif.

[0265] The one or more drugs added to the cells grown in said discrete volumes of said hydrogel matrix comprise the drug(s) used for anticancer standard of care (SoC) treatment of the patient.

[0266] According to a preferred embodiment, the biopsy or resection can additionally be processed for histological analysis and/or omics testing (e.g. NGS) to establish the baseline for reference. The results of these additional analyses can also be used for comparing and/or correlating the above ex vivo and in vivo tests.

[0267] Based on this method, it is possible to reliably assess whether the applied anticancer standard of care (SoC) treatment is suitable, or whether a different drug treatment regime tested ex vivo as described above might be more promising. Thus, with the present invention the cancer treatment can be personalized and optimized. Functional in vitro data can be generated that may increase accuracy of treatment decisions by health care providers.

Patient-Derived Organoids (PDO) in Precision Medicine

[0268] Cancer is a multifactorial disease that results from both genetic and epigenetic transformation of normal cells, leading to abnormal proliferation. Conventional cancer treatments include surgical resection, radiotherapy, non-specific or targeted chemotherapies and immunotherapy to inhibit cell division or induce apoptosis of cancer cells.

[0269] Different cancer types respond to treatment in different ways, and therefore some cancer types can be treated better than others. Despite the development of potent chemotherapeutics and oncogene-specific targeted drugs, durable or long-lasting cure of this disease has not been achieved for many patients.

[0270] Recent improvements of DNA sequencing technologies allow the fast identification of specific genome mutations of patient tumors with the potential of tailoring cancer therapies based on molecular profiles of tumors. Significant improvements were shown in the treatment of leukemia, lung and melanoma cancers (Druker et al. (N Engl J Med, Vol. 344, No. 14 (2001), 1031), Lynch et al. (N Engl J Med 350; 21 (2004), 2129), Flaherty et al. (N Engl J Med 2010; 363:809-19). However, the clinical benefit of genome-guided precision medicine is still highly debatable (Le Tourneau et al. (www.thelancet.com/oncology, Published online Sep. 3, 2015, http://dx.doi.org/10.1016/S1470-2045(15)00188-6), Prasad (Nature 537 (2016), S63), Letai (NATURE MEDICINE, VOLUME 23|NUMBER 9|September 2017, 1028). Recent clinical trials assessing the rate of assigning patients with solid tumors to targeted therapies showed that only part of them (10-50%) bear mutations matching available clinically validated and approved therapies (Letai 2017, Sicklick (Nature Medicine https://doi.org/10.1038/s41591-019-0407-5 (2019)). Besides this, two fundamental biological aspects impair the efficiency of genome-guided precision medicine: [0271] resistance to the specific therapy due to the intra-patient genetic and phenotypic heterogeneity of cancer cells (presence of divergent subclones) (Tannock et al. (N Engl J Med 375; 13 (2016), 1289), Flavahan et al., (Science 357, 266 (2017)); [0272] insufficient biological understanding of the tumor microenvironment effect in modulating the drug response (Friedmann et al. (Nature Reviews Cancer AOP, published online 5 Nov. 2015; doi:10.1038/nrc4015 2015)).

[0273] Screens of drugs on patient-derived cells (functional precision medicine) could address these limitations and be complementary to genomics and pathological data to support the prediction of patient outcome and therefore guiding the decision-making therapy process.

[0274] Novel in vitro tumor biology models that recapitulate the in vivo tumor microenvironment, such as Patient Derived Organoids (PDO), have the advantage of growing in a 3D environment, reproducing the spatial architecture of the original tissue. Organoids are miniature 3D in vitro structures grown from patient-derived cells that mimic key features and functions of its original healthy or diseased tissue. A variety of PDO have been established for many tumors including—but not limited to—colorectal cancer (Sato et al. (Nature vol. 469 (2011), 415), van de Wetering et al. (Cell 161 (2015), 933-945), pancreas ductal adenocarcinoma (Boj et al. (Cell 160, 324-338, Jan. 15, 2015), Huang et al. (Nature medicine, published online 26 Oct. 2015; doi:10.1038/nm.3973), breast cancer (Sachs et al. (Cell 172 2018, 1-14) and lung cancer (Sachs et al. (The EMBO Journal e 100300|2019). Overall, these studies showed that PDO can maintain the same genetic driver mutations identified in the primary tumor.

[0275] Recently, patient organoids derived from different locations of the same tumor were used to study the nature and extent of intra-tumor heterogeneity and to assess their response to a panel of drugs (Roerink et al. Nature 556, 457-462, 2018). Significant differences in responses to drugs between closely related cells of the same tumor were observed.

[0276] Prospective use of organoids as functional diagnostic tool in clinic has been shown already for rectal cancer (Ganesh et al., Nature Medicine, 10, 1067-1614(2019)) metastatic colorectal cancer (Vlachogiannis et al., Science 359, 920-926 (2018) and Ooft et al., Science Translational Medicine, 11, (2019), DOI: 10.1126/scitranslmed.aay2574), pancreatic cancer (Tiriac, CANCER DISCOVERY, SEPTEMBER 2018, DOI: 10.1158/2159-8290.CD-18-0349) and appendiceal cancer (Votanopoulos et al., Ann Surg Oncol (2019) 26:139-147). In these studies, the drug response of the PDOs correlates with the outcome of the same treatment on patients from which the organoids were derived.

[0277] Despite these promising results of the PDO drug responses matching the corresponding patient outcomes, these studies are confined to a limited number of patients, and the methods used rely on a basal membrane extract (BME) with undefined composition and batch to batch variability, such as Matrigel®. This represents a significant limitation in the standardization of the PDO for translation to clinically relevant applications. Also, only single culture conditions were employed for each type of cancer, regardless of possible differences in genetic and/or phenotypic tumor features, which also include, but is not limited to the biomarker expression, that may require different extracellular matrix conditions. This may favour the growth of specific cell populations or inducing the expression of only some phenotypes during ex vivo cell expansion (WO 2010/090513 A2; WO 2016/015158 A1; WO 2015/173425 A1) and therefore failing to mimic in vivo tumor characteristics and drug responses. This has been outlined above with respect to lung cancer cells, preferably non-small cell lung cancer cells, overexpressing c-Met.

[0278] In order to overcome the limitations of naturally-derived matrices such as Matrigel®, fully-defined and also synthetic hydrogels have been already investigated to grow a variety of tissues, including intestinal and colon organoids from mouse and human origins (Gjorevski et al., Designer matrices for intestinal stem cell and organoid culture, Nature, Vol 539, 24 Nov. 2016, 560-56, WO 2017/037295 A1; or Cruz-Acuna et al., Synthetic hydrogels for human intestinal organoid generation and colonic wound repair, Nature cell biology, advanced online publication published online 23 Oct. 2017; DOI: 10.1038/ncb3632, 1-23, WO 2018/165565 A1) and more recently from appendiceal, pancreatic and mesothelioma cancer patient cells (Votanopoulos 2019 (above), Broguiere et al., Adv. Mater. 2018, U.S. Pat. No. 1,801,621 (2018), Mazzocchi et al., SCIENTIFIC Reports (2018) 8:2886 DOI:10.1038/s41598-018-21200-8).

[0279] Although some of these studies are using a fully-defined or fully synthetic matrix, they still rely on the use of single culture condition regardless of the tumor feature.

Pancreatic Cancer

[0280] With the present invention, it is possible to culture patient pancreatic cells, preferably pancreatic ductal adenocarcinoma (PDAC) cells, under conditions that sustain the growth and expansion of these cells. Subsequently, the cells are exposed to different drug treatments in order to select an efficient drug treatment for the cancer patient.

[0281] According to the present invention, it could be shown that PDAC cells could be well cultured and tested using a combination of a fully defined soft (50-1000 Pa stiffness) or medium (1000-2000 Pa) or hard (2000-3000 Pa) non self-degradable hydrogel matrix comprising at least one RGD adhesion motif and a culture medium, preferably comprising Wnt agonists such as R-spondin and Wnt 3a.

[0282] Thus, according to this embodiment, the present invention is related to a method of testing the influence of drugs on pancreatic ductal adenocarcinoma (PDAC) cells, comprising the steps of: [0283] a) providing preselected extracellular matrix conditions comprising a fully defined non self-degradable hydrogel matrix array with discrete volumes having a stiffness in the range of 50 to 3000 Pa, preferably 50 to 2000 Pa and most preferably 50 to 1000 Pa, and prepared by crosslinking, onto a substrate or into discrete volumes of a substrate, preferably a multiwell plate, different combinations of one or more different PEG hydrogel precursor molecules, in the presence of optionally one or more biologically active molecules, at least one crosslinking agent, and said pancreatic ductal adenocarcinoma cells, so as to create fully defined three-dimensional extracellular matrix conditions that differ from each other in their biological, biophysical and/or biochemical characteristics; [0284] b) allowing said pancreatic ductal adenocarcinoma cells to grow in said discrete volumes of said hydrogel matrix in the presence of one or more different culture media, preferably comprising Wnt agonists such as R-spondin and Wnt 3a; [0285] c) adding one or more drugs to the cells grown in said discrete volumes of said hydrogel matrix;

[0286] wherein at least one of said crosslinking agent and/or said optional bioactive agent comprises a RGD motif.

[0287] Preferably, said PEG hydrogel precursor molecule is PEG-VS (Polyethylene glycol with terminal vinylsulfone moieties), especially preferable 4-arm or 8-arm PEG-VS.

[0288] More preferably, said fully defined non self-degradable hydrogel matrix array is prepared by crosslinking PEG-VS, especially preferable 4-arm or 8-arm PEG-VS, with a peptide comprising at least two, preferably two cysteine moieties as a crosslinking agent, wherein said crosslinking agent may comprise a RGD motif.

[0289] As an optional bioactive ligand, a ligand comprising a bioactive motif including a RGD adhesion motif may be used. Examples of suitable RGD motifs are RGD, RGDS, RGDSP, RGDSPG, RGDSPK, RGDTP, RGDSPASSKP, PHSRNSGSGSGSGSGRGDSPG or any cyclic RGD motifs such as cyclo(RGDfC), but principally any known and successfully employed RGD sequences, in the field of hydrogels and cell culture, could be used.

[0290] Preferably, said optional bioactive ligand is selected from the group consisting of natural laminins, for example laminin-111, in particular mouse laminin-111, recombinant laminin isoforms, and biofunctional fragments thereof. Examples of suitable recombinant laminin isoforms are laminin-111, laminin-211, laminin-332, laminin-411, laminin-511, or laminin-521, laminin-511 being preferred.

[0291] As an optional bioactive ligand, a ligand comprising a bioactive motif including a collagen peptide motif may be used. Example of a suitable collagen peptide is DGEA.

[0292] As an optional bioactive ligand, a ligand comprising glycosaminoglycans such as hyaluronic acid and hyaluronan may be used. Examples of hyaluronic acid are hyaluronic acid 50k, hyaluronic acid 1000k, hyaluronate thiol 50k or hyaluronate thiol 1000k.

