Method for Differentiating Epithelial Stem Cells

Abstract

The subject matter of the present invention is a method for differentiating epithelial stem cells, comprising culturing one or more epithelial stem cells in contact with an extracellular matrix in the presence of an expansion medium, a bovine pituitary extract, a receptor tyrosine kinase ligand, a supernatant of primary fibroblasts and optionally, a Rho kinase inhibitor.

Claims

1. A method for differentiating bladder epithelial stem cells, wherein said method comprises: culturing one or more epithelial stem cells in contact with an extracellular matrix in the presence of an expansion medium, the expansion medium comprising a basal medium for animal or human cells to which is added a bovine pituitary extract (BPE), a receptor tyrosine kinase ligand, a supernatant of bladder primary fibroblasts, and optionally, a Rho kinase inhibitor (rho-associated protein kinase inhibitor or ROCK inhibitor).

2. A method according to claim 1, wherein, in the expansion medium, the receptor tyrosine kinase ligand is selected from the group consisting of FGF, HGF and EGF.

3. A method according to claim 1, wherein, in the expansion medium, the receptor tyrosine kinase ligand is EGF.

4. The method according to claim 1, wherein, in the expansion medium, the ROCK inhibitor is selected from the group consisting of Fasudil, Ripasudil, Netarsudil, RKI-1447, Y-27632, GSK429286A, C21H16F4N4O2 or Y-30141.

5. The method according to claim 1, wherein, in the expansion medium, the ROCK inhibitor is Y-27632.

6. The method according to claim 1, wherein the supernatant of bladder primary fibroblasts is a supernatant of bladder primary fibroblast originating from human bladder tissue.

7. The method according to claim 1, wherein the epithelial stem cells are from human bladder.

8. An expansion medium, comprising a basal medium for animal or human cells to which is added a bovine pituitary extract (BPE), a receptor tyrosine kinase ligand, a supernatant of bladder primary fibroblasts.

9. The expansion medium of claim 8, wherein the ROCK inhibitor is selected from the group consisting of Fasudil, Ripasudil, Netarsudil, RKI-1447, Y-27632, GSK429286A, C21H16F4N4O2 or Y-30141.

10. The expansion medium of claim 9, wherein the ROCK inhibitor is Y-27632.

11. The expansion medium of claim 8, wherein the receptor tyrosine kinase ligand is selected from the group consisting of FGF, HGF and EGF or their combinations.

12. The expansion medium of any one of claim 8, wherein the supernatant of primary fibroblasts is a supernatant of bladder fibroblasts.

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23. A method of preparation of an organoid culture of human bladder tissue, in particular of preparation of an organoid culture of normal bladder tissue or of cancer bladder tissue, comprising the steps of: a. Providing a suspension of cells comprising urothelial cells including epithelial adult stem cells originating from said human bladder tissue in a suspension medium which comprises a basal medium for culture of animal or human cells, a ROCK inhibitor and optionally bovine pituitary extract (BPE) and/or a receptor tyrosine kinase ligand selected among the group of EGF, FGF and HGF and wherein said suspension medium is devoid of supernatant of primary fibroblast and, b. Culturing the cells provided in step above (a.) in an extracellular matrix and an expansion medium suitable for differentiation of said human epithelial adult stem cells, in conditions allowing cell expansion and cell differentiation of the epithelial adult stem cells originating from said human bladder tissue until a three dimensional tissue is obtained, wherein such step of culturing encompasses one or multiple passages or the cells, advantageously 2, 3, 4, or 5 passages, in particular up to 10 or up to 15 passages, c. Allowing the three dimensional tissue obtained in step above (b.) to grow as an organoid culture and optionally performing cellular and/or molecular characterization of the obtained organoids, wherein the expansion medium comprises at least a basal medium for culture of animal or human cells and a supernatant of primary fibroblast and wherein the composition of the expansion medium is adjusted during cell culture step as disclosed in b. so that in the each passage of the cell culture the expansion medium initially additionally comprises bovine pituitary extract (BPE) and/or a receptor tyrosine kinase ligand selected among the group of EGF, FGF and HGF and, optionally a ROCK inhibitor, when the expansion medium in said each passage is changed or replenished it is devoid of the ROCK inhibitor, in particular the expansion medium is as defined in any one of claims 1 to 12 for replenishment during successive cell passages.

