Ultra low attachment plate

The enrichment of OCSCs and establishment of single cell-forming OCSCs were performed as described previously [19 (link)]. In brief, ovarian cancer cell lines were cultured in ultra-low attachment plates (Corning®, Corning, NY, USA) in RPMI-1640 (Thermo Fisher Scientific, Waltham, MA, USA) supplied with 1% nonessential amino acids (Thermo Fisher Scientific, Waltham, MA, USA), sodium pyruvate (Thermo Fisher Scientific, Waltham, MA, USA), 10% fetal bovine serum (FBS, Biological Industries, Kibbutz Beit−Haemek, Israel), 10 μg/mL insulin (Thermo Fisher Scientific, Waltham, MA, USA), basic fibroblast growth factor (bFGF; PeproTech, Rehovot, Israel), and human recombinant epidermal growth factor (EGF; PeproTech, Rehovot, Israel). The cells were cultured in suspension, and starting from 14 days, the cultures were examined every day for sphere formation. Spheres were then dissociated and passaged at least eight times in 2 months to generate spheres, which are henceforth referred to as OCSC (SR1 and SR2) cells. For OCSCs differentiation, we transferred OCSCs from ultra-low attachment plates into standard cell culture dishes (Corning®, Corning, NY, USA) and removed growth factors bFGF and EGF. The cells were harvested and extracted for DNA and RNA at Day 8 (AD1), 21 (AD2), 25 (AD3), and 42 (AD4) after OCSCs adhesion.

To obtain OCICs, we subcutaneously injected cells of the ovarian cancer cell line A2780 into nude mice (2×10⁶ Cells per mouse). After a tumor diameter reached about 1.5 cm (usually at four weeks after injection), we removed the tumor tissue, cut it into small pieces, and digested it with collagenase to prepare single cell suspensions. Then the collected single cells were cultured in serum-free DMEM-F12 (Invitrogen) supplemented with 5 µg/ml of insulin (Sigma), 20 ng/ml of human recombinant epidermal growth factor (EGF; Invitrogen), 10 ng/ml of basic fibroblast growth factor (b-FGF; Invitrogen), and 0.4% bovine serum albumin (BSA; Sigma) in Ultra Low Attachment plates (Corning). OCICs and the control cells were all separated from other cells using continuous density gradient centrifugation. The control cells were also obtained by injecting A2780 cells into nude mice and the separation methods were similar to those used for OCICs. They were cultured in Ultra Low Attachment plates (Corning) and the medium was similar with OCICs except that the culture medium included 10% fetal bovine serum (FBS). All media included penicillin (10 units/ml) and streptomycin (10 ng/ml) and cells were grown in a humidified incubator at 37°C with 5% CO₂. Medium was changed every 3 days.

For the tumor-sphere formation assay, BC cells were resuspended as a single cell suspension in a 1:1 ratio of serum-free keratinocyte growth medium (Gibco, Waltham, MA, USA) and growth factor-reduced Matrigel (BD Biosciences, Mountain View, CA, USA) and then plated into ultra-low-attachment plates (Costar, Corning, NY, USA). The size of the tumor spheres was measured 7 days after the first plating. For quantification, the perimeter of the tumor spheres was measured in eight randomly chosen representative areas selected from each group using ImageJ software (National Institute of Mental Health, Bethesda, MD, USA).
For the limiting dilution assay, BC cells were diluted to a density of 1 cell per well and plated into 96-well plates in 50 μl of culture medium. Fresh culture medium was added every 2 days, and the plated cells were cultured for 10 days after plating. The number of colonies was calculated for quantitative analysis.

Human prostate cancer cell lines PC3 (CRL-1435) and LNCaP (CRL-1740), DU145 (HTB-81) were obtained from American Type Culture Collection (ATCC) and cultured in Ham's F-12K (Invitrogen) or RPMI medium 1640 (Invitrogen). The 22Rv1 cell line (CRL-2505) was obtained from Prof. Martin Lackmann (Department of Biochemistry, Monash University, Melbourne, Australia). Media for all cell lines was supplemented with 10% Fetal Bovine Serum (FBS) (Invitrogen 10100-147). The usual androgen concentration within FBS (0.1–1 nM Di Hydro Testosterone) is sufficient to efficiently maintain androgen-sensitive cells such as LNCaP and 22RV1 [36 (link)].
For spheroid culture, cells were grown in serum-free DMEM/F-12 medium (Invitrogen 10565) supplemented with 1% N2 (Invitrogen 17502-048), 2% B27 (Invitrogen 17504-044), 20 ng/mL human epidermal growth factor (EGF Invitrogen PHG0311), 20ng/mL human fibroblast growth factor-basic (FGF-b Invitrogen PHG0026) and 100 units of Penicillin-Streptomycin (Invitrogen) in ultra-low attachment plates (Costar).

A total of 1 × 10³ cells were seeded in triplicate in 24-well ultra-low-attachment plates (Costar) containing 1 ml of MammoCult basal medium (Stem Cell Technologies) supplemented with 10% MammoCult proliferation supplement, 4 μg/ml heparin, 0.48 μg/ml hydrocortisone, penicillin and streptomycin. After 5–10 days, depending on the cell line, the number of primary tumorspheres formed was measured using an inverted microscope (Olympus IX-71). Subsequently, secondary tumorspheres were generated by collecting the medium containing the primary tumorspheres, centrifuging for 5 min at 900 rpm (91 g), trypsinizing for 5 min, and centrifuging again at 900 rpm (91 g) for 5 min. Finally, the cells were resuspended in the same complete supplemented MammoCult medium, and 1 ml per well was seeded in a 24-well low-attachment plate. The plates were incubated for 4–5 days, and the number and size of the tumorspheres were again evaluated and quantified.

A total of 5000 cells were seeded in triplicate in 24-well Ultra-Low Attachment Plates (Costar) containing 1 ml of MammoCult basal medium (Stem Cell Technologies) supplied with 10% MammoCult proliferation supplement, 4 μg/ml heparin, 0.48 μg/ml hydrocortisone, penicillin, and streptomycin. After 5–10 days, depending on the cell line (AA in 5–7 days, AW in 6–10 days, BG in 7–8 days), the number of primary tumorspheres formed was measured using an inverted microscope (Olympus IX-71).
Subsequently, the secondary tumorspheres were generated by collecting the medium with the tumorspheres, centrifuging for 5 min at 900 r.p.m., trypsinizing for 5 min, and centrifuging again at 900 r.p.m. for 5 min. Finally, all the cells trypsinized were resuspended in fresh, complete, supplemented MammoCult medium and 1 ml/well was seeded in a 24-well, low-stick plate.They were incubated for more 4–5 days and the number and size of the tumorspheres were measured again and quantified.