METHODS FOR PRODUCING CANCER STEM CELL SPHEROIDS

Abstract

The invention provides a method for producing a population of ready-to-use spheroid forming cancer cells, comprising: (i) growing cancer cells in suspension culture in a first culture medium on one or more first low-adhesion tissue culture plates thereby forming cancer cell spheroids enriched in cancer stem cells; (ii) disaggregating said cancer cell spheroids to form a suspension of single cells enriched in cancer stem cells; (iii) plating said suspension of single cells in a second culture medium on one or more second low-adhesion tissue culture plates; and (iv) freezing said suspension of single cells in said one or more second tissue culture plates, thereby producing a population of ready-to-use spheroid forming cancer cells. Also provided are cell populations produced by the method and kits for growing cancer cell spheroids, including for use in screening of test compound.

Claims

1. A method for producing a population of ready-to-use spheroid forming cancer cells, comprising: (i) growing cancer cells in suspension culture in a first culture medium on one or more first low-adhesion tissue culture plates thereby forming cancer cell spheroids enriched in cancer stem cells; (ii) disaggregating said cancer cell spheroids to form a suspension of single cells enriched in cancer stem cells; (iii) plating said suspension of single cells in a second culture medium on one or more second low-adhesion tissue culture plates; and (iv) freezing said suspension of single cells in said one or more second tissue culture plates, thereby producing a population of ready-to-use spheroid forming cancer cells.

2. The method according to claim 1, wherein said cancer cells comprise cells of a cancer cell line or primary cell culture derived from a tumour.

3. The method according to claim 2, wherein the cancer cell line is selected from the group consisting of: human breast carcinoma MDA-MB-436; human glioblastoma U87MG; human colon carcinoma HCT116; human ovarian SK-OV-3; human lung NCI-H446; human lung A549 carcinoma; human pancreatic PANC1; human pancreatic Capan-1; human MCF-7 breast carcinoma; human BT474 breast carcinoma; human OVCAR ovarian carcinoma; human LNCaP prostate carcinoma; and human CaCo2 colon carcinoma.

4. The method according to claim 2, wherein said primary culture comprises a patient derived xenograft (PDX).

5. The method according to any one of the preceding claims, wherein said first culture medium comprises a mixture of Dulbecco's modified Eagle's medium (DMEM) and Ham's F-12 medium, supplemented with B27 supplement, epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF).

6. The method according to claim 5, wherein the medium does not comprise methylcellulose.

7. The method according to claim 5 or claim 6, wherein the EGF and bFGF supplementation is added only once at the beginning of the culture and is not added repeatedly thereafter.

8. The method according to any one of the preceding claims, wherein said one or more first low-adhesion tissue culture plates and/or said one or more second low-adhesion tissue culture plates comprise a coating of poly-2-hydroxyethylmethacrylate or comprise Corning Ultra-Low Attachment plates.

9. The method according to any one of the preceding claims, wherein said one or more second low-adhesion tissue culture plates comprise more wells per plate than said one or more first low-adhesion tissue culture plates.

10. The method according to any one of the preceding claims, wherein: said one or more second low-adhesion tissue culture plates comprise 96-well plates or 384-well plates; and/or said one or more first low-adhesion tissue culture plates comprise 6-well plates or 12-well plates.

11. The method according to any one of the preceding claims, wherein said second culture medium comprises a cryopreservation medium.

12. The method according to claim 11, wherein said cryopreservation medium comprises CELLBANKER cell freezing medium.

13. The method according to any one of the preceding claims, wherein growing said cancer cells in step (i) comprises plating the cells at a density of between 1000 cells/ml and 100000 cells/ml.

14. The method according to claim 13, wherein the cancer cells comprise breast carcinoma MDA-MB-436 cells and the growing said cancer cells in step (i) comprises plating the cells at a density of between 10000 cells/ml and 30000 cells/ml, optionally at a density of about 25000 cells/ml.

15. The method according to claim 13, wherein the cancer cells comprise glioblastoma U87MG cells and the growing said cancer cells in step (i) comprises plating the cells at a density of between 5000 cells/ml and 15000 cells/ml, optionally at a density of about 10000 cells/ml.

