Poly hema

Ovarian cancer cell lines were cultured under standard conditions in RPMI 1640 (Thermo Fisher Scientific, Waltham, MA, USA) containing 10% foetal bovine serum (FBS) (Biowest Nuaillé, France)). Normal immortalized mesothelial cell line MeT5A (ATCC, American Type Culture Collection) was maintained in Medium 199 (Thermo Fisher Scientific) containing 10% FBS, 3.3 nM epidermal growth factor (EGF) (PeproTech, London, UK), 400 nM hydrocortisone (Sigma, St. Louis, MO, USA), 870 nM Bovine insulin (Sigma) and 20 nM HEPES (Thermo Fisher Scientific). Cell lines were maintained at 37 °C and 5% CO₂. All cell lines were authenticated using short tandem repeat (STR) profiling and regularly tested for the absence of mycoplasma. For the 3D cultures, polyHEMA (Poly(2-hydroxyethyl methacrylate)) (Sigma) coated plates were prepared by dissolving 120 mg/mL of polyHEMA in 95% ethanol, then adding 100 µL of the solution to 96-well round-bottom plates and drying for 48 h at 55 °C. Ovarian cancer aggregates were generated by plating 4 × 10³ cells per well and incubated for four days.

Cell proliferation was monitored semi-quantitatively in a sodium 3′ [1-(phenylyamino-carbonyl)-3, 4-tetrazolium]-bis-(4-methoxy-6-nitro)-benzene sulfonic acid hydrate (XTT)-based colorimetric assay using the cell proliferation kit II (Roche). To estimate anchorage-dependent and anchorage-independent growth, 5000 cells/well were seeded into untreated 96-well plates (BD Falcon) in a volume of 150 μl medium or into poly-HEMA coated 96-well plates, respectively. For the preparation of poly-HEMA coated 96-well plates, 100 υl of a 5 mg/ml stock solution of poly-HEMA (Sigma-Aldrich) dissolved in 96% ethanol by mixing at room temperature for 4 h were added to the wells and allowed to dry for 72 h at 37°C. Growth of cells was determined in triplicate experiments 48 h after the transient knock-down. Formazan formation was determined spectrophotometrically. The optical density of the medium without cells was subtracted from all probe values to obtain the final results. The statistical significance was calculated using ANOVA and Tukey's post hoc tests.

To prepare the 1.2% poly-HEMA (Poly-2-hydroxyethyl methacrylate) solution, 1.2 g poly-HEMA (Sigma, USA) was dissolved in 100 mL 96% ethanol by rotating overnight to dissolve the polymers completely. The solution was centrifuged for 30 min at 400×g to remove unsolved particles and then filtered by 0.22 µm filters. The tissue culture dishes were coated with poly-HEMA solution (1.2 mL per each well of six well plate or 2.5 mL per T25 tissue culture flask) under the biosafety laminar flow hood at room temperature for an overnight to evaporate ethanol completely. Finally, the plates were washed with phosphate buffer saline (PBS) and stored at 37 °C incubator for future use.

Low cell attachment plates were prepared by incubating 24-well plates with 0.5 ml of 20 mg/ml poly-HEMA (Sigma, USA) solution prepared in 95% EtOH followed by overnight evaporation in the cell-culture hood. EV-LTC-250, BIK-LTC-0, and BIK-LTC-250 single cells were plated in replicate in poly-HEMA coated 24-well plates at 5 cells/mm³ suspension (500 µl suspension volume per well) in DMEM/F12 (1:1) supplemented with 20 ng/mL FGF-2 (Sigma, USA), 20 ng/mL EGF (PeproTech), 2% B27 without vitamin A (GIBCO, USA) and 1 × ITS (insulin-transferrin-selenium, GIBCO). 0.5% Methylcellulose (Sigma, USA) was used to prevent cell aggregation, allowing the growth of mammospheres in different z-planes of the medium. Every 3 days, 500 µl of fresh medium was added to each well without removing the old medium. Mammospheres were imaged in brightfield (Zeiss AxioObserver.Z1 Microscope) on days 4, 8, and 12 (on day 28 for MDA-MB-231 LTC). Mammosphere formation efficiency was determined on day 12 (day 28 for MDA-MB-231 LTC) and was calculated from the number of spheres per well, divided by the number of cells plated, multiplied by 100 (to convert it to percentage).

ESCC cell lines (KYSE30, KYSE450 and KYSE510) were provided by Dr. Y. Shimada (Kyoto University, Kyoto, Japan) [19 (link)]. These cells and two ESCC sublines (30-U/D) derived from KYSE-30 cells [11 (link)] were cultured in RPMI1640 with 10% FBS, streptomycin (100 mg/ml) and penicillin (100 U/ml). HEK293T cells were obtained from ATCC (Manassas, VA, USA) and cultured in DMEM with 10% FBS, streptomycin (100 mg/ml), and penicillin (100 U/ml). All cells were cultured under humanized condition (37°C, 5% CO₂). STR analysis was carried out to authorize ESCC cells.
Akt inhibitors, LY294002 and Wortmannin, were purchased from Cell Signaling Technology (Danvers, MA, USA) and the cells were pre-treated for 4 hr before the transwell assay. PolyHEMA was obtained from Sigma-Aldrich (Shanghai, China) and coat of 6-well dishes with 2 ml PolyHEMA (10 mg/ml in ethanol) was repeated twice and these treated dishes were air-dried in the sterilized safety cabinet before addition of cells.