24. The method according to claim 23, wherein the organoid culture is prepared from a bladder tissue previously obtained from a patient bladder tumour wherein the method comprises the following additional steps before first step (a.) of claim 23: i. providing a sample of bladder tumour tissue of a patient and dissecting said tissue in order to recover urothelium separated from submucosa, and recovering a suspension enriched with urothelial cells in particular as a supernatant following agitation of the medium comprising the dissociated cells; ii. centrifugating the suspension enriched with urothelial cells of i. and recovering the centrifugation pellet that contains urothelial cells.

25. The method according to claim 23, wherein the organoid culture is prepared from a patient derived tumour xenograft (PDX) of a bladder and wherein the method comprises the following additional steps before first step (a.) of claim 23: i. Providing bladder tumour tissue previously obtained from the xenograft, dissecting said bladder tumour tissue into pieces that may allow cells to be in contact with a dissociation solution, in particular bladder tumour tissue pieces of about 5 mm3; ii. Optionally incubating the obtained bladder tumour tissue of i. with a stripping solution to break intercellular junctions between urothelial cells and submucosal cells and recovering a suspension enriched with urothelial cells in particular as a supernatant following agitation of the suspension comprising the dissociated cells; iii. centrifugating the suspension enriched with urothelial cells of ii. and recovering the centrifugation pellet that contains urothelial cells.

26. The method according to claim 23, wherein the organoid culture is prepared from a bladder tissue previously obtained from a healthy bladder tissue of a human subject wherein the method comprises the following additional steps before first step (a.) of claim 23: i. Providing bladder tissue of a human subject and dissecting said tissue in order to recover urothelium separated from submucosa, and optionally incubating the bladder tumour tissue with a stripping solution to break intercellular junctions between urothelial cells and submucosal cells and recovering a suspension enriched with urothelial cells in particular as a supernatant following agitation of the suspension comprising the dissociated cells; ii. centrifugating the suspension enriched with urothelial cells of i. and recovering the centrifugation pellet that contains urothelial cells.

27. The method according to claim 23 wherein in step a. the expansion medium contains 80% to 40% of volume of a basal medium or of a basal medium supplemented with BPE and receptor tyrosine kinase ligand and receptor tyrosine kinase ligand in particular complete KSFM medium and 20% to 60% of volume of primary fibroblasts supernatant (PFS), and wherein in culture step b. during 2 to 3 days after the onset of the culture the expansion medium also comprises a ROCK inhibitor and during the subsequent cell culture a fresh expansion medium or a replenished medium is provided that comprises 80% to 40% of volume of a basal medium or of a basal medium supplemented with BPE and receptor tyrosine kinase ligand, with one volume of primary fibroblast supernatant (PFS), and said fresh expansion medium or said replenished medium is devoid of a ROCK inhibitor.

28. The method according to claim 23 wherein the extracellular matrix is provided to the cell suspension prior to the expansion medium and the extracellular matrix is allowed to polymerize in order to allow cells to be embedded therein prior to the addition of the expansion medium.

29. The method of claim 23, wherein organoids in the culture show viability for at least 10 days, while being maintained in the extracellular matrix, such as matrigel, and in the culture medium.

30. The method according to claim 1 wherein the organoid culture comprises organoids that have a size in the range of at least 30 μm.

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Description

[0124] Other subjects will emerge from reading of the description, the examples and the figures which follow.

[0125] FIGS. 1 and 2: describe cultures of healthy bladder organoids in presence of different culture media. a) Medium KSFM+BPE+hEGF 10×, b) medium KSFM+BPE+hEGF+fibroblast supernatant, 20×.

[0126] FIG. 3: describes tumoural organoids, 10×. a) bilobed organoids, b) organoids in cluster.

[0127] FIGS. 4 and 5: show a morphological comparison between healthy c) and tumoural d) organoids at D15, magnitude 10×.

[0128] FIG. 6 describes the IF characterization of organoids from a healthy bladder 1 actin, 2 Ck5, 3 Ck17, 4 Ck20, 5 UPK3A and 6 Dapi.

[0129] FIGS. 7 to 10: show immunofluorescence performed on BLHOU-40 tissues, 1) healthy tissue, 2) cancer tissue.

