OVARIAN CANCER ORGANOID CULTURE

Abstract

The present invention relates to a method for the production of a culture, e.g. an organoid culture of ovarian cancer or cancer precursor cells, particularly of high grade serous ovarian carcinoma cells. By means of this method, an organoid culture of ovarian cancer or cancer precursor cells and a biobank comprising a plurality of different organoid cultures of ovarian cancer or cancer precursor cells may be generated. Further, a culture medium suitable for the long-term culture of ovarian cancer or cancer precursor cells is provided. Furthermore, use of the organoid culture and the biobank for medical applications, e.g. in the field of diagnostics, and therapy and in the field of drug screening is described.

Claims

1. A method for the production of an ovarian cancer or cancer precursor cell culture, said method comprising the steps: (a) cultivating ovarian cancer or cancer precursor cells in a suitable cell culture medium which comprises at least one compound which stimulates BMP signaling, e.g. via BMP2, and (b) optionally obtaining a cell culture from the cultivation step (a), wherein the cell culture is an organoid culture, and wherein the culture medium is free of Noggin.

2. The method of claim 1, wherein the culture medium comprises at least one compound which stimulates BMP signaling selected from mitogenic growth factors such as EGF or/and FGF, a BMP, e.g. BMP2, or/and a Rspondin, e.g. Rspondin1, wherein the medium particularly comprises (i) EGF or/and a BMP, or (ii) EGF, FGF or/and a Rspondin.

3. The method of claim 1, wherein the culture medium is free from exogenously added Wnt protein, e.g. from an exogenously added Wnt3a protein.

4. The method of claim 1, wherein the culture medium is free of an exogenously added compound which inhibits BMP signaling.

5. The method of claim 1, wherein the ovarian cancer cells are primary ovarian cancer or cancer precursor cells, more particularly primary ovarian cancer or cancer precursor cells derived from a human patient.

6. The method of claim 1, wherein the ovarian cancer is a high-grade serous ovarian carcinoma.

7. An ovarian cancer or cancer precursor cell organoid culture which can be stably propagated.

8. The organoid culture of claim 7, as obtainable by the method of claim 1.

9. A biobank, comprising a plurality of different organoid cultures of claim 7.

10. A method for using one or more organoid cultures of claim 7, (i) for the determination of the efficacy of a compound in the treatment of ovarian cancer, or (ii) for the identification of a compound suitable in the treatment of ovarian cancer.

11. Use of a cell culture medium which comprises at least one compound which stimulates BMP signaling, e.g. via BMP2, for the production of a culture, e.g. an organoid culture, from ovarian cancer or cancer precursor cells, wherein the cell culture medium is free of Noggin.

12. A method for the determination of the efficacy of a compound in the treatment of ovarian cancer, particularly of ovarian cancer in an individual patient, comprising the steps (a) providing an ovarian cancer or cancer precursor cell organoid culture according to claim 7, (b) contacting at least one compound with the organoid culture provided in step (a), said at least one compound being selected from therapeutically active agents suitable for the treatment of ovarian cancer, (c) determining growth or/and propagation of the organoid culture after being contacted with the at least one compound, and (d) selecting at least one compound which inhibits growth or/and propagation of the organoid culture, as determined in step (c).

13. A screening method for the identification of a compound suitable in the treatment of ovarian cancer, particularly of ovarian cancer in a plurality of different patients, comprising the steps (a) providing at least one ovarian cancer or cancer precursor cell organoid culture according to claim 7, (b) contacting at least one compound with the at least one organoid culture provided in step (a), said at least one compound being selected from candidate active agents presumed to be suitable for the treatment of ovarian cancer, (c) determining growth or/and propagation of the at least one organoid culture after being contacted with the at least one compound, and (d) selecting at least one compound which inhibits growth or/and propagation of at least one organoid culture, as determined in step (c).

14. A compound selected from active agents suitable for the treatment of ovarian cancer, for use in the treatment of a patient suffering from ovarian cancer, said treatment comprising the steps (a) providing an ovarian cancer or cancer precursor cell organoid culture according to claim 7 from said patient, (b) contacting at least one compound with the organoid culture provided in step (a), said at least one compound being selected from active agents suitable for the treatment of ovarian cancer, (c) determining growth or/and propagation of the organoid after being contacted with the at least one compound, (d) selecting a compound which inhibits growth or/and propagation of the organoid culture, as determined in step (c), and (e) treating the patient with compound selected in step (d).

