Mdah 2774

MDAH-2774 (CRL-10303), TOV-112D (CRL-11731) cells were from ATCC while OVSAHO (JCRB1046) and KURAMOCHI (JCRB0098) cells were from JCRB Cell Bank. All cell lines were maintained in RPMI-1640 medium (Sigma–Aldrich Co.) containing 10% heat-inactivated FBS, 100 U/ml penicillin and streptomycin (complete medium) at 37 °C in a 5% CO₂ atmosphere. Cisplatin-resistant (MI-res) isogenic cells were generated by treating EOC parental cells for growth for 2 hours with a cisplatin dose 10-fold higher than the calculated IC50 and then allowing to re-grow in drug-free complete medium (pulse treatment). The subsequent drug treatment was administrated when the cells reached again a 70–80% of confluence. In total MI-res cells received 20 pulse treatments. All the experiments were performed with cells kept in cisplatin-free medium for ≥2 months unless otherwise stated.

SKOV3, A2780CR, and MDAH2774 cell lines were obtained from ATCC (Manassas, VA, USA). OVCAR-8 cells were obtained from the National Cancer Institute (Bethesda, MD, USA). SKOV3, A2780CR and MDAH2774 cells were cultured in DMEM medium and OVCAR-8 cells were cultured in RPMI1640 medium. Culture media were supplemented with 10% FBS and 1% penicillin/streptomycin. All cells were grown in 5% (v/v) CO₂ at 37 °C.

The cancer cell lines A2780, SK-OV-3, PA-1, TOV21G, TOV112D, OVCAR3, MDA-MB-231, MC38, 4T1, CT26, SCC-9, B16-F10, and MDAH-2774 were acquired from ATCC. The Kuramochi, A1847, SNU251, COV362, ID8, and Pan02 cell lines were from Cobier. The ID8-Luc cell line was from Pharmalegacy. The mouse KPC cell line was purchased from Modelorg. The mouse KPL cell line was a gift from Ji et al. [62 (link)]. All cells were maintained in RPMI1640 or DMEM (Basalmedia) supplemented with 10% fetal bovine serum (FBS) (Gibco). IN10018 was provided by InxMed. The chemotherapeutic drugs used for screening were purchased from Medchemexpress. Doxorubicin and defactinib were purchased from SuperLan-chem, and PLD for animal studies was from Jingyuan. FAK siRNA was synthesized by Genepharma, and the sequence is listed in the Supplementary Table 3. The antibodies used in the study are listed in Supplementary Table 4.

Human ovarian cancer cell lines TOV112D, TOV21G, OV90, SKOV3, MDAH2774, and ES2 were obtained from ATCC (Rockville, MD, USA). BR and BG1 cells were described previously.^{10 (link)} Cancer cells were cultured in Dulbecco's modified Eagle's medium/F-12 supplemented with 10% fetal bovine serum and antibiotics at 37 ^oC with 5% CO2. Bortezomib (Millennium Pharmaceuticals, Cambridge, MA, USA) was dissolved in medical injectable water at the concentration of 10 mM. Another proteasome inhibitor MG132 (Sigma, St Louis, MO, USA, M7449-200UL) was dissolved in DMSO at 10 mM. Autophagy inhibitor 3-methyladenine (Sigma, M9281-100MG) was dissolved in medical injectable water at 50 mM. Cycloheximide (Sigma, C1988-1G) and chloroquine (Sigma, C6628-25G) were dissolved in medical injectable water at 10 mM. Rapamycin (Sigma, R8781-200UL) was dissolved in DMSO at 10 mM, and PD98059 (Sigma, P215-1MG) was dissolved in DMSO at 100 mM. Cisplatin was available at 0.5 mg/ml (Fresenius Kabi, Raleigh, NC, USA).

The research was carried out on human ovarian endometrioid adenocarcinoma cell line MDAH-2774, purchased from ATCC^® (Manassas, VA, USA) originating from a patient who did not receive chemotherapy or radiation prior to the collection of cancer cells [42 (link)]. Cell culture was maintained at 37 °C, 5% CO₂, 95% air humidity in RPMI-1640 medium (GlutaMAX^TM GIBCO; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS, Sigma-Aldrich, St. Louis, MO, USA). Cell passages were carried out 2–3 times a week when confluency was about 80–90%. Cells were then removed from the flasks by trypsinization (trypsin 0.25% and EDTA 0.02%; Sigma-Aldrich, St. Louis, MO, USA) and washed with DPBS buffer (Sigma-Aldrich, USA).

MDAH-2774, SKOV3, TOV112D, TOV21G, OV90, MDAH-MB-468, BT-549, FaDu, CAL27 and 293T17 cells were purchased from ATCC; OVCAR8 from NCI; Immortalized Human Ovarian Epithelial cells (HuNoEOC) from ABM; COV318 from ECACC; 293FT from Invitrogen. Fresh tumor specimens were used to generate primary EOC cultures.

TOV‐112D (CRL‐11731), MDAH‐2774 (CRL‐10303) cells and hTERT‐immortalized human foreskin fibroblasts BJ‐hTERT (CRL‐4001) were from ATCC (Manassas, VA, USA) while OVSAHO (JCRB1046) cells were from JCRB Cell Bank (Tokyo, Japan). BJ‐hTERT cells were cultured in Dulbecco's modified Eagle's medium, whereas TOV‐112D, MDAH‐2774, and OVSAHO were cultured in RPMI‐1640 medium. All media were supplemented with 10% FBS (Cambrex, Verviers, Belgium) heat‐inactivated, 50 units per mL penicillin (Cambrex), 500 g·mL⁻¹ streptomycin (Cambrex), and glutamine 4 mm. The cells were grown in a humidified atmosphere composed of 95% air and 5% CO₂ at 37 °C.
CDDP‐resistant cancer cells (referred as Pt‐res cells) were generated as previously described and include pooled resistant populations (pool) and individual clones, as indicated in the text [6, 7, 8]. Primary cells (OV102, OV202, OV199, OV200) were established from ascites of EOC patients, collected by the CRO‐Aviano National Cancer Institute Institutional Biobank with a written informed consent from patients. The CRO‐Aviano Internal Review Board approved this study (#IRB‐06/2011).
Primary cells were maintained in OCMI medium (M199 and Ham's 1 : 1) supplemented with 2% FBS, EGF (10 ng·mL⁻¹), hydrocortisone (500 ng·mL⁻¹), cholera toxin (25 ng·mL⁻¹), and insulin (20 µm·mL⁻¹). Negative mycoplasma cultures were confirmed by monthly mycoplasma tests.