[0130] FIG. 11: L987 PDX model histological analysis. The analysis of the PDX model was performed in comparison of the patient's tumour. The following characteristics were observed [0131] + Cancer type and subtype: High grade epidermoid urothelial carcinoma+high grade papillary carcinoma; recurrence of a high grade (G3) urothelial carcinoma [0132] + Tumor stage: pTNM: pT4a [0133] + Basal-like phenotype [0134] + FGFR3 Mutation: R248C [0135] + PPARg mutation: no mutation

[0136] FIG. 12: Pharmacology assay in L987. The evaluation was performed using erlotinib (30 mg/kg) or a vehicle (10 mg/kg) administered per os to mice engrafted with the PDX. No antitumor response of L987 is shown.

[0137] FIG. 13: Pharmacology assay in L987. The evaluation was performed using erlotinib (100 mg/kg) administered per os or with a vehicle (10 mg/kg) administered through intraperitoneal route. An antitumor response is obtained with erlotinib with tumour growth inhibition (TGI) at a level of 62.4% when compared to control.

[0138] FIG. 14: Study Design for a screening assay (cell viability) on cancer organoid model from PDX

The assay is suitable to establish a proof of concept for using organoid model as a preclinical tool

Read-outs:

[0139] Immunofluorescence CK5, CK17, CK20, GATA3, Upk3 and Ki67 [0140] Viability assay reflected by metabolic activity (concentration of ATP by luminescence) in organoids culture: using the kit Cell Titer Glo 3D (Promega).
Pharmacological treatment (triplicates)— 2 Runs [0141] Treatment for 5 days [0142] Compound: Erlotinib (Run 1: 0.04-0.2-1-5-10-20 μM; Run 2: 0.02-0.04-0.1-0.2-0.5-1 μM) [0143] Vehicle: DMSO 2%

[0144] FIG. 15: Cellular composition of the organoid line BLOU-026: Immunofluorescence staining with the specific marker Uroplakin 3: Upk3 labeling (orange), DAPI nuclei labeling (cyan), actin labeling (green), Organoids were analyzed with Opera Phenix (Perkin Elmer) (×20 N.A=1.20). One confocal stack in the core of one selected organoid from representative z-stack slice showing Upk3 expression. [0145] A—Maximal intensity projection of all z-stack slices [0146] B—One confocal stack in the core of one selected organoid: Representative z-stack slice showing entire structure (nuclei and actin) [0147] C—Merged representation for Upk3, actin and nuclei labeling.

[0148] Scale Bar=100 μm

[0149] FIG. 16: Cellular composition of the organoid line BLOU-026: Immunofluorescence staining with the specific luminal marker: GATA3 labeling (red), DAPI nuclei labeling (cyan), actin labeling (green), Organoids were analyzed with Opera Phenix (Perkin Elmer) (×20 N.A=1.20). [0150] A—One confocal stack in the core of one selected organoid: Representative z-stack slice showing GATA3 expression. Maximal intensity projection of all z-stack slices [0151] B—Merged representation for GATA3, actin and nuclei labeling. Scale Bar=100 μm

[0152] FIG. 17: Cellular composition of the organoid line BLOU-026: Immunofluorescence staining with the specific markers CK20 (luminal) and CK17 (basal): CK20 labeling (orange), CK17 labelling (red), DAPI nuclei labeling (cyan), actin labeling (green), Organoids were analyzed with Opera Phenix (Perkin Elmer) (x20 N.A=1.20). [0153] A—One confocal stack in the core of one selected organoid: Representative z-stack slice showing nuclei. [0154] B—One confocal stack in the core of one selected organoid: Representative z-stack slice showing actin labeling. [0155] C—One confocal stack in the core of one selected organoid: Representative z-stack slice showing CK20 expression. [0156] D—One confocal stack in the core of one selected organoid: Representative z-stack slice showing CK17 expression. [0157] E—Merged representation for CK20, CK17, actin and nuclei labeling. Scale Bar=100 μm

[0158] FIG. 18: Cellular composition of the organoid line BLOU-026: Immunofluorescence staining with the specific marker CK5 (basal) and the proliferation marker Ki67: CK5 labeling (red), Ki67 labelling (orange), DAPI nuclei labeling (cyan), actin labeling (green), Organoids were analyzed with Opera Phenix (Perkin Elmer) (×20 N.A=1.20). [0159] Scale Bar=100 μm

[0160] FIG. 19: pharmacological assay using the BLOU-026 line [0161] Treatment with erlotinib was performed during 5 days [0162] Erlotinib was administered in DMSO 2% as a vehicle and the doses used were as follows: Erlotinib (Run 1: 0.04-0.2-1-5-10-20 μM; Run 2: 0.02-0.04-0.1-0.2-0.5-1 μM).
Drug response of organoid line (viability test by reference to ATP activity) is shown on the figure. Dose response curves for BLOU-026 organoids treated with the EGFR inhibitor erlotinib. Each data point corresponds to three biological replicates; error bars correspond to one standard error to the mean.