Description

FIGURE LEGENDS

[0203] FIG. 1: Example of an ovarian cancer cell organoid culture line (OVK ORG 11) which grows in medium A

[0204] Although some cell aggregates were initially formed in the SM standard medium, the culture underwent growth arrest in the next passage. Organoids cultivated in medium A reached much bigger size (˜200-500 μm) and could be propagated stably.

[0205] FIG. 2: Addition of soluble BMP2 improves organoid growth in high-grade serous carcinoma (HGSC) 3D cultures

[0206] The plot is representative of formed organoid counts per field view from 3 different patient isolates which were cultivated in parallel in medium −/+BMP2.

[0207] FIG. 3: Patient-derived organoid cell line (OVK ORG 2) shows established stable long term growth in medium B

[0208] The upper part shows the absence of organoid formation from cancer cells in standard SM medium, while healthy fallopian tube cells from the same patient gave rise to a stable organoid culture. The lower part shows the formation of organoids from cancer cells in medium B.

[0209] FIG. 4: Confocal images of a patient derived organoid cell line (OVK ORG 11) showing strong nuclear accumulation of p53 which is characteristic in cells harboring p53.

[0210] Organoids are positive for p53, EpCAM and PAX8 and have highly polymorphic nuclei which is hallmark of HGSC.

[0211] FIG. 5: Individual response curve to carboplatin treatment in organoid cell lines ORG1 and ORG2

[0212] Treatment of organoid cancer lines with carboplatin, a standard component of the chemotherapy treatment for HGS patients revealed differences in responsiveness and kinetics. Cell vialiability (y axis) was determined proportional to the strength of luminiscence signal (Cell titer.sup.R Glow 3D Promega). The test also showed only minor technical variability among triplicates (bars of different color) which makes it suitable for quantitative studies.

EXAMPLE

[0213] Methods

[0214] All samples processed in the study were obtained as fragments of solid tumor deposits from chemotherapy naïve high grade serous ovarian cancer (HGSC) patients undergoing debulking surgery. Tumor samples were removed during the surgery. Initial assessment of the tumor content was performed by the operating clinician. Samples were transported on ice within 2 hours of the procedure and subjected to a cell isolation procedure.

[0215] The following steps were performed to ensure proper quality analysis (phenotypic and genotypic) of the processed tissue:—a 3 mm.sup.3 fragment was shock frozen in liquid nitrogen and stored at −80° C.; a further 3 mm.sup.3 fragment was fixed at room temperature (RT) for 24 h in 3.7% paraformaldehyde (PFA) followed by paraffin embedding. The remaining tissue was used for cell isolation.

[0216] In the following, an experimental procedure including all the steps required for establishment of long term organoid culture from HGSC patients tissue is described. As Matrigel® is highly selective and supports growth of epithelial cells no absolute exclusion of stroma cells is necessary to obtain pure organoid lines which are 100% EpCam positive.

[0217] Step 1: Isolation and 2D Cultivation of Cancer Cells [0218] Adv. +++: Advanced F12 Medium+HEPES (6 ml)+Glutamax (5 ml)+5% fetal calf serum (FCS) [0219] Adv. ++: Advanced F12 Medium+HEPES (6 ml)+Glutamax (5 ml) [0220] Cut tissue with a scissor in small pieces and then mince further with scalpel for 3-5 minutes [0221] Collect all fragments, incubate with 2 ml Collagenase Type 1+2 ml Collagenase type II and transfer to a 6 well plate, [0222] Incubate fragments for 45 min at 37° C. 5% CO.sub.2, [0223] Add 5 ml Adv +++ and transfer in to a 50 ml Falcon tube, [0224] Vortex vigorously for 30 seconds, [0225] Let pieces settle down, take supernatant and transfer in to a fresh tube, [0226] Add again 5 ml Adv +++, mix with a pipette, [0227] Let pieces settle down, take supernatant and transfer in to a fresh tube, [0228] Centrifuge for 5 min at 400 rpm, [0229] Discard supernatant, [0230] Prepare 10 ml Adv +++, add 30 μl Y-27632 and 10 μl hEGF (final conc. 10 ng/ml), [0231] Resuspend cell pellet in medium, [0232] Seed cell in a flask, [0233] Incubate at 5% CO.sub.2 at 37° C. and grow cells 3-5 days prior to transfer to 3D culture.