16. The method according to any one of the preceding claims, wherein said plating said suspension of single cells in step (iii) comprises plating the cells at a density of between 1000 cells/ml and 2000 cells/ml, optionally at a density of about 1600 cells/ml.

17. The method according to any one of claim 16, wherein the cancer cells comprise colon carcinoma HCT116 cells or glioblastoma U87MG cells.

18. The method according to any one of claims 4 to 12, wherein the cancer cells comprise PDX cells and the growing said cancer cells in step (iii) comprises plating the cells at a density of between 5000 cells/well and 15000 cells/well, optionally at a density of about 10000 cells/well.

19. The method according to any one of the preceding claims, wherein said plating said suspension of single cells in step (iii) comprises plating the cells into multiple wells at different cell densities.

20. The method according to claim 19, wherein wells at the edge of the one or more second low-adhesion tissue culture plates are left blank or receive cells at a different density to wells not at the plated edge.

21. The method according to any one of the preceding claims, further comprising packing, labelling and/or shipping the one or more second tissue culture plates comprising the frozen population of ready-to-use spheroid forming cancer cells.

22. The method according to any one of the preceding claims, further comprising: (v) thawing the frozen population of ready-to-use spheroid forming cancer cells by adding a third culture medium to some or all of the wells of the one or more second tissue culture plates and warming the one or more second tissue culture plates at least until the frozen cells are thawed.

23. The method according to claim 22, wherein the third culture medium is the same as the first culture medium.

24. The method according to claim 22 or claim 23, further comprising: (vi) growing the thawed population of ready-to-use spheroid forming cancer cells until a plurality of spheroids form.

25. The method according to claim 24, wherein step (vi) comprises incubating the cells for between 5 and 7 days, optionally for about 6 days.

26. The method according to claim 24, wherein the cancer cells comprise PDX cells and wherein step (vi) comprises incubating the cells for between 4 and 6 days.

27. The method according to any one of claims 22 to 26, comprising adding at least one test compound to one or more of the wells prior to the formation of spheroids in order to assess the effect of the at least one test compound on spheroid formation.

28. The method according to any one of claims 24 to 27, comprising adding at least one test compound to one or more of the wells once spheroids have formed in order to assess the effect of the at least one test compound on spheroid viability.

29. A population of ready-to-use spheroid forming cancer cells produced or producible by a method as defined in any one of claims 1 to 21.

30. The population of cells according to claim 29, wherein the cells are frozen in one or more low-adhesion multi-well tissue culture plates.

31. The population of cells according to claim 30, wherein the one or more plates are sealed and/or labelled for shipping.

32. The population of cells according to any one of claims 29 to 31, wherein the cells are cell line cells selected from the group consisting of: human breast carcinoma MDA-MB-436; human glioblastoma U87MG; human colon carcinoma HCT116; human ovarian SK-OV-3; human lung NCI-H446; human lung A549 carcinoma; human pancreatic PANC1; human pancreatic Capan-1; human MCF-7 breast carcinoma; human BT474 breast carcinoma; human OVCAR ovarian carcinoma; human LNCaP prostate carcinoma; and human CaCo2 colon carcinoma.

33. The population of cells according to any one of claims 29 to 31, wherein the cancer cells are PDX cells.

34. A kit of parts, comprising: one or more low-adhesion multi-well tissue culture plates having plated therein a frozen population of ready-to-use spheroid forming cancer cells produced or producible by a method as defined in any one of claims 1 to 21; and a sealed container comprising cell culture medium that is suitable for growing cancer cell spheroids.

35. The kit according to claim 34, wherein the cell culture medium comprises a mixture of Dulbecco's modified Eagle's medium (DMEM) and Ham's F-12 medium, supplemented with B27 supplement, epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF).

Description

BRIEF DESCRIPTION OF THE FIGURES

[0050] FIG. 1 shows the number of spheres per ml for different media formulations and supplement schedules on the breast carcinoma cell line MDA-MB-436.

