Stempro hesc sfm

The human PCa cell lines PC3, DU145, LNCaP, C42B,, 22Rv1 and human umbilical vein endothelial cells (HUEVEC) were obtained from American Type Culture Collection (Manassas, VA). PC3, LNCaP, C42B, and 22Rv1 cells were grown in RPMI-1640 medium (Invitrogen Inc., CA, USA) supplemented with 10% fetal bovine serum (FBS) and 100 U/ml penicillin/streptomycin. DU145 were cultured in Dulbecco’s modified Eagle medium (Invitrogen Inc.) containing 10% FBS and 100 U/ml penicillin/streptomycin. HUVEC cells were grown in human large vessel endothelial cell basal medium (catalog no. M200500, Thermofisher Scientific) with low serum growth supplement (catalog no. S00310, ThermoFisher Scientific). The human LuCaP147 patient derived xenograft (PDX) was generously provided by Dr. Eva Corey [22 (link)]. Human LuCaP147 spheroids were maintained in StemPro^® hESC SFM (Invitrogen Inc.) medium supplemented with 10 nM of R1881 and 2 μM of Y-27632 as previously described [23 (link)]. All cells were maintained in a humidified 5% CO₂ incubator at 37 °C and periodically monitored for mycoplasma contamination by polymerase chain reaction (PCR) PCR. LuCaP147 spheroids passaged less than 15 times and PCa cell lines with passages fewer than 20 were used in this study.

Adapted from previously published protocols [9 (link),18 ], iPSC maintenance medium was changed in one confluent well of a six-well plate and replaced with 1.5 ml of StemPro hESC SFM (Gibco) supplemented with 50 ng ml⁻¹ BMP4 (R&D), 50 ng ml⁻¹ VEGF (R&D) and 20 ng ml⁻¹ SCF (Life Technologies). Cells were passaged into two wells with 2.25 ml of fresh media using the EZPassageTM tool and embryoid bodies (EBs) were formed in suspension for 4 days (supplemented with cytokines on day 2). 10–15 EBs were transferred per well to a gelatin-coated six-well plate in X-VIVO15 media (3 ml per well) supplemented with 100 ng ml⁻¹ CSF1 (BioLegend), 25 ng ml⁻¹ IL3 (Peprotech), 2 mM Glutamax (Gibco), 0.05 mM β-mercaptoethanol (Gibco) and 1% v/v Penicillin-Streptomycin (Life Technologies) and media were replaced every three to four days for three weeks. Monocyte-like precursors were harvested from the supernatant (every three to four days for up to three months) and plated into untreated bacteriological plates or six-well plates in X-VIVO15 media supplemented with 100 ng ml⁻¹ CSF1 (BioLegend), 2 mM Glutamax (Gibco) and 1% Penicillin-Streptomycin (Life Technologies) for 9–11 days.

Fresh dissected breast tumor tissues were transported within DMEM/F12 culture media (Gibco) and subsequently processed as previously described [23 (link)]. The isolation of patient-derived microtumors was adapted from Kondo et al. [24 (link)]. Briefly, tumors were washed in HBSS (Gibco), fragmented with forceps, and digested with Liberase DH (Roche) for 2 h at 37 °C. The digested tissue was filtered through a 500 µm stainless steel mesh (VWR) followed by a 40 µm cell strainer (Corning). Tumor fragments retained by the cell strainer were washed in HBSS and cultured in suspension in StemPro® hESC SFM (Gibco) supplemented with 8 ng/ml FGF-basic (Gibco), 0.1 mM β-mercaptoethanol (Gibco), 1.8% BSA (Gibco) and 100 µg/ml Primocin (Invivogen) in a cell-repellent culture dish (60 × 15 mm) (Corning). The single-cell filtrate was used for the expansion of tumor-infiltrating lymphocytes in Advanced RPMI 1640 (GIBCO) supplemented with 2 mM glutamine (Gibco), 1% MEM vitamins (Gibco), 5% human serum (SigmaAldrich) and 100 µg/ml Primocin (Invivogen). IL-2 (100 U/ml), IL-7 (10 U/ml) and IL-15 (23.8 U/ml) (Peprotech) were freshly added to the culture media. CD3/CD28 Dynabeads (Milteny Biotech) were added for expansion.