[0293] Preferably, said culture medium is characterized by the presence of Wnt agonists such as R-spondin and Wnt 3a. According to a preferred embodiment, a culture medium may be used that is described in Boj et al. (Cell 160, 324-338, Jan. 15, 2015), p. 335, right col., 2.sup.nd para, or Huang et al. (Nature medicine, published online 26 Oct. 2015; doi:10.1038/nm.3973). Especially preferred is the culture medium adapted from Boj et al., which comprises AdDMEM/F12 medium supplemented with HEPES, Glutamax, penicillin/streptomycin, B27, Primocin, N-acetyl-L-cysteine, Wnt3a-conditioned medium [50% v/v] or recombinant protein [100 ng/ml], RSPO1-conditioned medium [10% v/v] or recombinant protein [500 ng/ml], Noggin-conditioned medium [10% v/v] or recombinant protein [0.1 μg/ml], epidermal growth factor [EGF, 50 ng/ml], Gastrin [10 nM], fibroblast growth factor 10 [FGF10, 100 ng/ml], Nicotinamide [10 mM], Prostaglandin E2 [PGE2, 1 μM] and A83-01 [0.5 μM].

[0294] Especially preferable, said pancreatic ductal adenocarcinoma cells are from freshly isolated or frozen cells from a biopsy or tissue resection of a human or from patient-derived xenograft (PDX) tissue.

[0295] With said method, it is possible to grow, expand and subsequently test said pancreatic ductal adenocarcinoma cells in a selected medium under extracellular matrix conditions that recapitulate drug results observed in vivo.

[0296] According to an especially preferred embodiment, the extracellular matrix conditions are chosen such that the use of a naturally-derived matrix such as Matrigel® can be completely avoided.

[0297] According to this embodiment, the present invention is also related to a kit of parts for testing the influence of drugs on pancreatic ductal adenocarcinoma cells, comprising: [0298] a) components for preparing a fully defined non self-degradable hydrogel matrix array having a stiffness in the range of 50 to 3000 Pa, preferably 50 to 2000 Pa and most preferably 50 to 1000 Pa, so as to create fully defined three-dimensional extracellular matrix conditions that differ from each other in their biological, biophysical and/or biochemical characteristics, said components comprising [0299] one or more different PEG hydrogel precursor molecules, preferably PEG-VS, especially preferable 4-arm or 8-arm PEG-VS, [0300] at least one crosslinking agent, preferably a peptide comprising at least two, preferably two, cysteine moieties, [0301] optionally one or more biologically active molecules, wherein at least one of said crosslinking agent and/or said optional bioactive agent comprises a RGD motif; [0302] b) one or more different culture media, preferably comprising Wnt agonists such as R-spondin and Wnt 3a.

[0303] According to this embodiment, the present invention is also related to a kit of parts for testing the influence of drugs on pancreatic ductal adenocarcinoma cells, comprising: [0304] a) components for preparing a fully defined non self-degradable hydrogel matrix array having a stiffness in the range of 50 to 3000 Pa, preferably 50 to 2000 Pa and most preferably 50 to 1000 Pa, so as to create fully defined three-dimensional extracellular matrix conditions that differ from each other in their biological, biophysical and/or biochemical characteristics, said components comprising [0305] one or more different PEG hydrogel precursor molecules, preferably PEG-VS, especially preferable 4-arm or 8-arm PEG-VS, [0306] at least one crosslinking agent, preferably a peptide comprising at least two, preferably two, cysteine moieties, [0307] optionally one or more biologically active molecules, wherein at least one of said crosslinking agent and/or said optional bioactive agent comprises a RGD motif; [0308] b) one or more different culture media, preferably comprising Wnt agonists such as R-spondin and Wnt 3a; [0309] c) optionally, cells from a cell repository/biobank that have been created using the same extracellular matrix conditions.

[0310] The preselected extracellular matrix conditions of this embodiment can also be used in a method for testing the efficiency of therapies for a cancer patient, comprising the steps: [0311] a) providing freshly isolated or frozen pancreatic ductal adenocarcinoma cells from a biopsy or a tissue resection of a cancer patient; [0312] b) establishing and expanding organoids from said cells, and applying one or more drugs to said organoids by the method described above; [0313] c) comparing the activity of the one or more drugs applied in step b) with the result of the treatment of said patient with one of said drugs applied in step b); [0314] d) and/or providing drug activity results on patient organoids and corresponding genetic and phenotypic data of the disease to support physician in making decisions on how to treat the patient.

[0315] Patient biopsies or resections dedicated for the isolation of pancreatic ductal adenocarcinoma (PDAC) cells to establish organoids in step b), can be collected during a standard diagnostic procedure and subsequently transported to the site where step b) is to be conducted.

[0316] Step b) of this method is conducted as described above, i.e. by providing preselected extracellular matrix conditions comprising a fully defined non self-degradable hydrogel matrix array with discrete volumes having a stiffness in the range of 50 to 3000 Pa, preferably 50 to 2000 Pa and most preferably 50 to 1000 Pa and prepared by crosslinking, onto a substrate or into discrete volumes of a substrate, preferably a multi-well plate, different combinations of one or more different PEG hydrogel precursor molecules in the presence of optionally one or more biologically active molecules, at least one crosslinking agent, and said pancreatic ductal adenocarcinoma cells, so as to create fully defined three-dimensional extracellular matrix conditions that differ from each other in their biological, biophysical and/or biochemical characteristics;

[0317] allowing said pancreatic ductal adenocarcinoma cells to grow in said discrete volumes of said hydrogel matrix in the presence of one or more different culture media preferably comprising Wnt agonists such as R-spondin and Wnt 3a;

[0318] and adding one or more drugs to the cells grown in said discrete volumes of said hydrogel matrix;

[0319] wherein at least one of said crosslinking agent and/or said optional bioactive agent comprises a RGD motif.

[0320] The one or more drugs added to the cells grown in said discrete volumes of said hydrogel matrix comprise the drug(s) used for anticancer standard of care (SoC) treatment of the patient.

[0321] According to a preferred embodiment, the biopsy or resection can additionally be processed for histological analysis and/or omics testing (e.g. NGS) to establish the baseline for reference. The results of these additional analyses can also be used for comparing and/or correlating the above ex vivo and in vivo tests.

[0322] Based on this method, it is possible to reliably assess whether the applied anticancer standard of care (SoC) treatment is suitable, or whether a different drug treatment regime tested ex vivo as described above might be more promising. Thus, with the present invention the cancer treatment can be personalized and optimized. Functional in vitro data can be generated that may increase accuracy of treatment decisions by health care providers.

Co-Culturing of PDAC Cells

[0323] According to another preferred embodiment of the present invention, cancer cells and preferably pancreatic ductal adenocarcinoma (PDAC) cells can be co-cultured in combination with stromal cells, preferably fibroblasts. For this embodiment, the hydrogel matrix is preselected as being a preferably non self-degradable PEG hydrogel having a stiffness in the range of 50 to 3000 Pa, preferably 50 to 2000 Pa and most preferably 50 to 1000 Pa, wherein at least one of the crosslinking agents comprises an enzymatically degradable motif, preferably a MMP-sensitive motif, and wherein at least one of the crosslinking agent and/or said optional bioactive agent comprise a RGD motif.

[0324] Preferably, the culture medium to be used in said embodiment comprises Wnt agonists such as R-spondin and Wnt 3a and preferably additionally FBS.

[0325] Thus, according to this embodiment, the present invention is related to a method of testing cancer cells, preferably pancreatic ductal adenocarcinoma (PDAC) cells, that have been co-cultured with stromal cells, preferably fibroblasts, comprising the steps of: [0326] a) providing preselected extracellular matrix conditions comprising a fully defined, preferably non self-degradable hydrogel matrix array with discrete volumes having a stiffness in the range of 50 to 3000 Pa, preferably 50 to 2000 Pa and most preferably 50 to 1000 Pa and prepared by crosslinking, onto a substrate or into discrete volumes of a substrate, preferably a multi-well plate, different combinations of one or more different PEG hydrogel precursor molecules, in the presence of optionally one or more biologically active molecules, at least one crosslinking agent, and said cancer cells, preferably pancreatic ductal adenocarcinoma (PDAC) cells, and said stromal cells, preferably fibroblasts, so as to create fully defined three-dimensional extracellular matrix conditions that differ from each other in their biological, biophysical and/or biochemical characteristics; [0327] b) allowing said pancreatic ductal adenocarcinoma cells and stromal cells, preferably fibroblasts, to grow in said discrete volumes of said hydrogel matrix array in the presence of one or more different culture media, preferably comprising Wnt agonists such as R-spondin and Wnt 3a, and preferably additionally FBS; [0328] c) adding one or more drugs to the cells grown in said discrete volumes of said hydrogel matrix;

[0329] wherein the at least one crosslinking agent comprises an enzymatically degradable motif, preferably a MMP-sensitive motif, and wherein at least one of the crosslinking agent and/or said optional bioactive agent comprise a RGD motif.

[0330] According to a preferred embodiment, said method is carried out such that at least two different arrays are provided, wherein the arrays differ with respect to the presence or absence of an enzymatically degradable motif, preferably a MMP-sensitive motif. In the array where said enzymatically degradable motif, preferably MMP-sensitive motif, is present, the PDAC cells can be co-cultured with the stromal cells, preferably fibroblasts. In the array where said enzymatically degradable motif, preferably MMP-sensitive motif, is not present, the PDAC cells are grown in a single culture that impairs the growth of stromal cells such as fibroblasts.

[0331] Preferably, said PEG hydrogel precursor molecule is PEG-VS (Polyethylene glycol with terminal vinylsulfone moieties), especially preferable 4-arm or 8-arm PEG-VS. According to another preferred embodiment, a self-degradable PEG may be prepared from one or more PEG-Acr precursor molecules and used alone or in combination with a PEG-VS precursor molecule.

[0332] More preferably, said fully defined, preferably non self-degradable hydrogel matrix array is prepared by crosslinking PEG-VS, especially preferable 4-arm or 8-arm PEG-VS, with a peptide comprising at least two, preferably two cysteine moieties as a crosslinking agent, wherein said crosslinking agent comprises an enzymatically degradable motif, preferably a MMP-sensitive motif, and additionally may comprise a RGD motif.

[0333] As an optional bioactive ligand, a ligand comprising a bioactive motif including a RGD adhesion motif may be used. Examples of suitable RGD motifs are RGD, RGDS, RGDSP, RGDSPG, RGDSPK, RGDTP, RGDSPASSKP, PHSRNSGSGSGSGSGRGDSPG or any cyclic RGD motifs such as cyclo(RGDfC), but principally any known and successfully employed RGD sequences, in the field of hydrogels and cell culture, could be used.

[0334] Preferably, said optional bioactive ligand is selected from the group consisting of natural laminins, for example laminin-111, in particular mouse laminin-111, recombinant laminin isoforms, and biofunctional fragments thereof. Examples of suitable recombinant laminin isoforms are laminin-111, laminin-211, laminin-332, laminin-411, laminin-511, or laminin-521, laminin-511 being preferred.

[0335] As an optional bioactive ligand, a ligand comprising a bioactive motif including a collagen peptide motif may be used. Example of a suitable collagen peptide is DGEA.