Example 1: Studied Population

[0163] Organoid cultures of human bladder were performed from bladder specimens collected intraoperatively during cystectomy for urothelial bladder cancer of human patients from 50 to 70 years old.

[0164] After extraction of the surgical specimen, a vertical incision was made on the anterior surface of the bladder and two samples were taken: One in a macroscopically “healthy” zone (control group—culture A) and one in a macroscopically “tumoral” zone (tumor organoids—culture B). Two tumors were classified as muscle invasive (at least pT2 or more) and poorly differentiated (high grade) and two were non-invasive (pT1) after cystectomy and anatomo-pathological analysis of the tumour (according to the 1973 and 2004 WHO grading classification).

TABLE-US-00001 Type of Histol- Histology Number Chemo- chemo- ogy after Sample of TR BCG therapy therapy of TR cystectomy BLHOU-33 1 0 1 MVAC >pT2 + pT3a CIS BLHOU-34 1 0 1 MVAC pT2 G3 pT3b BLHOU-36 3 1 0 0 pT2 pT1 BLHOU-40 1 0 1 MVAC pT4 pT1 MVAC = Methotrexate, Vinblastin, Adriamicin, Cisplatin BCG = Bacille of Calmette and Guerin (0 = no instillation/1 = a series of 6 instillations in the bladder) CIS = cisplatine TR = Transuretral Resection of the bladder

Example 2 Obtention of Bladder Cells

[0165] The bladder samples were dissected to eliminate adipose and connective tissue and to retain the urothelium and the submucosa. A “stripping” solution (Southgate et al., 2002, Culture of Epithelial Cells, Second Edition) is injected into the submucosa. The urothelium is incubated in the “stripping solution” at 4° C. overnight, with gentle shaking to break the intercellular junctions. The next day, the urothelium is removed from the muscularis/stroma using dissecting forceps and placed in collagenase IV solution (100U/ml) for 20 minutes at 37° C. Then, to stop the dissociation, 3 ml of KSFM medium (Keratinocyte Serum-Free Medium—Thermo Fischer) supplemented with Y27632 (10 μM—Sigma Aldrich, France) is added. Following strong manual agitation, the supernatant enriched in urothelial cells is centrifuged at 250×g for 5 minutes at 4° C. The supernatant is discarded and the pellet containing urothelial cells resuspended in KSFM supplemented medium containing 50 ng/mL of BPE and 5 ng/mL of EGF. After centrifugation and suspension in a volume of Matrigel (Matrigel hESC-qualified Matrix, BD Biosciences) at 4° C., a concentration of 2×10.sup.6 cells/mL of Matrigel is obtained.

Example 3: Culture Cells in Slides

[0166] 8-well Lab-teks (Lab-tek II Chamber Slides system, Dutscher) or 96-well μ-angiogenesis-Ibidi plates are used (60 000 cells in 25 □L of Matrigel for 8-well labteck and 25 000 cells seeded in 10 □L of Matrigel for 96-well plate). Matrigel is polymerized for 20 minutes at 37° C. and then the complete medium is added (one volume of complete KSFM medium and one volume of primary fibroblast supernatant (PFS) isolated from the culture of human bladder fibroblasts). The supernatant of fibroblasts is obtained after 3 to 4 days of culture of the fibroblasts (ATCC-PCS-420-013) in a fibroblast basal medium (ATCC-PCS-201-030) supplemented with a fibroblast growth kit-low serum (ATCC-PCS-201-041). During the first 3 days of culture, this complete expansion medium is supplemented with Y27632 (10 μM). The medium is changed every 2-3 days with fresh expansion medium which corresponds to the complete medium without Y27632 (250 μL/well in the 48 well plates and labtek, 100 μL/well in the 96-well plates). Plates are incubated at 37° C. in normoxia condition (5% CO2).