[0234] Step 2: Seeding Cancer Cells in 3D Culture [0235] Remove Medium from the flask and wash one time with PBS, [0236] Add 1 ml TrypLE and incubate 10 min until cells are completely detached, [0237] Remove cells with 5 to 10 ml of Adv ++ and transfer in a 15 ml Falcon tube, [0238] Centrifuge at 1200 rpm for 5 min, [0239] Remove supernatant, [0240] Add 1 ml add Adv ++ and count cells, [0241] Keep cells on ice during this time; [0242] Seeding of cells: [0243] For a 24 well plate—20.000 cells per 50 μl Matrigel [0244] For a 48 well plate—10.000 cells per 25 μl Matrigel [0245] Seed cells in prewarmed plate, [0246] Let Matrigel solidify for 30 min in the incubator, [0247] Add growth medium, [0248] For a 24 well plate—500 μl, [0249] For a 48 well plate—250 μl, [0250] Change Medium twice times per week.

[0251] Step 3: Splitting of the Cancer Organoid Culture

[0252] Splitting should be carried out about once per 3 weeks. [0253] Remove medium from all wells, [0254] Add 1 ml ice cold Adv +++ to the Matrigel droplet, [0255] Scrape with pipet tip on the bottom of well, [0256] As Matrigel dissolves transfer organoids in the medium to 15 ml Falcon tube, [0257] Wash the splitting well with 500 μl cold medium and put the medium thereafter into the same Falcon tube, [0258] Fill up to 5 ml with Adv ++ (cold) and spin down (1000 rpm, 5 min), [0259] Remove supernatant and add 1 ml TrypLE, [0260] Incubate for 10 min at 37 C, vortex shortly 2-3 times for 20 seconds, [0261] Fill up to 5 ml with cold Adv ++, [0262] Centrifuge at 4° C. for 5 min at 1200 rpm, [0263] Remove the supernatant, [0264] If impossible, wash the pellet again, [0265] For 1 well dissolve cells in 50 μl Matrigel, seed as an drop per well, [0266] Incubate for 30 min at 37° C., [0267] Add culture medium, [0268] Change medium every 3-4 days.

[0269] Results

[0270] We report culture conditions and procedures for establishing a stable long term organoid culture from primary tumor deposits of high grade serous ovarian cancer.

[0271] There are substantial changes in the niche conditions which need to be provided to ensure in vitro cultivation of patient tumor samples, compared to the already described niche conditions for adult stem cells from the healthy epithelium (Kessler et al., 2015, supra). Significantly, we found that exogenous supplementation of Wnt 3a is detrimental for ovarian cancer stem cells as all of the established lines grew in Wnt3a-negative media. However, some individual samples continued to benefit from supplementation of Wnt agonist.

[0272] In contrast to the healthy oviductal epithelium, and other healthy mucosal models, we found that a HGSC organoid culture is dependent on active BMP signaling. This may be achieved by omission of the Noggin from the media (see medium A). As Noggin is a potent chelating agent for endogenous BMPs which are produced in the organoids this leads to the activation of downstream BMP signaling. Interestingly, none of the tested cancer tissues was able to give rise to an organoid culture in standard 3D medium (SM). Organoids could not form at all, or experienced growth arrest during cultivation (FIG. 1).

[0273] Cancer cells seeded in medium A, containing only basic medium components plus EGF and BMP2, gave rise to a stable and expandable long term culture. Although endogenous activation of the BMP pathway occurs as direct consequence of the absence of Noggin in the medium it was tested if increasing the amount of BMP confers further growth advantage to the organoids. Indeed, organoid formation efficiency in the HGSC organoid cultures was clearly improved by exogenous supplementation of soluble BMP2 (final concentration 10 ng/ml) to the growth medium (FIG. 2). Thus, BMP2 was included in the standard composition of the medium A.

