Lt07 118

Written informed consent for patient participation was obtained by a qualified investigator (protocol 12-N-0095 approved by the National Institute of Neurological Disorders and Stroke, National Institutes of Health). α-Dystroglycanopathy patient hiPSCs were reprogrammed from dermal fibroblasts using an hOKSML mRNA reprogramming kit (Stemgent, 00-0067). Control-1 hiPSCs were reprogrammed in the same manner from BJ foreskin fibroblasts (ATCC, CRL-2522). Immunocytochemical validation of germ layer differentiation was performed off-site (Stemgent). Control-2 hiPSCs were reprogrammed from control foreskin fibroblasts (ATCC, CRL-2097) using the CytoTune-iPS 2.0 Sendai reprogramming kit (Thermo Fisher, A16517). Control-3 hiPSCs (NC15) were previously generated by lentiviral reprogramming of adult dermal fibroblasts (Grunseich et al., 2014 (link)). Karyotype analysis was performed after at least 10 passages (WiCell), and all cell lines were routinely tested for mycoplasma contamination (LT07-118, Lonza).

Human gastric cancer cell lines were obtained from the Cancer Cell Line Encyclopedia (CCLE) core facility (BROAD Institute, Cambridge), which obtained them directly from commercial sources and authenticated the lines using standard short tandem repeat analysis. RPE and MDA-MB-468 cells were obtained from ATCC. RPE1 cells were grown in DMEM-F12 (Life Technologies, #10565042) supplemented with 10% FBS and 1% penicillin/streptomycin, HGC27 cells were grown in DMEM (Life Technologies, #11965118) supplemented with 10% FBS and 1% penicillin/streptomycin. AGS, KE39, GSU, NUGC3 and MDA-MB-468 were grown in RPMI 1640 (Life Technologies, #11875119) supplemented with 10% FBS and 1% penicillin/streptomycin. Cells lines were maintained in humidified 37 °C the incubator with 5% CO2 and routinely tested for mycoplasma contamination (Lonza #LT07-118).

For routine cell culture, CHO cells (ATCC #CCL-61) were cultured in 100 mm cell culture dishes (Corning Falcon #353003) at 37 °C, in a humidified incubator with 5% CO₂ in F-12K medium (Kaighn’s modification of Ham’s F-12 medium, ATCC #30-2004) supplemented with 10% fetal bovine serum (Gibco #16140-071) and penicillin (100 U/ml) and streptomycin (100 U/ml) (Gibco #15140122). Cells were obtained from ATCC, and routinely tested for mycoplasma contamination (Lonza #LT-07-118).

Organoids were established from tumors collected and processed under IRB approval from both participating institutions University of Pittsburgh and the Royal College of Surgeons in Ireland. Organoid lines were generated from tumors following Sachs et al.’s protocol^{69 (link)} with the addition of estradiol supplementation for ER+ tumors. Established organoids were dissociated into single cells and seeded in organoid media with 5% of Cultrex® Reduced Growth Factor Basement Membrane Matrix, type 2 (BME, Trevigen, 3533-001-02) for the intervention experiment. 24hrs after seeding, organoids were treated with vehicle or niraparib (N = 4–8). Cell viability was measured 7 days post-treatment using CellTiter-Glo® 3D Cell Viability assay (Promega). MDA-MB-436 (ATCC) cells were utilized as positive control. Cells used were authenticated (SourceBioScience) and regularly tested for mycoplasma contamination (LT07-118, Lonza).

The MCF7 cells were obtained from ATCC and cultured in Minimum Essential Medium Eagle (MEM) (M2279, Sigma) supplemented with 2 mM l-glutamine (G7513, Sigma) and 10% fetal calf serum (FCS) (F7524, Sigma). The LY2 cells were a gift from Robert Clarke (Georgetown, USA) and were cultured as previously described [17 (link)]. Each cell line was tested for mycoplasma contamination (LT07-118, Lonza) and genotyped (Source BioScience) according to ATCC guidelines. T347 cells were derived from an ER+ PR− HER2+ brain metastatic tumour from a breast cancer patient as previously described [18 (link)]. AI resistant LetR cells were generated as previously described [19 (link)].

Human prostate tumor cell line CW22Rv1 (22Rv1) was purchased from ATCC and grown in RPMI-1640 supplemented with either 10% fetal bovine serum (FBS) or 3% charcoal-stripped FBS (for palmitic acid and cholesterol treatment where indicated), and 1% penicillin/streptomycin (all components from Thermo Fisher). Human androgen-refractory prostate cancer cells of the ARCaP_M cell line were gifted to us from Leland Chung (Cedars-Sinai Medical Center), and grown in DMEM, supplemented with 5% FBS and 1% penicillin/streptomycin [24 (link)].
Human primary fibroblasts were grown from prostatectomy specimens at Cedars-Sinai Medical Center or the Greater Los Angeles Veterans Affairs under their respective institutional review boards [42 (link)]. The tumor-inductive status of fibroblasts was determined by tissue recombination with BPH1 (a non-tumorigenic human prostate epithelial cell line, as previously described [32 (link)]). CAF was cultured in DMEM/F12 supplemented with 5% FBS, 5% Nu-Serum, 1% penicillin/streptomycin, 10⁻⁹ M testosterone (Sigma-Aldrich), and 4 μg/mL insulin (12585014, Fisher Scientific). All cells were grown in a humidified incubator at 37 °C with 5% CO₂. All cells were tested for mycoplasma (LT07118, Lonza, Rockland, ME, USA) every 1 month and were negative.