[0336] As an optional bioactive ligand, a ligand comprising glycosaminoglycans such as hyaluronic acid and hyaluronan may be used. Examples of hyaluronic acid are hyaluronic acid 50k, hyaluronic acid 1000k, hyaluronate thiol 50k or hyaluronate thiol 1000k.

[0337] Preferably, said culture medium is characterized by the presence of Wnt agonists such as R-spondin and Wnt 3a, and preferably additionally FBS. According to a preferred embodiment, a culture medium may be used that is described in Boj et al. (Cell 160, 324-338, Jan. 15, 2015), p. 335, right col., 2.sup.nd para, or Huang et al. (Nature medicine, published online 26 Oct. 2015; doi:10.1038/nm.3973). Especially preferred is the culture medium adapted from Boj et al., which comprises AdDMEM/F12 medium supplemented with HEPES, Glutamax, penicillin/streptomycin, B27, Primocin, N-acetyl-L-cysteine, Wnt3a-conditioned medium [50% v/v] or recombinant protein [100 ng/ml], RSPO1-conditioned medium [10% v/v] or recombinant protein [500 ng/ml], Noggin-conditioned medium [10% v/v] or recombinant protein [0.1 μg/ml], epidermal growth factor [EGF, 50 ng/ml], Gastrin [10 nM], fibroblast growth factor 10 [FGF10, 100 ng/ml], Nicotinamide [10 mM], Prostaglandin E2 [PGE2, 1 μM] and A83-01 [0.5 μM].

[0338] Especially preferable, said pancreatic ductal adenocarcinoma cells are from freshly isolated or frozen cells from a biopsy or tissue resection of a human or from patient-derived xenograft (PDX) tissue, or from patient-derived organoids (PDO), optionally pre-established in BME, such as Matrigel®.

[0339] Preferably, said stromal cells are isolated from patient. Especially preferable, said stromal cells are fibroblasts.

[0340] With said method, it is possible to grow, expand and subsequently test said pancreatic ductal adenocarcinoma cells in co-culture with stromal cells in a selected medium under extracellular matrix conditions that recapitulate drug results observed in vivo.

[0341] According to an especially preferred embodiment, the extracellular matrix conditions are chosen such that the use of a naturally-derived matrix such as Matrigel® can be completely avoided.

[0342] According to this embodiment, the present invention is also related to a kit of parts for testing the influence of drugs on cancer cells, preferably pancreatic ductal adenocarcinoma (PDAC) cells, that have been co-cultured with stromal cells, preferably fibroblasts, comprising: [0343] a) components for preparing a fully defined, preferably non self-degradable hydrogel matrix array having a stiffness in the range of 50 to 3000 Pa, preferably 50 to 2000 Pa and most preferably 50 to 1000 Pa, so as to create fully defined three-dimensional extracellular matrix conditions that differ from each other in their biological, biophysical and/or biochemical characteristics, said components comprising [0344] one or more different PEG hydrogel precursor molecules, preferably PEG-VS, especially preferable 4-arm or 8-arm PEG-VS, [0345] at least one crosslinking agent, preferably a peptide comprising at least two, preferably two, cysteine moieties, [0346] optionally one or more biologically active molecules, wherein the at least one crosslinking agent preferably comprises an enzymatically degradable motif, preferably a MMP-sensitive motif, and wherein at least one of the crosslinking agent and/or said optional bioactive agent comprise a RGD motif; [0347] b) one or more different culture media, preferably comprising Wnt agonists such as R-spondin and Wnt 3a, preferably additionally FBS.

[0348] According to this embodiment, the present invention is also related to a kit of parts for testing the influence of drugs on cancer cells, preferably pancreatic ductal adenocarcinoma (PDAC) cells, that have been co-cultured with stromal cells, preferably fibroblasts, comprising: [0349] a) components for preparing a fully defined, preferably non self-degradable hydrogel matrix array having a stiffness in the range of 50 to 3000 Pa, preferably 50 to 2000 Pa and most preferably 50 to 1000 Pa, so as to create fully defined three-dimensional extracellular matrix conditions that differ from each other in their biological, biophysical and/or biochemical characteristics, said components comprising [0350] one or more different PEG hydrogel precursor molecules, preferably PEG-VS, especially preferable 4-arm or 8-arm PEG-VS, [0351] at least one crosslinking agent, preferably a peptide comprising at least two, preferably two, cysteine moieties, [0352] optionally one or more biologically active molecules, wherein the at least one crosslinking agent preferably comprises an enzymatically degradable motif, preferably a MMP-sensitive motif, and wherein at least one of the crosslinking agent and/or said optional bioactive agent comprise a RGD motif; [0353] b) one or more different culture media, preferably comprising Wnt agonists such as R-spondin and Wnt 3a, preferably additionally FBS [0354] c) optionally, cells from a cell repository/biobank that have been created using the same extracellular matrix conditions.

[0355] The preselected extracellular matrix conditions of this embodiment can also be used in a method for testing the efficiency of therapies for a cancer patient, comprising the steps: [0356] a) providing freshly isolated or frozen cancer cells, preferably pancreatic ductal adenocarcinoma cells, from a biopsy or a tissue resection of a cancer patient, and providing stromal cells isolated from patient, so as to establish a co-culture system; [0357] b) establishing and expanding cells from said co-culture system, and applying one or more drugs to said cells by the method described above; [0358] c) comparing the activity of the one or more drugs applied in step b) with the result of the treatment of said patient with one of said drugs applied in step b); [0359] d) and/or providing drug activity results on patient organoids and corresponding genetic and phenotypic data of the disease to support physician in making decisions on how to treat the patient.

[0360] Patient biopsies or resections dedicated for the isolation of pancreatic ductal adenocarcinoma (PDAC) cells to establish organoids in step b), can be collected during a standard diagnostic procedure and subsequently transported to the site where step b) is to be conducted.

[0361] Step b) of this method is conducted as described above, i.e. by providing preselected extracellular matrix conditions comprising a fully defined, preferably non self-degradable hydrogel matrix array with discrete volumes having a stiffness in the range of 50 to 3000 Pa, preferably 50 to 2000 Pa and most preferably 50 to 1000 Pa and prepared by crosslinking, onto a substrate or into discrete volumes of a substrate, preferably a multi-well plate, different combinations of one or more different PEG hydrogel precursor molecules in the presence of optionally one or more biologically active molecules, at least one crosslinking agent, and said cancer cells, preferably pancreatic ductal adenocarcinoma cells, co-cultured with stromal cells, preferably fibroblasts, so as to create fully defined three-dimensional extracellular matrix conditions that differ from each other in their biological, biophysical and/or biochemical characteristics;

[0362] allowing said cancer cells, preferably pancreatic ductal adenocarcinoma cells, and said stromal cells, preferably fibroblasts, to grow in said discrete volumes of said hydrogel matrix in the presence of one or more different culture media preferably comprising Wnt agonists such as R-spondin and Wnt 3a, preferably additionally FBS;

[0363] and adding one or more drugs to the cells grown in said discrete volumes of said hydrogel matrix;

[0364] wherein the at least one crosslinking agent preferably comprises an enzymatically degradable motif, preferably a MMP-sensitive motif, and wherein at least one of the crosslinking agent and/or said optional bioactive agent comprise a RGD motif.

[0365] The one or more drugs added to the cells grown in said discrete volumes of said hydrogel matrix comprise the one or more drugs used for anticancer standard of care (SoC) treatment of the patient.

[0366] According to a preferred embodiment, the biopsy or resection can additionally be processed for histological analysis and/or omics testing (e.g. NGS) to establish the baseline for reference. The results of these additional analyses can also be used for comparing and/or correlating the above ex vivo and in vivo tests. Based on this method, it is possible to reliably assess whether the applied anticancer standard of care (SoC) treatment is suitable, or whether a different drug treatment regime tested ex vivo as described above might be more promising. Thus, with the present invention the cancer treatment can be personalized and optimized. Functional in vitro data can be generated that may increase accuracy of treatment decisions by health care providers.

Colorectal Cancer

[0367] Colorectal cancer (CRC) cells are known to exhibit heterogeneity (Roerink et al., (Nature, published online https://doi.org/10.1038/s41586-018-0024-3 (2018))). Significant differences in responses to drugs between closely related cells of the same tumor were observed. The same discussion as before for the pancreatic cells applies here.

[0368] With the present invention, it is possible to culture patient colorectal cancer (CRC) cells under conditions that sustain the growth and expansion of these cells. Subsequently, the cells are exposed to different drug treatments in order to select an efficient drug treatment for the cancer patient. Thus, the present invention provides a precision medicine platform enabling the growth and drug testing of CRC tissues in different microenvironments and therefore captures the specificities of multiple clones inside a single tumor.

[0369] According to the present invention, it could be shown that colorectal cancer (CRC) cells could be well cultured and tested using a combination of a fully defined soft or medium (50-2000 Pa stiffness) hydrogel matrix comprising at least one RGD adhesion motif and optionally laminin, preferably laminin-111 or laminin-511, and especially preferable natural mouse laminin-111 or recombinant human laminin-511, as an additional bioactive ligand, and a culture medium, preferably comprising Wnt agonists such as R-spondin and Wnt 3a.

[0370] Thus, according to this embodiment, the present invention is related to a method of testing the influence of drugs on colorectal cancer (CRC) cells, comprising the steps of: [0371] a) providing preselected extracellular matrix conditions comprising a fully defined hydrogel matrix array with discrete volumes having a stiffness in the range of 50 to 2000 Pa and prepared by crosslinking, onto a substrate or into discrete volumes of a substrate, preferably a multi-well plate, different combinations of one or more different PEG hydrogel precursor molecules, in the presence of optionally one or more biologically active molecules comprising laminin, preferably laminin-111 or laminin-511, and especially preferable natural mouse laminin-111 or recombinant human laminin-511, at least one crosslinking agent, and said colorectal cancer cells, so as to create fully defined three-dimensional extracellular matrix conditions that differ from each other in their biological, biophysical and/or biochemical characteristics; [0372] b) allowing said colorectal cancer cells to grow in said discrete volumes of said hydrogel matrix in the presence of one or more different culture media, preferably comprising Wnt agonists such as R-spondin and Wnt 3a; [0373] c) adding one or more drugs to the cells grown in said discrete volumes of said hydrogel matrix;

[0374] wherein at least one of said crosslinking agent and/or said bioactive agent comprises a RGD motif.

[0375] Preferably, said PEG hydrogel precursor molecules are PEG-VS (Polyethylene glycol with terminal vinylsulfone moieties), especially preferable 4-arm or 8-arm PEG-VS, and/or PEG-Acr (Polyethylene glycol with terminal acrylate moieties), especially preferable 4-arm or 8-arm PEG-Acr.

[0376] More preferably, said fully defined self-degradable hydrogel matrix array is prepared by crosslinking a 50:50 mixture of PEG-VS, especially preferable 4-arm or 8-arm PEG-VS, and PEG-Acr, especially preferable 4-arm or 8-arm PEG-Acr, with a peptide comprising at least two, preferably two cysteine moieties as a crosslinking agent, wherein said crosslinking agent may comprise a RGD motif.