Example 4: Morphological Studies of “Healthy” Organoids

[0167] a) Comparison of culture media:

[0168] Culture wells of healthy control cells (A cultures) are made with and without PFS in the culture. Then, the cells are monitored and compared at day 14.

[0169] A larger number of structures as well as a substantially greater diameter for the organoids subjected to a culture medium supplemented with PFS are observed at D14 (FIG. 2) (approximately 40 organoids per well of 80 μm against a dozen structures of 20 microns in diameter with the KSFMc (FIG. 1)). This was observed with all the cultures of organoids performed.

[0170] The culture medium supplemented with primary fibroblast supernatant (PFS) thus seemed to promote the proliferation and growth of organoids compared to the KSFM culture medium complemented with EGF and BPE alone. [0171] b) Growth monitoring of organoids:

[0172] A total of 6 cultures of healthy organoids were carried out with the culture medium supplemented by PFS. We then compared the median organoid areas (solid structures) for these cultures at 14 days. There was a significant increase in the median organoid area on Day 14 (p=0.0013).

Example 5: Comparative Study of the Morphology of Organoids from Healthy and Tumour Bladders

[0173] The growth and morphology of organoids derived from patients with healthy control organoids (A cultures) or bladder cancer (B cultures) were monitored at day 7.

[0174] The observations were made under a wide-field microscope at 10× magnification (Apotome Zeiss, 10× objective). When acquiring images, the choice of fields was randomly made. The size of the organoids has been evaluated using the image processing software Image J.

[0175] In tumor cultures, solid organoids classically observed in healthy cultures but also new morphologies were found. Indeed, the new solid structures for tumor cultures can be classified as full multilobed organoids, “bilobed” (FIG. 3a) or “in clusters” (FIG. 3b). These different forms have never been observed in healthy cultures.

[0176] The size of the tumor organoids appeared to be higher than the healthy organoids, as shown between the healthy (A) and tumor (B) cultures (FIGS. 4 and 5). The comparison was made measuring 340 organoids, 170 healthy and 170 tumours.

Example 6: Immunolabeling of Organoids

[0177] Immunolabeling on tissue sections

[0178] Immunolabeling is performed on frozen bladder sections. Fresh samples were included in OCT (Optimal Cutting Temperature), cut longitudinally, and immediately frozen on dry ice and stored at −80° C. They are then cut with cryostat in 5□m sections, deposited on slides (Superfrost Plus, Thermo Scientific) and fixed with 3.7% formaldehyde for 20 minutes at room temperature or with 50% acetone/50% methanol until evaporation, depending on the antibody used. Blocking of nonspecific antigenic sites and permeabilization were achieved by a solution containing PBS/1% BSA/0.5% Triton ×100. Samples are incubated with diluted primary antibodies in blocking buffer overnight at 4° C. in a humid chamber. After washing with PBS without calcium/magnesium 1×, they are incubated for 1 hour with the secondary antibodies in the dark, at room temperature. The different antibodies used are summarized in Table 1 below. DAPI (4′,6-diamidino-2-phenylindole) nuclei labeling (diluted 1/200, incubated 20 min at room temperature) is performed and the slides are mounted with a drop of Prolong gold mounting medium and a coverslip.

TABLE-US-00002 TABLE 1 Antibodies used for immunolabeling Ref Tissue Antibody Species Provider Dilution fixation Cytokeratin 5 Rabbit ref 905501 /100 50/50 acetone Biolegend methanol Cytokeratin 17 Rabbit Ref ab10972 /100 50/50 acetone 5Abcam methanol Cytokeratin 20 Mouse Ref M7019  /50 FA 3.7% Dako Uroplakine 3a Rabbit Ref abin1688372  /50 50/50 acetone Santa cruz methanol GATA 3 Rabbit Ref AAB106625 /800 Formaldehyde Abcam 3.7% Ki 67 Mouse Ref M7240 /100 Formaldehyde Dako 3.7% Alexa A568 anti Donkey Ref A10037 /500 mouse Life Technologies Alexa A555 anti Donkey Ref A31572 rabbit Life technologies [0179] Immunolabeling on organoids

[0180] Organoids are fixed at culture day 7 or 14 with 3.7% formaldehyde for 40 minutes (labteks) or 20 minutes (96-well) at room temperature. They are permeabilized with 0.5% Triton (in PBS) and the aspecific antigenic sites are blocked for 1.5 hours at 37° C. with a 3% BSA solution in PBS. Then the organoids are incubated with the primary antibodies at 4° C. overnight followed by one hour at 37° C. After washes, the secondary antibodies are incubated for one hour at 37° C. in the dark. DAPI nuclei labeling (dilution 1/200e) and actin labeling by Alexa Fluor-488 phalloidin (diluted 1:500 in DAPI solution) is performed. After incubation for 20 minutes in the dark at room temperature, washes are performed and a drop of Vectashield mounting medium (Vector Labs) without DAPI is deposited in each well.