[0274] We found stable long term propagation of HGSC organoids in basic medium (see table) supplemented solely with EGF and BMP2 (designated medium A). However two of the generated cultures required additional growth support which was provided by Rspondin 1 and FGF (medium B) in the medium. Both media lack Noggin and thus have activated BMP signaling contrary to the requirements of a healthy organoid culture. As a control comparison, a Fallopian tube organoid culture from the same patient efficiently cultivated in SM medium, showing that new properties are restricted to the malignant tissue.

TABLE-US-00001 TABLE 1 Basic Components of components standard medium (present in all for healthy Components Components media) FT (SM) of medium A of medium B Advance F12++, EGF 10 ng/ml, EGF 10 ng/ml, EGF 10 ng/ml, B27 2%, FGF 100 ng/ml, BMP2 10 FGF 100 ng/ml, Nicotinamide Noggin 100 ng/ml Rspondin, 10% 1 mM, N2 1%, ng/ml, conditioned Y-27632 9 Rspondin 10% medium μM, TGFβ RI conditioned 0.5 μM medium inhibitor Wnt 25% conditioned medium

[0275] In order to find out whether Rspondin is required for achieving stable long term cultivation, each patient sample should be subjected to parallel testing under two different conditions in the presence and absence of Rspondin: for example, medium A and (general organoid culture supplements+BMP2+EGF) and medium B (general organoid culture supplements+EGF+FGF+Rspondin1).

[0276] Patient-derived organoid cultures from ovarian cancer can be propagated stably in a long term culture (>6 months) with regular splitting intervals of e.g. 3-4 weeks. The cultures can be routinely frozen and thawed which is a key prerequisite for biobanking storage.

[0277] Immunofluorescence analysis of the key structural and functional biomarkers confirmed that organoids match native tumor tissue in phenotype, e.g. PAX8 confirming Mullerean lineage, EpCAM, p53 and p16 (FIG. 4). All tested organoid cultures were matched in phenotype to the fragment of native tumor processed and stained in standard procedure which was used for clinical diagnosis. Ovarian cancer cancer organoids mostly lack central cavities in contrast to fallopian tube organoids which are built of polarized epithelial monolayers. This is expected as histological atypia and stratification are present already in the early stages of the carcinogenesis in the fallopian tube (Shaw et al 2009, Modern Pathology 22, 1135-1138). Panel sequencing of paired organoid culture and tissue confirmed that the tested organoids recapitulate cancer tissue composition also on the genomic level. Importantly cultures can be expanded to multi-well format to enable high throughput experimental set up. Pilot drug test experiments with carboplatin, first line compulsory agent in the treatment of the HGSC patients revealed that organoids show individual patient profiles in their response to the drug which can be determined by precise quantitative measurements.

[0278] Therefore it can be concluded that ovarian cancer organoids fatefully reproduce tumor cells in vitro and thus represent an adequate biological model which opens a potential to advance treatment approaches in several major categories: 1) in vitro testing of drug response (complementary to the in silico predictions based on NGS data); 2) drug screening of new drug candidates; 3) in vitro study of resistance mechanisms; 4) identification of new biomarkers, as tissue based approaches are limited by a high background due to complexity of in vivo system; 5) identification of novel neo-antigens for improving efficiency of immunotherapies; 6) development of assays to study antigen specific immune responses in vitro such are direct cytotoxicity assays (CTL assays) by using patient cancer organoids; 7) investigating mechanisms to improve efficiency of the immunotherapy by genetically modifying cancer organoids.

[0279] Within this study we have so far generated 15 patient organoid lines. The production method could be further improved by optimizing clinical sampling during surgery as most of the patients undergo surgery at FIGO (International Federation of Gynecology and Obstetrics) stage III where metastatic peritoneum deposits are abundant and any large tumor mass is likely heterogeneous. The procedure described herein sets a major milestone which enables creation of live tumor biobanks for high number of patients.

[0280] Taking into account very narrow therapeutic options which are currently available to ovarian cancer patients, and wide spread platinum resistance which ultimately develops in all patients, the organoid culture represents an attractive model to test in advance the therapeutic response of each patient to different classes of approved drugs and provide the clinician with valuable information which could improve decision making process at the late stages. This type of application is a promising methodology for improvement of personalized therapy.