[0377] More preferably, said fully defined non self-degradable hydrogel matrix array is prepared by crosslinking PEG-VS, especially preferable 4-arm or 8-arm PEG-VS, with a peptide comprising at least two, preferably two cysteine moieties as a crosslinking agent, wherein said crosslinking agent may comprise a RGD motif.

[0378] As an optional bioactive ligand, a ligand comprising a bioactive motif including a RGD adhesion motif may be used. Examples of suitable RGD motifs are RGD, RGDS, RGDSP, RGDSPG, RGDSPK, RGDTP, RGDSPASSKP, PHSRNSGSGSGSGSGRGDSPG or any cyclic RGD motifs such as cyclo(RGDfC), but principally any known and successfully employed RGD sequences, in the field of hydrogels and cell culture, could be used.

[0379] Preferably, said optional bioactive ligand is selected from the group consisting of natural laminins, for example laminin-111, in particular mouse laminin-111, recombinant laminin isoforms such as recombinant human laminin-511, and biofunctional fragments thereof. Examples of suitable recombinant laminin isoforms are laminin-111, laminin-211, laminin-332, laminin-411, laminin-511, or laminin-521.

[0380] Preferably, said culture medium is characterized by the presence of Wnt agonists such as R-Spondin and Wnt 3a. According to a preferred embodiment, a culture medium may be used that is described in Vlachogiannis et al., Science 359, 920-926 (2018) (see e.g. supplementary material, p. 5, Human PDO culture media). Alternatively, the commercially available culture medium Intesticult® may be used. Especially preferred is the culture medium described in Vlachogiannis et al., which comprises Advanced DMEM/F12, supplemented with B27 additive, N2 additive, BSA, L-Glutamine, penicillin-Streptomycin, EGF, Noggin, R-Spondin 1, Gastrin, FGF-10, FGF-basic, Wnt-3A, Prostaglandin E 2, Y-27632, Nicotinamide, A83-01, SB202190 and optionally HGF.

[0381] Especially preferable, said colorectal cancer cells are from freshly isolated or frozen cells from a biopsy or tissue resection of a human or from patient-derived xenograft (PDX) tissue.

[0382] With said method, it is possible to grow, expand and subsequently test said colorectal cancer cells in a selected medium under conditions that recapitulate drug results observed in vivo.

[0383] According to an especially preferred embodiment, the extracellular matrix conditions are chosen such that the use of a naturally-derived matrix such as Matrigel® can be completely avoided.

[0384] According to this embodiment, the present invention is also related to a kit of parts for testing the influence of drugs on colorectal cancer cells, comprising: [0385] a) components for preparing a fully defined hydrogel matrix array having a stiffness in the range of 50 to 2000 Pa, so as to create fully defined three-dimensional extracellular matrix conditions that differ from each other in their biological, biophysical and/or biochemical characteristics, said components comprising [0386] one or more different PEG hydrogel precursor molecules, preferably PEG-VS, especially preferable 4-arm or 8-arm PEG-VS, and/or PEG-Acr, especially preferable 4-arm or 8-arm PEG-Acr, [0387] at least one crosslinking agent, preferably a peptide comprising at least two, preferably two, cysteine moieties, [0388] optionally one or more biologically active molecules comprising laminin, preferably laminin-111 or laminin-511, and especially preferable natural mouse laminin-111 or recombinant human laminin-511,  wherein at least one of said crosslinking agent and/or said bioactive agent comprises a RGD motif; [0389] b) one or more different culture media, preferably comprising Wnt agonists such as R-spondin and Wnt 3a.

[0390] According to this embodiment, the present invention is also related to a kit of parts for testing the influence of drugs on colorectal cancer cells, comprising: [0391] a) components for preparing a fully defined hydrogel matrix array having a stiffness in the range of 50 to 2000 Pa, so as to create fully defined three-dimensional extracellular matrix conditions that differ from each other in their biological, biophysical and/or biochemical characteristics, said components comprising [0392] one or more different PEG hydrogel precursor molecules, preferably PEG-VS, especially preferable 4-arm or 8-arm PEG-VS, and/or PEG-Acr, especially preferable 4-arm or 8-arm PEG-Acr, [0393] at least one crosslinking agent, preferably a peptide comprising at least two, preferably two, cysteine moieties, [0394] optionally one or more biologically active molecules comprising laminin, preferably laminin-111 or laminin-511, and especially preferable natural mouse laminin-111 or recombinant human laminin-511,  wherein at least one of said crosslinking agent and/or said bioactive agent comprises a RGD motif; [0395] b) one or more different culture media, preferably comprising Wnt agonists such as R-spondin and Wnt 3a; [0396] c) optionally, cells from a cell repository/biobank that have been created using the same extracellular matrix conditions.

[0397] The preselected extracellular matrix conditions of this embodiment can also be used in a method for testing the efficiency of therapies for a cancer patient, comprising the steps: [0398] a) providing freshly isolated or frozen colorectal cancer cells from a biopsy or a tissue resection of a cancer patient; [0399] b) establishing and expanding organoids from said cells, and applying one or more drugs to said organoids by the method described above; [0400] c) comparing the activity of the one or more drugs applied in step b) with the result of the treatment of said patient with one of said drugs applied in step b); [0401] d) and/or providing drug activity results on patient organoids and corresponding genetic and phenotypic data of the disease to support physician in making decisions on how to treat the patient.

[0402] Patient biopsies or resections dedicated for the isolation of colorectal cancer cells to establish organoids in step b), can be collected during a standard diagnostic procedure and subsequently transported to the site where step b) is to be conducted.

[0403] Step b) of this method is conducted as described above, i.e. by providing preselected extracellular matrix conditions comprising a fully defined hydrogel matrix array with discrete volumes having a stiffness in the range of 50 to 2000 Pa and prepared by crosslinking, onto a substrate or into discrete volumes of a substrate, preferably a multi-well plate, different combinations of one or more different PEG hydrogel precursor molecules in the presence of optionally one or more biologically active molecules comprising laminin, preferably laminin-111 or laminin-511, and especially preferable natural mouse laminin-111 or recombinant human laminin-511, at least one crosslinking agent, and said colorectal cancer cells, so as to create fully defined three-dimensional extracellular matrix conditions that differ from each other in their biological, biophysical and biochemical characteristics;

[0404] allowing said colorectal cancer cells to grow in said discrete volumes of said hydrogel matrix in the presence of one or more different culture media preferably comprising Wnt agonists such as R-spondin and Wnt 3a;

[0405] and adding one or more drugs to the cells grown in said discrete volumes of said hydrogel matrix;

[0406] wherein at least one of said crosslinking agent and/or said optional bioactive agent comprises a RGD motif.

[0407] The one or more drugs added to the cells grown in said discrete volumes of said hydrogel matrix comprise the drug(s) used for anticancer standard of care (SoC) treatment of the patient.

[0408] According to a preferred embodiment, the biopsy or resection can additionally be processed for histological analysis and/or omics testing (e.g. NGS) to establish the baseline for reference. The results of these additional analyses can also be used for comparing and/or correlating the above ex vivo and in vivo tests.

[0409] Based on this method, it is possible to reliably assess whether the applied anticancer standard of care (SoC) treatment is suitable, or whether a different drug treatment regime tested ex vivo as described above might be more promising. Thus, with the present invention the cancer treatment can be personalized and optimized. Functional in vitro data can be generated that may increase accuracy of treatment decisions by health care providers.

Breast Cancer

[0410] There are distinct breast cancer subtypes, which require different culture conditions. The same discussion as before for the pancreatic cells applies here.

[0411] With the present invention, it is possible to culture breast cancer cells, for example, but not limited to the triple negative (TNBC) or HER2+ receptor status under conditions that sustain the growth and expansion of these cells. Subsequently, the cells are exposed to different drug treatments in order to select an efficient drug treatment for the cancer patient. Thus, the present invention provides a precision medicine platform enabling the growth and drug testing of breast cancer tissues in different microenvironments and therefore captures the specificities of multiple clones inside a single tumor.

[0412] According to the present invention, it could be shown that breast cancer cells could be well cultured and tested preferably using a fully defined enzymatic-degradable hydrogel matrix and preferably a culture medium comprising FBS (serum) or Wnt agonist such as R-spondin, preferably under hypoxic (low oxygen 5% O.sub.2) conditions. For some subtypes (in particular TNBC subtype) at least one RGD adhesion motif and optionally laminin, preferably laminin-111 and especially preferably natural mouse laminin-111, as an additional bioactive ligand were preferable.

[0413] Thus, according to this embodiment, the present invention is related to a method of testing the influence of drugs on breast cancer cells, comprising the steps of: [0414] a) providing preselected extracellular matrix conditions comprising a fully defined, preferably enzymatic-degradable hydrogel matrix array with discrete volumes and prepared by crosslinking, onto a substrate or into discrete volumes of a substrate, preferably a multi-well plate, different combinations of one or more different PEG hydrogel precursor molecules, in the presence of optionally one or more biologically active molecules, at least one crosslinking agent, and said breast cancer cells, so as to create fully defined three-dimensional extracellular matrix conditions that differ from each other in their biological, biophysical and/or biochemical characteristics; [0415] b) allowing said breast cancer cells to grow in said discrete volumes of said hydrogel matrix in the presence of one or more different culture media, preferably comprising FBS (serum) or Wnt agonist such as R-spondin; [0416] c) adding one or more drugs to the cells grown in said discrete volumes of said hydrogel matrix;

[0417] wherein said at least one crosslinking agent comprises preferably an enzymatically degradable motif, preferably a MMP-sensitive motif.

[0418] Preferably, said hydrogel matrix array has a soft or medium stiffness in the range of 50-2000 Pa.

[0419] Preferably, said PEG hydrogel precursor molecules are PEG-VS (Polyethylene glycol with terminal vinylsulfone moieties), especially preferable 4-arm or 8-arm PEG-VS, and/or PEG-Acr (Polyethylene glycol with terminal acrylate moieties), especially preferable 4-arm or 8-arm PEG-Acr.

[0420] More preferably, said fully defined hydrogel matrix array is prepared by crosslinking PEG-VS, especially preferable 4-arm or 8-arm PEG-VS, or a 50:50 mixture of PEG-VS, especially preferable 4-arm or 8-arm PEG-VS, and PEG-Acr, especially preferable 4-arm or 8-arm PEG-Acr, with a peptide comprising at least two, preferably two cysteine moieties as a crosslinking agent, wherein said crosslinking agent may comprise an enzymatically degradable motif, preferably a MMP-sensitive motif.

[0421] As an optional bioactive ligand, a ligand comprising a bioactive motif including a RGD adhesion motif may be used. Examples of suitable RGD motifs are RGD, RGDS, RGDSP, RGDSPG, RGDSPK, RGDTP, RGDSPASSKP, PHSRNSGSGSGSGSGRGDSPG or any cyclic RGD motifs such as cyclo(RGDfC), but principally any known and successfully employed RGD sequences, in the field of hydrogels and cell culture, could be used.