[0181] The organoids and slides were observed using the Zeiss LSM 710 confocal microscope with 20× and 40× objectives. For each experiment, sections or control wells are imaged either without any antibodies (control of autofluorescence) and with the secondary antibodies alone. The control images were acquired with the same microscopy parameters and processed identically using the Image J software.

[0182] The organoids were also imaged with an automated imaging device (Opera Phenix, Perkin Elmer) with 20× water objectives. The images were processed with the Harmony software that drives the device.

Example 7: Characterization of Healthy Organoids by Immunofluorescence (IF)

[0183] These structures were observed using a Zeiss LSM 710 confocal microscope at day 14. The epithelial markers Ck5 and Ck17 are physiologically expressed in undifferentiated basal cells. Ck20 is a predominant epithelial marker on superficial, differentiated cells. There was then a peripheral disposition of undifferentiated cell markers with a predominance of Ck20 at the center of the structures. In addition, Uroplakin-3A spots are visualized in the center of the structures. Uroplakin-3A (UPK3A) is a protein that plays a major role in urothelial barrier function and is therefore characteristic of differentiated cells. Cell differentiation therefore seems hierarchical, occurring from the outside to the inside of these organoids (see FIG. 6).

Example 8: Characterization of Tumor Tissues and Organoids by Immunofluorescence

[0184] We performed labeling on sections of healthy bladders and tumours with different urothelial markers (FIGS. 7 and 8). We found a similar distribution of these markers on samples A and B. Indeed, Ck17 was found preferentially at the level of the basement membrane and Ck20 in contact with the light (here facing upwards images). There was, however, unusual uptake of Ck17 in the superficial cells for tumor cutting (indicated by a white arrow). In the same way, there was an unusual fixation of Ck5 at the submucosal level on the tumor section (indicated by a white arrow). Uroplakin-3A was also observed in superficial cells in contact with bladder light. The tumor tissue sections used for labeling Ki67 (cell proliferation marker) and GATA3 (nuclear transcription factor) were probably located at the level of a papillary fringe, as evidenced by the histological aspect and the thickness of the urothelium. Overexpression of Ki67 and GATA3 was observed on these tumor sections, evoking an increase in cell proliferation.

[0185] The organoids were then observed on day 14 under a confocal microscope, where IF labeling was performed with the same antibodies as those used on tissue sections (FIGS. 9 and 10).

[0186] We then observed an expression of the different urothelial markers, including Upk3A, showing differentiated cell expression and confirming the urothelial character of these structures. It seems that cell differentiation is from the outside to the inside of the structure. Indeed, the Ck17 (basal marker) was observed mainly at the periphery of the structure while Ck20 or UPK3A (luminal markers) were expressed towards the center of these organoids whether in healthy cultures or tumours.

[0187] There is no specific difference between the IF labeling of organoids according to the healthy or tumor character under a confocal microscope.

Example 9: Pharmacological Responses of Organoids Following Treatments

[0188] The organoids were cultured in Matrigel in 96-well black plates for 15 days. Healthy and tumorous organoid cultures were treated with cisplatin and gemcitabine from the 8th day of culture. The concentrations used are 2 μM, 20 μM and 200 μM for cisplatin and 0.05 μM, 0.5 μM and 5 μM for gemcitabine. These molecules were added to the culture medium, the latter being changed every two days until the 15th day of culture. The gemcitabine and cisplatin are chemotherapy medications used to treat bladder cancers (Rouprêt et al., Progrès en Urologie, 2018, 28: S46-S78).