[0051] FIG. 2 shows spheroid diameter (m) for different media formulations and supplement schedules on the breast carcinoma cell line MDA-MB-436.

[0052] FIG. 3 shows spheroid circularity for different media formulations and supplement schedules on the breast carcinoma cell line MDA-MB-436.

[0053] FIG. 4 shows the number of spheres per ml for different media formulations and supplement schedules on the glioblastoma cell line U87MG.

[0054] FIG. 5 shows spheroid diameter (m) for different media formulations and supplement schedules on the glioblastoma cell line U87MG.

[0055] FIG. 6 shows spheroid circularity for different media formulations and supplement schedules on the glioblastoma cell line U87MG.

[0056] FIG. 7 shows sphere number plotted against cell density (cells/ml) for the breast carcinoma cell line MDA-MB-436. As can be see, sphere number initially increases in a relatively linear fashion with cell density, but then reaches a plateau at around 25000 cells/ml plating density.

[0057] FIG. 8 shows representative micrographs of spheroids formed upon cell density seeding on the breast carcinoma cell line MDA-MB-436. The cell densities are as indicated (1000, 5000, 10000, 25000, 50000, 100000 and 250000 cells/ml).

[0058] FIG. 9 shows sphere number plotted against cell density (cells/ml) for the glioblastoma cell line U87MG. As can be see, sphere number initially increases at a steep slope with cell density, but then the gradient decreases beyond 10000 cells/ml plating density.

[0059] FIG. 10 shows representative micrographs of spheroids formed upon cell density seeding on the glioblastoma cell line U87MG. The cell densities are as indicated (2000, 5000, 10000, 25000, 50000 and 100000 cells/ml).

[0060] FIG. 11 shows spheroid number plotted against plating cell density in multi-well plates. As can be seen, the effect of cell seeding density on multi-well spheroid number production from spheroid enriched cultures on multi-well plates using the colon carcinoma cell line HCT116 allowed 1600 cells/ml to be selected as the optimum density.

[0061] FIG. 12 shows spheroid diameter (m) plotted against plating cell density for spheroid enriched cultures on multi-well plates using the colon carcinoma cell line HCT116.

[0062] FIG. 13 shows spheroid number plotted against plating cell density in multi-well plates. As can be seen, the effect of cell seeding density on multi-well spheroid number production from spheroid enriched cultures on multi-well plates using the glioblastoma cell line U87MG allowed 1600 cells/ml to be selected as the optimum density. For this cell line, seeding density higher than 1600 cells/ml produced fewer spheres due to aggregation of existing spheres.

[0063] FIG. 14 shows spheroid diameter (m) plotted against plating cell density for spheroid enriched cultures on multi-well plates using the glioblastoma cell line U87MG.

[0064] FIG. 15 shows spheroid number plotted against days in culture after seeding at a density of 1600 cells/ml on multi-well plates using the colon carcinoma cell line HCT116. As can be seen, the highest number of spheroids was observed on day 6.

[0065] FIG. 16 shows spheroid diameter (m) plotted against days in culture after seeding at a density of 1600 cells/ml on multi-well plates using the colon carcinoma cell line HCT116.

[0066] FIG. 17 shows spheroid number plotted against days in culture after seeding at a density of 1600 cells/ml on multi-well plates using the glioblastoma cell line U87MG. Spheroid number increased up to day 8 and then decreased, indicating aggregation of spheroids. The optimum recording date was therefore selected as day 6.

[0067] FIG. 18 shows spheroid diameter (m) plotted against days in culture after seeding at a density of 1600 cells/ml on multi-well plates using the glioblastoma cell line U87MG. Spheroid diameter increased exponentially indicating aggregation of spheroids beyond day 8.

[0068] FIG. 19 shows a schematic overview of the process workflow according to an embodiment of the present invention. A 2D monoculture is grown to bulk 3D culture spheroids, which are then disaggregated to form a single cell preparation in a culture media suitable for cell freezing. The cancer stem cell (CSC) enriched single cell population is then dispensed into 96-well plates, which are packaged and labelled appropriately before freezing at 80 C. The frozen ready to use spheroid-forming plate may then be shipped to an end user. The end user thaws the plate by adding warm cell culture medium and grows the cells for 5-7 days. The cells re-form spheroids.