[0422] As an optional bioactive ligand, a ligand comprising a bioactive motif may be used. Preferably, said optional bioactive ligand is selected from the group consisting of natural laminins, for example laminin-111, in particular mouse laminin-111, recombinant laminin isoforms, and biofunctional fragments thereof. Examples of suitable recombinant laminin isoforms are laminin-111, laminin-211, laminin-332, laminin-411, laminin-511, or laminin-521.

[0423] As an optional bioactive ligand, a ligand comprising glycosaminoglycans such as hyaluronic acid and hyaluronan may be used. Examples of hyaluronic acid are hyaluronic acid 50k, hyaluronic acid 1000k, hyaluronate thiol 50k or hyaluronate thiol 1000k.

[0424] According to a preferred embodiment, a culture medium may be used that is described in Sachs et al. (Cell 172 2018, 1-14 (see e.g. supplementary material, table S2)). Alternatively, the commercially available culture media Intesticult™, Mammocult™, WITP™, MEBM™, or StemPro™ hESC SFM may be used. Especially preferred is the culture medium described in Sachs et al., which comprises R-Spondin 1 conditioned medium or R-Spondin 3, Neuregulin 1, FGF 7, FGF 10, EGF, Noggin, A83-01, Y-27632, SB202190, B27 supplement, N-Acetylcysteine, Nicotinamide, GlutaMax 100x, Hepes, Penicillin/Streptomycin, Primocin and Advanced DMEM/F12. Other media such as IMDM+FBS (serum), or those described in Liu et al. (Sci Rep 2019, (9):622), or Lancaster et al. (Nat Biotechnol 2017 35(7): 659-666) may be used.

[0425] According to a preferred embodiment, hypoxic (low oxygen 5% O.sub.2) conditions are preferred.

[0426] Especially preferable, said breast cancer cells are from freshly isolated or frozen cells from a biopsy or tissue resection of a human or from patient-derived xenograft (PDX) tissue.

[0427] With said method, it is possible to grow, expand and subsequently test said breast cancer cells in a selected medium under conditions that recapitulate drug results observed in vivo.

[0428] According to an especially preferred embodiment, the extracellular matrix conditions are chosen such that the use of a naturally-derived matrix such as Matrigel® can be completely avoided.

[0429] According to this embodiment, the present invention is also related to a kit of parts for testing the influence of drugs on breast cancer cells, comprising: [0430] a) components for preparing a fully defined, preferably enzyuratic-degradable hydrogel matrix array, so as to create fully defined three-dimensional extracellular matrix conditions that differ from each other in their biological, biophysical and/or biochemical characteristics, said components comprising [0431] one or more different PEG hydrogel precursor molecules, preferably PEG-VS, especially preferable 4-arm or 8-arm PEG-VS, and/or PEG-Acr, especially preferable 4-arm or 8-arm PEG-Acr, [0432] at least one crosslinking agent, preferably a peptide comprising at least two, preferably two, cysteine moieties, [0433] optionally one or more biologically active molecules, wherein said at least one crosslinking agent preferably comprises an enzymatically degradable motif, preferably a MMP-sensitive motif; [0434] b) one or more different culture media, preferably comprising FBS (serum) or Wnt agonist such as R-spondin.

[0435] According to this embodiment, the present invention is also related to a kit of parts for testing the influence of drugs on breast cancer cells, comprising: [0436] a) components for preparing a fully defined, preferably enzymatic-degradable hydrogel matrix array, so as to create fully defined three-dimensional extracellular matrix conditions that differ from each other in their biological, biophysical and/or biochemical characteristics, said components comprising [0437] one or more different PEG hydrogel precursor molecules, preferably PEG-VS, especially preferable 4-arm or 8-arm PEG-VS, and/or PEG-Acr, especially preferable 4-arm or 8-arm PEG-Acr, [0438] at least one crosslinking agent, preferably a peptide comprising at least two, preferably two, cysteine moieties, [0439] optionally one or more biologically active molecules, wherein said at least one crosslinking agent preferably comprises an enzymatically degradable motif, preferably a MMP-sensitive motif; [0440] b) one or more different culture media, preferably comprising FBS (serum) or Wnt agonist such as R-spondin; [0441] c) optionally, cells from a cell repository/biobank that have been created using the same extracellular matrix conditions.

[0442] The preselected extracellular matrix conditions of this embodiment can also be used in a method for testing the efficiency of therapies for a cancer patient, comprising the steps: [0443] a) providing freshly isolated or frozen breast cancer cells from a biopsy or a tissue resection of a cancer patient; [0444] b) establishing and expanding organoids from said cells, and applying one or more drugs to said organoids by the method described above; [0445] c) comparing the activity of the one or more drugs applied in step b) with the result of the treatment of said patient with one of said drugs applied in step b). [0446] d) and/or providing drug activity results on patient organoids and corresponding genetic and phenotypic data of the disease to support a physician in making decisions on how to treat the patient.

[0447] Patient biopsies or resections dedicated for the isolation of breast cancer cells to establish organoids in step b), can be collected during a standard diagnostic procedure and subsequently transported to the site where step b) is to be conducted.

[0448] Step b) of this method is conducted as described above, i.e. by providing preselected extracellular matrix conditions comprising a fully defined, preferably enzymatic-degradable hydrogel matrix array with discrete volumes prepared by crosslinking, onto a substrate or into discrete volumes of a substrate, preferably a multi-well plate, different combinations of one or more different PEG hydrogel precursor molecules in the presence of optionally one or more biologically active molecules, at least one crosslinking agent, and said breast cancer cells, so as to create fully defined three-dimensional extracellular matrix conditions that differ from each other in their biological, biophysical and biochemical characteristics;

[0449] allowing said breast cancer cells to grow in said discrete volumes of said hydrogel matrix in the presence of one or more different culture media preferably comprising FBS (serum) or Wnt agonist such as R-spondin;

[0450] and adding one or more drugs to the cells grown in said discrete volumes of said hydrogel matrix;

[0451] wherein at least one of said crosslinking agent preferably comprises an enzymatically degradable motif, preferably a MMP-sensitive motif.

[0452] The one or more drugs added to the cells grown in said discrete volumes of said hydrogel matrix comprise the drug(s) used for anticancer standard of care (SoC) treatment of the patient.

[0453] According to a preferred embodiment, the biopsy or resection can additionally be processed for histological analysis and/or omics testing (e.g. NGS) to establish the baseline for reference. The results of these additional analyses can also be used for comparing and/or correlating the above ex vivo and in vivo tests.

[0454] Based on this method, it is possible to reliably assess whether the applied anticancer standard of care (SoC) treatment is suitable, or whether a different drug treatment regime tested ex vivo as described above might be more promising. Thus, with the present invention the cancer treatment can be personalized and optimized. Functional in vitro data can be generated that may increase accuracy of treatment decisions by health care providers.

Prostate Cancer

[0455] This embodiment shows the benefits provided by the present invention with respect to the problem of selected growth of cancer cells over normal cells (e.g. wild-type healthy cells, stromal cells).

[0456] For some organs, patient-derived cancer cells that form tumor organoids tend to grow ex vivo more slowly than their healthy (wild-type) counterparts or associated stromal cells, and/or currently used ex vivo conditions based on Matrigel® or equivalent matrices are not able to select and favour the growth of one tissue type vs. the other.

[0457] Therefore, normal cells tend to overgrow the cancer organoids cultures, unless specific measures are taken. Despite the fact that in some cases modifications of the media composition could solve this issue, the overgrowth of normal cells (healthy cells) vs. cancer cells still remains a problem, for example for prostate cancer. This impairs the establishment of ex vivo growth of patient-derived cancer cells as physiological preclinical model e.g. to determine which drug or drug combination may work to treat the specific patient.

[0458] Overgrowth of normal cells has been particularly observed in the case of prostate cancer organoids (Drost et al., Development (2017) 144, 968-975). For example, less than ten prostate cancer cell lines currently exist (whereas for Colon cancer more than 50 cell lines exist), and none of them adequately reflects the correct cancer disease (ATCC and ECACC cell banks). Therefore, the ability to grow e.g. prostate cancer organoids while impeding the growth of normal cells, would significantly impact the development of new drugs by using more physiological preclinical models and enable the use of patient-specific cell models for personalized medicine applications.

[0459] Drost et al., Development (2017) 144, 968-975 (see pages 971-972 “Personalized cancer therapy”) summarizes how the selection between (i) cancer cells and (ii) normal cells (wild-type) is done to grow ex vivo pure cell populations of (i) vs. (ii). Briefly, where it is possible, this is achieved by adding or omitting chemicals/growth factors in cell culture Media. However, for prostate cancer this is not as easy, for example, as for certain colon cancers that harbour specific genetic mutations, which make their successful culture independent of certain chemicals in contrast to their wild-type counterparts. As described in Drost et al., Nature protocols, Vol. 11 no.2 (2016) 347 (see pages 347-348 “Limitation of the method”), their culture protocol, was not good enough for growing organoids derived from primary prostate cancers, most likely due to the fact that ex vivo tumor cells do not have a selective advantage over normal cells. Consequently, the normal prostate cells that are normally present within each cancer sample tissues, seem to overgrow the tumor cells (See also Karthaus et al., Cell 159, 163-175, Sep. 25, 2014, on page 171 last “Discussion” paragraph). Furthermore, a similar problem was also observed with sample biopsies from prostate metastasis in bone and soft tissues (Gao et al., Cell 159, 176-187, Sep. 25, 2014, see page 177-178 “Results”), where normal host tissue cells (e.g. stroma and/or epithelial cells) were overtaking the cancer cells.

[0460] Other examples of normal cell overgrowth that are less commonly reported include breast cancer and lung cancer ex vivo cultures. In a study published by Sachs et al. (Cell 172 2018, 1-14, demonstrating the establishment of >100 primary and metastatic breast cancer organoids, there are a couple of instances in which the pathology of the organoid is classified as normal while the original tissue pathology was classified as tumor (Sachs et al., Cell 172 2018, 1-14, Table S3). This can also be observed in lung cancer organoids, for example, derived from patients harbouring a mutation in p53, in which normal versus cancerous cells can be selected by adding chemicals to the cell culture Media (Sachs et al., The EMBO Journal e 100300|2019). However, if there is no p53 mutation, there is no way to prevent normal cell overgrowth.

[0461] According to this embodiment of the present invention, preselected extracellular matrix conditions were identified that promote the growth of prostate cancer cells while at the same time impeding the establishment of their normal counterpart. This allows the establishment of a screening method where cancer cells can be reliably evaluated that otherwise would be overgrown by their normal counterparts.

[0462] In detail, it was found that normal prostate cells are growing only in gel formulations containing a RGD adhesion motif, and their growth is better in soft gels compared to medium or hard ones. On the other hand, it was found that prostate cancer cells isolated from patient or from patient-derived xenograft (PDX) tumors show a similar growth in gels with and without the presence of a RGD motif. Some prostate cancer cells isolated from patient-derived xenograft (PDX) tumors grow even better in gel without RGD (soft or medium stiffness).