[0189] The cell viability was analyzed using the CellTiter-Glo®3D kit (Promega, #9683) at the fifteenth day of culture. Briefly, in order to dissociate the organoids, they were incubated with Tryple Express solution (Gibco) for 40 minutes at 37° C. by pipetting regularly. After dissociation of the structures, the CellTiter-Glo®3D reagent was added to the culture medium of each well (volume 1:1) and incubated for 30 minutes at room temperature, in the dark, with stirring, according to the manufacturer's instructions. Luminescence was measured with a luminometer (Varioskan Flash-Thermo Scientific, Illkirch, France). Three wells per condition were measured and analyzed. Data analyzes were performed using Excel software, and IC50 values were calculated using non-linear regression. Comparisons between groups were made using the paired or unpaired Student t-test. The level of significance retained was p<0.05. Statistical analysis was performed using PRISM (Graph Pad Prism® Version 5.0, Software, Inc., California, USA).

[0190] The IC50 of cisplatin for tumor culture was 1.75 μM while the IC50 for healthy culture was 4.2 μM (factor 2.4). These results show a greater effectiveness of treatment on tumor cells for the same concentration. The IC50 of Gemcitabine for tumor culture was 1.2 μM while the IC50 for healthy culture is 2.8 μM, also demonstrating a higher efficiency of chemotherapy on tumor culture (factor 2.3).

Example 10: Organoids from PDX

[0191] Organoids are developed from either fresh or frozen PDX obtained from bladder cancer. To obtain the organoids, the PDX tissue is handled as if it was the tumor tissue of a patient according to the protocol of example 2. Then, the same protocol as in examples 3, 5, 6, 8 and 9 is used to derive and obtain the organoids from the PDX.

Example 11: Pharmacological Results on Organoids Derived from PDX Model (Patient-Derived Tumor Xenograft Model) from Bladder Cancer

1.1. Description of the PDX Model L987

[0192] L987 is a basal-like model derived from a bladder carcinoma of high grade (G3) infiltrating the muscle of the bladder (pT4a). L987 has been developed as previously described (Lang H. et al, 2016, Oncotarget, Vol. 7, No. 37:59336-59359). L987 has been characterized at different levels to ensure the conservation of the patient's tumour characteristics. The different analyses were: [0193] Histology (H&E staining) and IHC [0194] Transcriptomic, [0195] Genomic (Single Tandem Repeat—STR, sequencing)
The histological profile of L987 (FIG. 11) and its parental tumour correlated with their molecular classification (basal tumour).
STR results (table 2) confirmed the high rate of identity between the PDX model observed after 3 passages (P3) and the patient's tumour. Both the patient's tumour and the PDX model harbour an FGFR3 mutation at position R284C

TABLE-US-00003 TABLE 2 Patient's tumor PDX tumor (P3) AMEL X Y X Y D10S1248 12 13 12 13 D12S391 22 23 22 23 D19S433 13 16.2 13 16.2 D1S1656 11 12 11 12 D22S1045 15 15 D2S1338 20 21 20 21 D2S441 11 15 11 15 D6S1043 12 14 12 14 TH01 6 7 6 7

1.2. Pharmacology Assay with L987

[0196] The inventors evaluated the anti-tumour response of an EGFR inhibitor, Erlotinib, on L987 PDX tumor. The PDX was engrafted on the back of Male Mice, Swiss Nude Mice (Charles River Laboratories) that were 4 weeks at delivery.
In a first experiment, mice were treated either with a vehicle per os (p. o.) 5 times a week or with Erlotinib at 30 mg/kg p.o., 5 days a week, for 4 weeks.
No effect of Erlotinib was observed at that dosing. (FIG. 12)
In a second experiment, mice were treated either with a vehicle by intraperitoneal route 4 times a week or with Erlotinib at 100 mg/kg per os, 6 days a week, for 4 weeks.
Results showed that the tumour responded to the treatment. The tumour growth inhibition was of 62.5% (FIG. 13)