[0069] FIG. 20 shows a schematic overview of assay processes according to some embodiments of the present invention. The frozen ready to use spheroid forming plate is thawed by adding stem cell media. At this point test compounds can be added in order to assess their effect on spheroid formation (left-hand branch: spheroid formation assay). Additionally or alternatively, test compounds may be added after spheroids have formed in order to assess their effect on 3D spheroid viability (right-hand branch: 3D viability assay).

[0070] FIG. 21 shows a photomicrograph of representative spheres obtained from passage 1 (T1) PDX-derived cells shown at day 13. Diameter bars are 150 m (lower left) and 182 m (upper right).

[0071] FIG. 22 shows representative whole well reconstruction images (2.5 magnification) from control (left) and thawed (right) Cell2Sphere plates at day 4 of spheroid growth at 10 000 cells/well.

DETAILED DESCRIPTION OF THE INVENTION

[0072] In describing the present invention, the following terms will be employed, and are intended to be defined as indicated below.

[0073] Cancer stem cell (CSC) are tumorigenic cancer cells capable of self-renewal. Typically CSCs are able to grow in suspension culture and form cancer cell spheroids.

[0074] Spheroid as used herein refers to a spherical or sphere-like structure formed of cancer cells typically enriched with CSCs. Also called tumorospheres (see Weiswald et al., Neoplasia, 2015, Vol. 17, No. 1, pp. 1-15, the content of which is expressly incorporated herein by reference), the spheroid may be a free-floating structure cultured in low-adherent conditions using a serum-free medium supplemented with certain growth factors.

[0075] Spheroid forming cells as used herein refers to cancer cells, including CSCs, that exhibit the propensity to form spheroids upon culturing under appropriate conditions for an appropriate time. Therefore, the spheroid forming cells need not be in the form of a spheroid. Typically, the spheroid forming cells are derived from a spheroid, e.g., following enzymatic and/or mechanical dissociation to yield a suspension of single cells. The spheroid forming cells may be enriched in CSCs. Moreover, the spheroid forming cells may in some cases be frozen, e.g. in a cryopreservation medium.

[0076] Test compound as used herein refers to any agent having or which is a candidate for having an effect on a cell, tissue, organ or system of interest. In particular, the test compound may be any drug (whether small molecule, protein, biologic, nucleic acid or carbohydrate) that may, for example, be employed in screening for anti-cancer properties. Of particular, interest are test compounds that show efficacy against spheroid formation, spheroid growth and/or spheroid viability.

[0077] The following is presented by way of example and is not to be construed as a limitation to the scope of the claims.

EXAMPLES

Example 1Effect of Cell Culture Medium Composition and Supplementation on Spheroid Formation

Methodology

[0078] Standard medium (SM): [0079] DMEM/F12 (1:2 mixture) [0080] Methylcellulose (final 5%) [0081] B27 supplement (final 2%) [0082] EGF (final 20 ng/ml) [0083] bFGF (final 20 ng/ml)

[0084] Standard supplementation procedure: [0085] 1. Mix base medium (DMEM+F12) [0086] 2. Add Methylcellulose [0087] 3. Add B27 [0088] 4. Mix [0089] 5. Re-suspend cells in mix.

[0090] Add growth factors (EGF+bFGF) directly to the culture well every 2 days.

[0091] Incubation conditions: 37 C., 5% CO.sub.2.

[0092] Disaggregation/dissociation of spheroids was enzymatic (5 min in 1:1 trypsin:DMEM solution at 37 C.) and mechanical (passing through a 25G needle (6 strokes).

[0093] As has been reported previously (e.g. [1] and [5]), certain human breast cancer cell lines are capable of forming spheroids (mammospheres). Cells are seeded in DMEM:F12 (2:1) medium without serum, supplemented with B27, EGF (20 ng/ml), bFGF (20 ng/ml) in six-well tissue culture plates that had been covered with poly-2-hydroxyethylmethacrylate (Sigma, St. Louis, Mo.) to prevent cell attachment, at a density of 1000 cells/ml. In order to prevent cell aggregation, methylcellulose was included in the medium at a final concentration of 0.5% (see [5]).