[0463] Thus, according to this embodiment, the present invention is related to a method of testing the influence of drugs on cancer cells that grow ex vivo more slowly than their normal counterparts or associated stromal cells, preferably prostate cancer cells, comprising the steps of: [0464] a) providing preselected extracellular matrix conditions comprising a fully defined hydrogel matrix array with discrete volumes, prepared by crosslinking, onto a substrate or into discrete volumes of a substrate, preferably a multi-well plate, different combinations of one or more different PEG hydrogel precursor molecules, in the presence of optionally one or more biologically active molecules, at least one crosslinking agent, and said cancer cells, so as to create fully defined three-dimensional extracellular matrix conditions that differ from each other in their biological, biophysical and/or biochemical characteristics; [0465] b) allowing said cancer cells to grow in said discrete volumes of said hydrogel matrix in the presence of one or more different culture media; [0466] c) adding one or more drugs to the cells grown in said discrete volumes of said hydrogel matrix;

[0467] wherein said crosslinking agent and said optional bioactive agent do not comprise any RGD motif.

[0468] Preferably, said PEG hydrogel precursor molecules are PEG-VS (Polyethylene glycol with terminal vinylsulfone moieties), especially preferable 4-arm or 8-arm PEG-VS, and/or PEG-Acr (Polyethylene glycol with terminal acrylate moieties), especially preferable 4-arm or 8-arm PEG-Acr.

[0469] More preferably, said fully defined self-degradable hydrogel matrix array is prepared by crosslinking PEG-Acr, especially preferable 4-arm or 8-arm PEG-Acr, or a 50:50 mixture of PEG-VS, especially preferable 4-arm or 8-arm PEG-VS, and PEG-Acr, especially preferable 4-arm or 8-arm PEG-Acr, with a peptide comprising at least two, preferably two cysteine moieties as a crosslinking agent.

[0470] Preferably, said hydrogel matrix array has a soft or medium stiffness in the range of 50-2000 Pa.

[0471] According to another preferred embodiment, said fully defined, non self-degradable hydrogel matrix array is prepared by crosslinking PEG-VS, especially preferable 4-arm or 8-arm PEG-VS, with a peptide comprising at least two, preferably two cysteine moieties as a crosslinking agent.

[0472] As an optional bioactive ligand, a ligand comprising a bioactive motif may be used. Preferably, said optional bioactive ligand is selected from the group consisting of Tenascin C and Glypican, natural laminins, for example laminin-111, in particular mouse laminin-111, recombinant laminin isoforms, and biofunctional fragments thereof. Examples of suitable recombinant laminin isoforms are laminin-111, laminin-211, laminin-332, laminin-411, laminin-511 or laminin-521.

[0473] Preferably, culture media were pre-selected to grow prostate cancer cells. Commercially available culture media, e.g. Mammocult™, WIT-P™, StemPro™ hESC SFM, PrEGM™ BulletKit™ from Lonza (ref. CC-3166), and NutriStem® hPSC XF may be used, as well as media as described in WO 2015/173425 A1 or Drost et al. (Nature Protocol 11, 347-358, January 2016) or Beshiri et al. (Clinical Cancer Research 24, 4332-4345), May 2018) or Puca et al. (Nature Communications 9:2404, 1-10, June 2018) or in Ince et al. (Cancer Cell 12, 160-170, August 2007), are suitable for the growth of cancer cells. It was found that the culture medium described in WO 2015/173425 A1 favours growth of cancer cells. Especially preferred is therefore a culture medium which comprises Glutamine, BSA, Transferrin, Noggin, FGF (2 or basic), FGF 10, EGF, R-Spondin conditioned medium or recombinant, Penicillin/Streptomycin, Glutathione, Nicotinamide, DHT, Prostaglandin E2, A83-01, Y-27632, N-acetylcysteine, SB202190 and Hepes.

[0474] Especially preferable, said cancer cells are from freshly isolated or frozen cells from a biopsy or tissue resection of a human or from patient-derived xenograft (PDX) tissue.

[0475] With said method, it is possible to grow and subsequently test said cancer cells in a selected medium under conditions that recapitulate drug results observed in vivo, without being overgrown by their normal counterparts or associated stromal cells.

[0476] According to an especially preferred embodiment, the extracellular matrix conditions are chosen such that the use of a naturally-derived matrix such as Matrigel® can be completely avoided.

[0477] According to this embodiment, the present invention is also related to a kit of parts for testing the influence of drugs on cancer cells that grow ex vivo more slowly than their normal counterparts or associated stromal cells, preferably prostate cancer cells, comprising: [0478] a) components for preparing a fully defined hydrogel matrix array, so as to create fully defined three-dimensional extracellular matrix conditions that differ from each other in their biological, biophysical and/or biochemical characteristics, said components comprising [0479] one or more different PEG hydrogel precursor molecules, preferably PEG-VS, especially preferable 4-arm or 8-arm PEG-VS, and/or PEG-Acr, especially preferable 4-arm or 8-arm PEG-Acr, [0480] at least one crosslinking agent, preferably a peptide comprising at least two, preferably two, cysteine moieties, [0481] optionally one or more biologically active molecules, wherein said crosslinking agent and said optional bioactive agent do not comprise any RGD motif; [0482] b) one or more different culture media.

[0483] According to this embodiment, the present invention is also related to a kit of parts for testing the influence of drugs on cancer cells that grow ex vivo more slowly than their normal counterparts or associated stromal cells, preferably prostate cancer cells, comprising: [0484] a) components for preparing a fully defined hydrogel matrix array, so as to create fully defined three-dimensional extracellular matrix conditions that differ from each other in their biological, biophysical and/or biochemical characteristics, said components comprising [0485] one or more different PEG hydrogel precursor molecules, preferably PEG-VS, especially preferable 4-arm or 8-arm PEG-VS, and/or PEG-Acr, especially preferable 4-arm or 8-arm PEG-Acr, [0486] at least one crosslinking agent, preferably a peptide comprising at least two, preferably two, cysteine moieties,  optionally one or more biologically active molecules, wherein said crosslinking agent and said optional bioactive agent do not comprise any RGD motif; [0487] b) one or more different culture media; [0488] c) optionally, cells from a cell repository/biobank that have been created using the same extracellular matrix conditions.

[0489] The preselected extracellular matrix conditions of this embodiment can also be used in a method for testing the efficiency of therapies for a cancer patient, comprising the steps: [0490] a) providing freshly isolated or frozen cancer cells from a biopsy or a tissue resection of a cancer patient; [0491] b) establishing and expanding organoids from said cells, and applying one or more drugs to said organoids by the method described above; [0492] c) comparing the activity of the one or more drugs applied in step b) with the result of the treatment of said patient with one of said drugs applied in step b); [0493] d) and/or providing drug activity results on patient organoids and corresponding genetic and phenotypic data of the disease to support physician in making decisions on how to treat the patient.

[0494] Patient biopsies or resections dedicated for the isolation of prostate cancer cells to establish organoids in step b), can be collected during a standard surgery or diagnostic procedure and subsequently transported to the site where step b) is to be conducted.

[0495] Step b) of this method is conducted as described above, i.e. by providing preselected extracellular matrix conditions comprising a fully defined hydrogel matrix array with discrete volumes, prepared by crosslinking, onto a substrate or into discrete volumes of a substrate, preferably a multi-well plate, different combinations of one or more different PEG hydrogel precursor molecules in the presence of optionally one or more biologically active molecules, at least one crosslinking agent, and said cancer cells, so as to create fully defined three-dimensional extracellular matrix conditions that differ from each other in their biological, biophysical and biochemical characteristics;

[0496] allowing said cancer cells to grow in said discrete volumes of said hydrogel matrix in the presence of one or more different culture media;

[0497] and adding one or more drugs to the cells grown in said discrete volumes of said hydrogel matrix;

[0498] wherein said crosslinking agent and said optional bioactive agent do not comprise any RGD motif.

[0499] The one or more drugs added to the cells grown in said discrete volumes of said hydrogel matrix comprise the drug(s) used for anticancer standard of care (SoC) treatment of the patient.

[0500] According to a preferred embodiment, the biopsy or resection can additionally be processed for histological analysis and/or omics testing (e.g. NGS) to establish the baseline for reference. The results of these additional analyses can also be used for comparing and/or correlating the above ex vivo and in vivo tests.

[0501] Based on this method, it is possible to reliably assess whether the applied anticancer standard of care (SoC) treatment is suitable, or whether a different drug treatment regime tested ex vivo as described above might be more promising. Thus, with the present invention the cancer treatment can be personalized and optimized. Functional in vitro data can be generated that may increase accuracy of treatment decisions by health care providers.

Dynamic Organoid Growth Image Analysis Method

[0502] There is a need to quantify accurately and easily different parameters of organoid growth (e.g. growth rate, organoid number, organoid size). As for conventional 2D cell culture, quantification of organoid growth can be achieved indirectly by using fluorometric, colorimetric or luminescent methods which measure the quantities of metabolites in culture wells (e.g. Alamar Blue, MTT, Cell Titer Glow 3D). All these indirect assays, even when they are not lethal for the cells, can affect the fitness of the organoids and totally prevent the possibility to further use the grown organoids (e.g. for drug testing, regenerative medicine).

[0503] Using non-invasive (and label-free) methods as light-microscopy to quantify organoid growth is therefore a favoured alternative in long-term culture and/or when the organoids need to be kept as “native” & “untouched” as possible (e.g. for regenerative medicine, biobanking). The need for quantification of high throughput imaging of 3D organoid cultures has led to the adaptation of 2D methods (Carpenter et al., Genome Biology 2006, 7:R100) or the development of new automatized detection and image segmentation algorithms which enable the counting and measurement of organoid in a fast, reproducible and unbiased manner. These methods are more easily and more accurately performed using fluorescent markers (Robinson et al., PLoSONE 10(12): e0143798. doi:10.1371/journal.pone.01437982015, Boutin et al., Nature scientific reports (2018)8:11135, DOI:10.1038/s41598-018-29169-0 2018), which, again is not compatible with the use of “untouched” & “label-free” patient-derived cells or re-implantation protocols, as they require immunofluorescence staining or fluorescent transgene expression.

[0504] Recently, in Borten et al., Nature scientific reports (2018) 8:5319, DOI:10.1038/s41598-017-18815-8 2018, a Matlab (from Mathworks company) based algorithm called OrganoSeg was developed to specifically analyse organoids from 3D brightfield images, thereby allowing to detect, segment (i.e. partitioning a digital image into specific set of pixels) and quantify many parameters from living native organoids grown in 3D (Borten 2018). This open-source software allows for identification and multiparametric morphometric classification of organoids based on size, sphericity and shape of the detected features at a given timepoint.

[0505] However, despite being accurate and powerful, this tool does not allow taking into account the time dimension and would require multiple analyses at different timepoints, and thus consequent compilation work, to properly assess the dynamics of organoid growth.