1.3. Generation and Characterization of the Organoid Line BLOU-026 from the PDX Model L987

[0197] The organoid line BLOU-026 was obtained following the same protocol as in examples 3, 5, 6, 8 and 9.
After 1 week culture, organoids observed were immature (cystic structures) or differentiated (budding structures).
Organoid dimensions varied from 40 to 500 μm, according to their differentiation state.
Organoid culture can be maintained for a long time, since organoids have been subjected to passaging more than 3 times.
Organoids obtained are representative of human bladder morphology, expressing specific markers of basal (CK5, CK17) and luminal (CK20, Upk3) cells.
The design of the study is shown on FIG. 14.
The cellular composition of the organoid line BLOU-026 has been examined using various markers. Uroplakin 3 is a marker specific for umbrella cells present in the luminal side of the bladder by contrast to markers expressed on cells that are on the basal side of the bladder. Uroplakin 3 is a marker of the late stage of the differentiation of the organoid showing that the obtained organoid would potentially encompass a lumen compartment. Other markers of the luminal compartment in the baldder were also detected which are GATA-3 and CK20. Additional markers were identified, providing proof for the presence of basal cells and of cells having proliferation capability, i.e., tumor cells. The identification of the markers provides proof of organoid formation with differentiated cells of the various urothelium cellular subtypes: superficial umbrella cells, intermediate cells and basal cells, the umbrella cells of the late differentiation stage showing that a mature organoid has been obtained.
FIG. 15 accordingly shows the cellular composition of the organoid line BLOU-26: Immunofluorescence staining with the specific marker Uroplakin 3: Upk3 labeling (orange), DAPI nuclei labeling (cyan), actin labeling (green), Organoids were analyzed with Opera Phenix (Perkin Elmer) (×20 N.A=1.20). [0198] A—One confocal stack in the core of one selected organoid: Representative z-stack slice showing CK5 expression [0199] B—One confocal stack in the core of one selected organoid: Representative z-stack slice showing Ki67 expression [0200] C—One confocal stack in the core of one selected organoid: Representative z-stack slice showing nuclei. [0201] D—One confocal stack in the core of one selected organoid: Representative z-stack slice showing actin labeling [0202] E—Merged representation for CK5, Ki67, actin and nuclei labeling
FIG. 16 discloses cellular composition of the organoid line BLOU-026 Immunofluorescence staining with the specific luminal marker: GATA3 labeling (red), DAPI nuclei labeling (cyan), actin labeling (green), Organoids were analyzed with Opera Phenix (Perkin Elmer) (×20 N.A=1.20). [0203] A—One confocal stack in the core of one selected organoid: Representative z-stack slice showing GATA3 expression. [0204] B—Maximal intensity projection of all z-stack slices [0205] C—Merged representation for GATA3, actin and nuclei labeling.
FIG. 17: Cellular composition of the organoid line BLOU-026 Immunofluorescence staining with the specific markers CK20 (luminal) and CK17 (basal): CK20 labeling (orange), CK17 labelling (red), DAPI nuclei labeling (cyan), actin labeling (green), Organoids were analyzed with Opera Phenix (Perkin Elmer) (x20 N.A=1.20). [0206] A—One confocal stack in the core of one selected organoid: Representative z-stack slice showing nuclei. [0207] B—One confocal stack in the core of one selected organoid: Representative z-stack slice showing actin labeling. [0208] C—One confocal stack in the core of one selected organoid: Representative z-stack slice showing CK20 expression. [0209] D—One confocal stack in the core of one selected organoid: Representative z-stack slice showing CK17 expression. [0210] E—Merged representation for CK20, CK17, actin and nuclei labeling.
The presence of basal (CK5, CK17) and luminal biomarkers (CK20, GATA3 and Upk3) suggested that the experimental conditions allowed correct differentiation of the organoids from BLOU-026 line. GATA3 was used as a marker of tumoral cells. The presence of Ki67 evidenced proliferation of the organoids.

1.4. Pharmacological Assay with BLOU-026

[0211] As for the PDX model L987 from which BLOU-026 is derived, a pharmacological assay using the BLOU-026 line has been set-up and validated and a concentration-response curve to Erlotinib has been constructed.
A viability assay reflected by metabolic activity in organoids culture was used based on the commercial kit Cell Titer Glo 3D (Promega), evaluating the concentration of ATP in organoids by luminescence.
Pharmacological treatment (triplicates)— 2 Runs [0212] Treatment for 5 days [0213] Vehicle: DMSO 2% [0214] Compound: Erlotinib (Run 1: 0.04-0.2-1-5-10-20 μM; Run 2: 0.02-0.04-0.1-0.2-0.5-1 μM)
The results are shown on FIG. 19.

1.5. Conclusion

[0215] In our experimental conditions, Erlotinib inhibits metabolic activity of organoid culture in a concentration-dependent manner. This result is in accordance with what has been observed in the corresponding PDX model L987.