Results

[0094] The effects of eliminating methylcellulose and changing growth factor (EGF and bFGF) supplementation to once at the beginning of the culture were assessed in relation to spheroid number, spheroid size and spheroid circularity using breast carcinoma and glioblastoma cell lines. As shown in FIGS. 1-6, it was found that methylcellulose can be eliminated from SM and growth factor supplementation carried out only once at the start of the culture while still maintaining a comparable number of spheroids (as compared with SM and standard supplementation procedure) and of approximately equal diameter and circularity (as compared with SM and standard supplementation procedure). By contrast, eliminating both methylcellulose and B27 led to lower numbers of spheroids for the cell lines investigated (MDA-MB-436 and U87MG).

Example 2Optimizing Conditions for Bulk-Phase (Phase 1) Spheroid Production

[0095] In order to enrich cell cultures for spheroid producing cells, spheroids are produced in bulk from single cells grown in monolayers (2D).

Cell Density Optimization

[0096] The number of cells/ml was tested against number of spheroids produced. We observed that upon a certain density spheroid number did not increased proportionally, indicating that spheroids aggregated. This can be seen from the supporting data on spheroid number and representative micrographs using breast carcinoma and glioblastoma cell lines presented in FIGS. 7-10. Therefore, cell density may optimally be maintained below this level, which is specific for each cell line.

[0097] As shown in FIGS. 7 and 8, the seeding density of MDA-MB-436 cells may optimally be 25000 cells/ml.

[0098] As shown in FIGS. 9 and 10, the seeding density of U87MG cells may optimally be 10000 cells/ml.

Example 3Optimizing Conditions for Spheroid Production Multi-Well Plates (Phase 2)

[0099] In accordance with certain embodiments of the present invention, spheroids prepared in phase 1 (as described above in Example 2) were mechanically and enzymatically disaggregated and filtered to render a single cell suspension. Cells were re-suspended in freezing medium (Cell Banker, AMS Biotechnology (Europe) Ltd, Milton Park, UK) and dispensed 20 l/well into 96-well plates. The plates may then be flash-frozen at 80 C. for storage. The cells are thawed by adding warm SM (or SM without methylcellulose) and grown in culture for 5-7 days to form spheroids.

[0100] The effect of plating cell density on spheroid production was assessed by plating cells at different densities and then measuring spheroid number and spheroid diameter after 7 days in culture.

[0101] As can be seen from FIGS. 11-14, the number of spheroids produced increased in proportion to the plating cell density. This was initially linear, but at least for U87MG cells, this flattened off above 1600 cells/ml due to aggregation of existing spheroids. Therefore, it was concluded that the optimum plating cell density in this case is around 1600 cells/ml.

[0102] In order to test the optimum day for assay data recording, cell number and size were measured every day using a starting cell seeding density of 1600 cells/ml. Spheroid number stabilizes at a certain number of days, when all spheroid forming cells have grown to the spheroid state. Diameter continues to increase as spheroids grow.

[0103] As shown in FIGS. 15-18, the spheroid number and diameter using colon carcinoma and glioblastoma cell lines was found to be optimal around day 6 of culture. Beyond this point spheroid aggregation became evident for HCT116 cells and U87MG cells.

Example 4Spheroid Production from Patient-Derived Xenograft (PDX) Cells in Multi-Well Plates

[0104] A sample colorectal tumor fragment was processed upon arrival into single cell suspension, obtaining a viability of 62.2% viable cells. 1210.sup.6 spare cells were seeded in bulk plates at a cell density of 20000 cells/ml in Cell2Sphere medium for enrichment in CSC (T1 spheroids). This is the same medium as described in detail above in Example 1. After 13 days, spheroids were disaggregated enzymatically and mechanically to obtain single cell suspensions. The method of enzymatic and mechanical disaggregation (trypsin/DMEM+25G needle) was the same as described in detail above in Example 1. A total of 23532 spheroids were collected.