[0506] According to the present invention, a new analysis method is provided. Based on a MATLAB code a new method was developed, which is able to align brightfield images acquired at different timepoints and automatically identify and segment organoids based on their intensity. This program uses the same method as OrganoSeg to segment objects from Brightfield images. The main difference resides in the use made of these segmented objects: While OrganoSeg uses the size and morphologies to classify different types of organoids at a given discrete timepoint, this new program allows for the dynamic follow up of organoid growth in one single analysis and thus the calculation of OFE/AIF and drug response. Accordingly, the program provides the following dynamic information about the organoid growth: [0507] Organoid Forming Efficiency (OFE) and, [0508] Area Increase Factor (AIF).

[0509] With the method of the present invention, it is also possible to generate aligned time-lapse videos for each well acquired, and “Time Projections” representing the overall growth of organoids along the culture duration in a single rendered image. Those time projections are easy to include in presentations and publications.

[0510] When single cells or small clusters of cells are encapsulated in 3D extracellular matrix, only a subset of these cells is able to grow and generate organoids; this is what is designed as the “Organoid Forming Efficiency” (OFE) of the culture. With the method of the present invention, the number of encapsulated cells at Day 0 is quantified. Moreover, the method allows the user to define a threshold for the size of what is considered to be an organoid. It then provides the OFE for any particular timepoint in the assay.

[0511] While the OFE gives an indication of the percentage of cells in the original culture capable of developing in organoids (e.g. stem cells), with the method of the present invention also the growth rate of the overall organoid culture is quantified by calculating the increase in area along time after segmentation of the time-lapse. This is the “Area Increase Factor” (AIF), which is corresponding to the ratio of the total area occupied by single cells at Day 0 to the total area occupied by the organoids at any given day. The method of the present invention allows for the selection of initial and final days to calculate the AIF.

[0512] Combining the OFE and AIF scores gives useful information on the fitness and performance of a given extracellular matrix condition.

[0513] This semi-automatized image analysis method according to the present invention allows for the temporal investigation and analysis of organoid growth in high throughput set-ups. It provides unbiased and reproducible scoring reflecting the fitness and performance of extracellular matrix conditions for any organoid cultures without the need of markers and/or detrimental assays. It also allows for semi-automatic quantification of patient-derived organoids drug test results (e.g. IC.sub.50-value determination).

Example 1: Testing of Lung Cancer Cells

Example 1a: Lung Cancer Cells Overexpressing the c-Met Receptor

[0514] From a patient, lung cancer cells overexpressing the c-Met receptor were obtained from PDX cells. Upon activation through ligand binding, the c-Met receptor autophosphorylates and activates several signaling cascades within the cell.

[0515] Treatment of patient-derived xenograft (PDX) cells of non-small cell lung cancer (NSCLC) model LXFA-1647 with a drug targeted against c-Met (c-Met inhibitor: PF-04217903, Selleck Chemicals) inhibits its autophosphorylation and induces tumor growth regression in vivo.

[0516] A PEG was used as a hydrogel precursor molecule for making a non self-degradable hydrogel. As a crosslinking agent, peptides containing at least two, preferably two cysteine moieties were used which varied in their amino acid sequence, in particular with respect to the presence or absence of a RGD adhesion motif and with respect to the presence or absence of a MMP degradation sequence. A further variation that was made to some hydrogels was the attachment of a bioactive ligand comprising a RGD adhesion motif or/and a ligand selected from the group consisting of natural laminins, recombinant laminin isoforms, and biofunctional fragments thereof. Examples of suitable recombinant laminin isoforms are laminin-111, laminin-211, laminin-332, laminin-411, laminin-511, or laminin-521. An array of hydrogels varying in the above preselected features was established, by the method described above.

[0517] The mechanical properties of the hydrogels were also varied (soft (50-1000 Pa), medium (1000-2000 Pa) or hard (2000-3000 Pa) gels).

[0518] For comparison, tests were also conducted in the undefined natural-derived matrix Matrigel®.

[0519] The culture medium was preselected to comprise the above described c-Met inhibitor PF-04217903 (Selleck Chemicals), i.e. a drug targeting c-met and inhibiting its autophosphorylation, or a drug used in standard of care (SoC) treatment of this cancer type (docetaxel). The respective drug was added to the culture media after 1 to 8 days of culture (1 to 8 days post cell encapsulation). The drug response was measured after 5 to 10 days post-drug addition. As a preferred culture medium, a medium was preselected that is characterized by the presence of FBS (serum) or Wnt agonists such as R-spondin. According to this example, a culture medium was used that was adapted from the medium described in Sachs et al. (The EMBO Journal e 100300|2019). The preferred culture medium comprised AdDMEM/F12 medium supplemented with glutamine, Noggin, EGF, fibroblast growth factor 7 and 10 [FGF7 and FGF10], HGF, R-spondin-conditioned medium, Primocin, penicillin/streptomycin, N-acetyl-L-cysteine, Nicotinamide, A83-01, SB202190 (p38-inhibitor), Y-27632 (rock inhibitor), B27 supplement and HEPES. Target expression (c-Met and Phospho c-Met) was detected by Western-blot in corresponding growth conditions.

[0520] The results are shown in FIGS. 1a, 1b and 1c.

[0521] With these preselected conditions, ex vivo growth (ex vivo culture, extracellular matrix) conditions that recapitulate drug results observed in vivo (i.e. activity of the c-Met inhibitor PF-04217903 (Selleck Chemicals)) were identified. These extracellular matrix conditions were qualified as “responder conditions”.

[0522] In this example, the most preferred responder conditions were the use of a non self-degradable hydrogel made from a respective PEG hydrogel precursor molecules and, as a crosslinking agent, a peptide containing two cysteine moieties without any RGD motif (either in the crosslinking agent or attached to the hydrogel). Said hydrogel has a soft stiffness in the range of 50-1000 Pa (example 1a), even more preferably 250-500 Pa.

[0523] In the same assay, other conditions were identified that result in drug resistance. Drug resistance was found to be dependent on the microenvironment, i.e. extracellular matrix or soluble factors. In particular, it could be shown that upon the attachment of 1 mM of a bioactive ligand comprising a RGD motif to the hydrogel, the tumor cells became resistant to the above described c-Met inhibitor PF-04217903 (Selleck Chemicals). Those conditions are qualified as “non-responder conditions” (example 1b).

[0524] Finally, it could be shown in the same assay that the use of Matrigel® as matrix also provided “non-responder conditions” (comparative example 1) in which the tumor cells did not respond to the above described c-Met inhibitor PF-04217903 (Selleck Chemicals).

[0525] In FIG. 1b, the effect of a standard of care (SoC) treatment with Docetaxel as well as the effect of treatment with the c-met-inhibitor PF-04217903 (Selleck Chemicals) under the conditions of example 1a are shown. Both drugs were clearly effective.

[0526] In contrast thereto, in FIG. 1c it is shown that under the conditions of comparative example 1 (Matrigel®), only an effect of a standard of care (SoC) treatment with Docetaxel could be observed. No effect of treatment with the c-met-inhibitor PF-04217903 (Selleck Chemicals) was observable. Accordingly, FIGS. 1a-1c) show that only under the preselection conditions of the present invention an effect of a c-met-inhibitor on the examined cells could be seen. When working under conventional conditions (i.e. using Matrigel®), the possible treatment with a c-met inhibitor would not have been recognized.

Example 1b: Lung Cancer Cells Overexpressing the EGFR Receptor

[0527] Example 1a was repeated with lung cancer cells overexpressing the EGFR receptor. These cells were obtained from PDX cells.

[0528] Using the “responder conditions” of example 1a (i.e. without any RGD motif (either in the crosslinking agent or attached to the hydrogel)), in example 1b no effect of treatment with a c-met inhibitor could be observed, as was expected due to the lack of autophosphorylation of the c-met receptor in the cells tested in example 1b (see FIG. 1d). On the other hand, the EGFR receptor as well as its phosphorylated form were overexpressed under these conditions (FIG. 1d), and drugs acting on the EGFR receptor (Erlotinib and Cetuximab) showed a clear effect (similar to conditions of comparative example 1 using Matrigel® (data not shown)), as well as the SoC treatment with Paclitaxel (FIG. 1e).

Example 2: Testing of Pancreatic Cancer Cells

[0529] Pancreatic ductal adenocarcinoma (PDAC) cancer cells from a patient were first expanded in mice as a PDX model. The PDX-derived cells were grown in a range of extracellular matrix conditions.

[0530] Treatment of patient-derived xenograft (PDX) cells of pancreatic ductal adenocarcinoma (PDAC) cancer model PAXF736 with a drug targeted against EGFR (EGFR inhibitor: Cetuximab) reduces the tumor growth in vivo.

[0531] A PEG was used as a hydrogel precursor molecule for making a non self-degradable hydrogel. As a crosslinking agent, peptides containing at least two, preferably two cysteine moieties were used which varied in their amino acid sequence, in particular with respect to the presence or absence of a RGD adhesion motif and with respect to the presence or absence of a MMP degradation sequence. A further variation that was made to some hydrogels was the attachment of a bioactive ligand comprising a RGD or a cyclic RGD adhesion motif, or alternatively a bioactive ligand comprising a DGEA motif. An array of hydrogels varying in the above preselected features was established, by the method described above.

[0532] The mechanical properties of the hydrogels were also varied (hard (2000-3000 Pa), medium (1000-2000 Pa) or soft (50-1000 Pa) gels).

[0533] A variety of different known, commonly employed and/or commercially available culture media was used.

[0534] In FIG. 2a and FIG. 2b, the results are shown for a soft non self-degradable PEG hydrogel with a crosslinking moiety without RGD motif, and with a bioactive ligand comprising a RGD adhesion motif (example 2a), as well as for a soft non self-degradable PEG hydrogel with a crosslinking moiety with RGD motif, and with a bioactive ligand comprising a DGEA adhesion motif (example 2b). For comparison, tests were also conducted in the undefined natural-derived matrix Matrigel® (comparative example 2).

[0535] It can be seen from FIG. 2a that all tested hydrogels led to a comparable growth of patient-derived xenograft (PDX) cells of pancreatic ductal adenocarcinoma (PDAC) cancer model PAXF736.

[0536] It can be seen from FIG. 2b that the hydrogel according to example 2a showed a drug sensitivity (for Cetuximab) comparable to that of Matrigel® (comparative example 2). On the other hand, the hydrogel according to example 2b showed a much higher drug sensitivity.

[0537] In FIG. 2c, it can be seen that when using a soft gel (50-1000 Pa, examples 2c and 2d) or medium gel (1000-2000 Pa, examples 2e and 2f) in the presence of a RGD motif and in the presence (examples 2c and 2e) or absence (examples 2d and 2f) of a MMP-sensitive motif, a very good growth of PDAC cells could be achieved, which was comparable to the growth of PDAC cells in Matrigel® (comparative example 2).

[0538] It was found that the presence of Wnt agonists such as R-spondin and Wnt 3a in the culture medium was important for cell growth. Also it was found that the hydrogel matrix should comprise at least one RGD motif.

[0539] This example shows the advantage of preselection according to the present invention. When working under conventional extracellular matrix conditions using Matrigel® (comparative example 2), no effect of an EGFR inhibitor on the tested cells was observable. Accordingly, a possibly effective treatment of this cancer type would not have been identified.

[0540] A comparison of examples 2a and 2b shows another advantage of the preselection according to the present invention. By using different preselection conditions that are principally suitable for a specific cell type (here presence of a RGD motif), it is possible to identify a possible resistance of the tested cells. In example 2a, the observed drug sensitivity against the EGFR inhibitor Cetuximab was much lower as compared to example 2b, indicating that treatment of this specific cancer cell type with an EGFR inhibitor alone might not be sufficient.

Example 3: Testing of Pancreatic Cancer Cells Co-Cultured with Fibroblasts

[0541] In a further experiment, co-culturing of PDAC cells (from PDO pre-established in Matrigel®) with different ratios of cancer associated fibroblasts (isolated from patient and pre-expanded in 2D culture) was examined in a range of extracellular matrix conditions. The fibroblasts in the co-culture were identified using a specific marker (CD90).

[0542] In FIG. 3, the results of co-culturing 33% PDAC cells with 67% fibroblasts are shown in a hydrogel comprising both a RGD motif and an enzymatically (MMP) degradable moiety (example 3a), in a hydrogel comprising only a RGD motif and no enzymatically degradable moiety (example 3b), in a hydrogel comprising no RGD motif and only an enzymatically degradable moiety (example 3c), and in a hydrogel comprising no RGD motif and no enzymatically degradable moiety (example 3d). For comparison, the results with the conventional undefined natural-derived matrix Matrigel® (comparative example 3) are shown.

[0543] It can be seen that the best co-culturing results were obtained in example 3a, i.e. in a preferably soft PEG hydrogel comprising both a RGD motif and an enzymatically degradable moiety.

[0544] This example shows that it is possible to preselect conditions depending on whether simultaneous growth of other cells such as fibroblasts should be permitted or not.

Example 4: Testing of Colorectal Cancer Cells

[0545] Colorectal cancer (CRC) cells from a patient and pre-established in Matrigel® were grown in a range of extracellular matrix conditions.

[0546] PEG was used as a hydrogel precursor molecule to provide a non self-degradable PEG hydrogel, or alternatively a 50:50 mixture of a non self-degradable PEG hydrogel and a self-degradable PEG hydrogel. As a crosslinking agent, peptides containing at least two, preferably two cysteine moieties were used which varied in their amino acid sequence, in particular with respect to the presence or absence of a RGD adhesion motif and with respect to the presence or absence of a MMP degradation sequence. A further variation that was made to some hydrogels was the attachment of a bioactive ligand comprising a RGD adhesion motif, or alternatively of a ligand selected from the group consisting of natural laminins, for example laminin-111, recombinant laminin isoforms such as recombinant human laminin-511, and biofunctional fragments thereof. Examples of suitable recombinant laminin isoforms are laminin-111, laminin-211, laminin-332, laminin-411, laminin-511, or laminin-521. An array of hydrogels varying in the above preselected features was established, by the method described above.

[0547] The mechanical properties of the hydrogels were also varied (hard (2000-3000 Pa), medium (1000-2000 Pa) or soft (50-1000 Pa) gels).

[0548] A variety of different known, commonly employed and/or commercially available culture media was used.

[0549] The results are shown in FIG. 4. FIG. 4 provides Brightfield images of human colon cancer organoids grown for 0 and 11 days.

[0550] In examples 4a to 4c hydrogels were used that were non self-degradable and non-enzymatically degradable. In example 4b, the crosslinking moiety comprised a RGD motif, wherein in examples 4a and 4c a bioactive ligand comprising a RGD motif was attached in a dangling manner. In example 4b, a bioactive ligand was attacked in a dangling manner that was laminin-111. The hydrogels according to examples 4a and 4b were soft (below 500 Pa), whereas the hydrogel according to example 4c was medium (above 1000 Pa). In example 4d, a hydrogel was used that was self-degradable, but non-enzymatically degradable, and had an initial stiffness in the range from 400 to 600 Pa. Said hydrogel had a crosslinking moiety that comprised a RGD motif, and laminin-111 as a bioactive ligand. For comparison, tests were also conducted in the undefined natural-derived matrix Matrigel® (comparative example 4).

[0551] It was shown that in the hydrogels according to examples 4a to 4d cell growth comparable to the standard Matrigel® was obtained, but in defined conditions (unlike Matrigel®).

[0552] On the other hand, in example 4e a hydrogel was used that was non self-degradable, but enzymatically degradable and furthermore did not comprise any RGD motif. Under these conditions, the tested CRC cells did not grow.

[0553] In example 4f, a self-degradable hydrogel with an initial stiffness around 400-600 Pa, an RGD motif (incorporated in the crosslinker) and recombinant human laminin-511 as bioactive agent was used. Very good growth of the tested CRC cells was observed.

[0554] It was found that the presence of Wnt agonists such as R-spondin and Wnt 3a in the culture medium was favorable for cell growth. Also it was found that the hydrogel matrix should comprise at least one RGD motif and optionally at least one bioactive ligand selected from the group consisting of natural laminins, for example laminin-111, recombinant laminin isoforms such as recombinant human laminin-511, and biofunctional fragments thereof.

Example 5: Testing of Breast Cancer Cells

[0555] Breast cancer cells derived from patients with distinct cancer subtypes (Triple Negative (TNBC) or HER2+ receptor status) were first expanded in mice as PDX models. The PDX-derived cells were then grown in a range of extracellular matrix conditions.

[0556] A PEG was used as a hydrogel precursor molecule to provide a non self-degradable hydrogel. As a crosslinking agent, peptides containing at least two, preferably two cysteine moieties were used which varied in their amino acid sequence, in particular with respect to the presence or absence of a RGD adhesion motif and the presence or absence of a MMP-sensitive motif.

[0557] A further variation that was made to some hydrogels was the attachment of a bioactive ligand comprising a RGD adhesion motif, or/and a ligand selected from the group consisting of natural laminins, for example laminin-111, recombinant laminin isoforms, and biofunctional fragments thereof. Examples of suitable recombinant laminin isoforms are laminin-111, laminin-211, laminin-332, laminin-411, laminin-511, or laminin-521. An array of hydrogels varying in the above preselected features was established, by the method described above.

[0558] The mechanical properties of the hydrogels were also varied (hard (2000-3000 Pa), medium (1000-2000 Pa) or soft (50-1000 Pa) gels).

[0559] A variety of different known, commonly employed and/or commercially available culture media was used.

[0560] Tests were performed under hypoxic (low oxygen 5% O.sub.2) or normoxic (18% O.sub.2) conditions.

[0561] It was found that the presence of FBS (serum) or Wnt agonist such as R-spondin in the culture medium was favorable for cell growth. Also it was found that the hydrogel matrix should be preferably enzymatically-degradable.

[0562] In FIG. 5, the results of growth of different breast cancer cell types are shown. Brightfield images of human primary or metastatic (Mets) breast cancer cells from four patients of either HER2+ or Triple Negative Breast Cancer (TNBC) (from patient-derived xenograft models) are reproduced (4× objective magnification).

[0563] It can be seen in the bottom row that the hydrogel according to example 5a (non self-degradable, enzymatically degradable soft (<500 Pa) PEG hydrogel comprising a RGD motif and a laminin-111 as bioactive ligand) after the same time provided growth conditions similar to comparative example 5 (Matrigel®) in the upper row for TNBC lung metastatic cells and for TNBC primary cells.

[0564] The hydrogel according to example 5b (non self-degradable, enzymatically degradable soft (<500 Pa) PEG hydrogel comprising a RGD motif, but no laminin bioactive ligand) provided growth conditions similar to comparative example 5 (Matrigel®) for TNBC brain metastatic cells.

[0565] The hydrogel according to example 5c (non self-degradable, enzymatically degradable medium (>1000 Pa) PEG hydrogel comprising no RGD motif and no laminin bioactive ligand) provided growth conditions similar to comparative example 5 (Matrigel®) for HER2+ skin metastatic cells.

[0566] In general, the TNBC subtype was more challenging to grow. Hypoxic conditions improved breast cancer organoid growth over normoxic conditions. In addition, the morphology of HER2+ and TNBC cells grown under the preselected extracellular matrix conditions matched that of previously published breast cancer organoids established in Matrigel® (Sachs et al., 2018, Cell 172, 1-14).

Example 6: Testing of Prostate Cancer Cells

[0567] Commercially available primary healthy prostate cells as well as prostate cancer cells from PDX cells were encapsulated within a range of different extracellular matrix conditions.

[0568] A PEG was used as a hydrogel precursor molecule to provide a non self-degradable hydrogel. As a crosslinking agent, peptides containing at least two, preferably two cysteine moieties were used which varied in their amino acid sequence, in particular with respect to the presence or absence of a RGD adhesion motif and the presence or absence of a MMP-sensitive motif.

[0569] A further variation that was made to some hydrogels was the attachment of a bioactive ligand comprising a RGD adhesion motif or/and a ligand selected from the group consisting of natural laminins, for example laminin-111, recombinant laminin isoforms, and biofunctional fragments thereof. Examples of suitable recombinant laminin isoforms are laminin-111, laminin-211, laminin-332, laminin-411, laminin-511, or laminin-521. An array of hydrogels varying in the above preselected features was established, by the method described above.

[0570] The mechanical properties of the hydrogels were also varied (hard (2000-3000 Pa), medium (1000-2000 Pa) or soft (50-1000 Pa) gels).

[0571] A variety of different known, commonly employed and/or commercially available culture media was used. Preferably, said culture medium is characterized by the presence of Wnt agonists such as R-spondin. According to a preferred embodiment, a culture medium may be used that is adapted from the medium described in Drost et al. (Nature Protocol 11, 347-358, January 2016) or Beshiri et al. (Clinical Cancer Research 24, 4332-4345), May 2018). The preferred culture medium comprises AdDMEM/F12 medium supplemented with glutamine, BSA, transferrin, Noggin, fibroblast growth factor 2 or basic, and FGF 10 [FGF2 or FGF-basic, and FGF10], EGF, R-spondin-conditioned medium, penicillin/streptomycin, glutathione, optionally N-acetyl-L-cysteine, Nicotinamide, DHT (dihydrotestosterone), insulin, prostaglandin E2, A83-01, SB202190 (p38-inhibitor), Y-27632 (rock inhibitor), and HEPES.

[0572] The results are shown in FIGS. 6a and 6b. The hydrogels in examples 6a and 6b were soft, enzymatically degradable hydrogels. Example 6a is a hydrogel that does not comprise a RGD motif. Example 6b is a hydrogel that comprises a RGD motif.

[0573] It was found that normal (healthy) cells were growing only in extracellular matrix containing the bioactive peptide RGD (FIG. 6a, Example 6b). Also, the growth of healthy cells was less pronounced with medium gels containing RGD compared to soft gels with RGD. On the other hand, in both examples 6a (without RGD) and 6b (with RGD) prostate cancer cells grew (FIG. 6b).

[0574] In contrast thereto, in the comparative example using naturally-derived matrix Matrigel®, no differentiation of growth of healthy prostate cells and prostate cancer cells could be achieved with either culture medium (FIGS. 6a and 6b).