[0105] T1 spheroids were then disaggregated, obtaining 5.310.sup.6 viable cells that were then seeded on P96 plates in two different cell densities (10000 cells/well and 20000 cells/well) rendering 6 plates in total. 1 plate of each set was used as control, the rest were frozen at 80 C. for storage.

[0106] After 3 days, frozen plates were thawed, and then the plates were analyzed for sphere number and viability at day 4 and day 9, following Cell2Sphere instructions. The image analysis was as described for cell lines in Examples 1-3 above. Data obtained is summarized below (Table 1). These are named T2 (for passage 2) spheroids.

TABLE-US-00001 TABLE 1 T2 spheroid characteristics Average Spheroid Average Spheroid Average Spheroid No (per well) sd Size (m) sd Circularity sd 10,000 cell/well Control 35.92 13.15 93.71 11.66 0.71 0.03 Frozen, Day 4 70.20 19.71 78.43 8.26 0.70 0.02 Frozen, Day 9 30.53 8.24 100.08 12.67 0.70 0.02 20,000 cell/well Control 59.40 10.84 100.49 8.23 0.72 0.02 Frozen, Day 4 82.09 22.22 87.31 13.56 0.71 0.02 Frozen, Day 9 27.27 12.18 125.20 26.31 0.71 0.03

[0107] Representative images of wells from the P96 plates are shown in FIG. 22.

[0108] Spheroid aggregates were observed when spheroids were grown for 9 days in Cell2Sphere plates, which explains the decrease in spheroid number from day 4 to day 9 (see Table 1 above). Without wishing to be bound by any particular theory, the present inventors believe that 4-6 days is optimum for PDX-derived spheroid number scoring.

[0109] Spheroid forming efficiency was calculated as 1/510 for T1 spheroids and 1/143 for T2 at 10000 cells/well and 1/244 at 20000 cells/well. Therefore, under the conditions tested the best enrichment was obtained at 10000 cells/well in T2 using Cell2Sphere technology.

[0110] The standard deviation seen in the present experiments was relatively high (e.g. 19.7% for spheroid number in frozen day 4 10000 cells/wellsee Table 1). However, it is expected that variability of cell number, and therefore standard deviation will decrease as industrial production processes are applied.

[0111] Using these conditions as estimation, it is possible to generate 8 plates from 1210.sup.6 single viable cells from fresh samples. Given that the average sample renders 2010.sup.6 single viable cells/gram of tumor, it will be possible to generate 13-14 Cell2Sphere plates per gram of fresh tumor tissue. Some variation is, however, expected from sample to sample.

CONCLUSIONS

[0112] Given the results described above, we conclude that fresh PDX samples can be used to produce frozen viable Cell2Sphere plates for spheroid generation for drug testing ex vivo.

REFERENCES

[0113] [1] Dontu G, Abdallah W M, Foley J M, et al. In vitro propagation and transcriptional profiling of human mammary stem/progenitor cells. Genes Dev 2003; 17:1253-70. [0114] [2] Ponti D, Costa A, Zaffaroni N, et al. Isolation and in vitro propagation of tumorigenic breast cancer cells with stem/progenitor cell properties. Cancer Res 2005; 65:5506-11. [0115] [3] Grimshaw M J, Cooper L, Papazisis K, et al. Mammosphere culture of metastatic breast cancer cells enriches for tumorigenic breast cancer cells. Breast Cancer Res 2008; 10:R52. [0116] [4] Fillmore C M, Kuperwasser C. Human breast cancer cell lines contain stem-like cells that self-renew, give rise to phenotypically diverse progeny and survive chemotherapy. Breast Cancer Res 2008; 10:R25. [0117] [5] Manuel Iglesias J, et al. Mammosphere formation in breast carcinoma cell lines depends upon expression of E-cadherein. PLoS ONE, 2013; 8(10); e77281.

[0118] All references cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual publication or patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

[0119] The specific embodiments described herein are offered by way of example, not by way of limitation. Any sub-titles herein are included for convenience only, and are not to be construed as limiting the disclosure in any way.