The procedure was adapted from Kondo et al. (2011) [11 (link)] and modified as follows. Tumor specimens were washed in HBSS (Gibco, Thermo Fisher Scientific, Waltham, MA, USA), minced with forceps, and digested with LiberaseTM DH [12 (link)] for 2 h at 37 °C. Digested tissue was centrifuged (300× g, 5 min), washed with HBSS and filtered through a stainless 500 μm steel mesh (VWR). The flow-through was again filtered through a 40 μm cell strainer (Corning, Corning, NY, USA). The filtrate containing the TIL fraction was resuspended in Advanced RPMI 1640 (Gibco) supplemented with 2 mM Glutamine (Gibco), 1% MEM Vitamins (Gibco), 5% human serum (Sigma-Aldrich, St. Louis, MO, USA) and 100 μg/mL primocin (Invivogen, San Diego, CA, USA). IL-2 (100 U/mL), IL-7 (10 U/mL) and IL-15 (23.8 U/mL) (Peprotech, East Windsor, NJ, USA) were freshly added to culture media. For expansion, CD3/CD28 dynabeads were added (Milteny Biotech, Auburn, CA, USA). PDM, held back by cell strainer, were washed in HBSS and cultured in suspension in StemPro^® hESC SFM (Gibco) supplemented with 8 ng/mL FGF-basic (Gibco), 0.1 mM β-mercaptoethanol (Gibco), 1.8% BSA (Gibco) and 100 μg/mL primocin (Invivogen) within cell-repellent culture dish (60 × 15 mm) (Corning).

We adapted the previously published differentiation protocol that resulted in optimal macrophage production^{18 ,44 (link)}. Maintenance media on one confluent well of a six-well plate was replaced with 1.5 ml of day 0 mix, which consisted of StemPro hESC SFM (Gibco) supplemented with BMP4 (50 ng/ml), VEGF (50 ng/ml) and SCF (20 ng/ml). Colonies were cut using the EZPassage tool into two wells with 2.25 ml of day 0 mix cultured in Ultralow Attachment 6-well plates (Greiner) for 4 days to induce embryoid body (EB) formation. In total, 10–15 EBs were transferred to each gelatin-coated well tissue-culture-grade six-well plates in 3 ml day 4 mix (X-VIVO15 media supplemented with M-CSF (100 ng/ml), IL3 (25 ng/ml), Penicillin-Streptomycin, Glutamax (2 mM) and β-mercaptoethanol (0.055 mM) and media was changed every 3–4 days. After 3 weeks, non-adherent monocyte-like precursors were harvested from the supernatant and plated into untreated bacteriological plates or six-well plates in Maturation mix (X-VIVO15 media supplemented with M-CSF (100 ng/ml), Glutamax (2 mM) and Penicillin-Streptomycin (1%)) for 9–11 days. Harvesting was repeated very 3–4 days for up to 3 months. To activate KLF1 in iKLF1.2-DMs, tamoxifen (100 nM) was added to the adherent macrophage population for the last 4 days of the differentiation process.

PDX tumors were extirpated from mice and digested with a Tumor Dissociation Kit and gentleMACS Dissociator (Miltenyi Biotec). Tumor fragments about 50 μm in diameter were separated by repeated low-speed centrifugation and embedded in 25 μL of medium with 10% Growth Factor Reduced-Matrigel (Corning) on 384-well ultra-low-attachment microplates (Corning) at a concentration of 100 to 150 fragments per well. After solidification of the Matrigel for 2 h at 37 °C, 25 μL fresh medium with samples was added to each well, and the plates were further incubated for 5 days. After the 5 days of culture, 25 μL of CellTiter-Glo 3D reagent (Promega) was added to each well and the luminescence was quantified with an Envision microplate reader (PerkinElmer). Depending on the assay or model, the following kinds of culture medium were used: DMEM/F12 supplemented with 10% embryonic stem-cell FBS (Thermo Fisher) or StemPro hESC SFM (Thermo Fisher) for PC-3 and PC-42 models; DMEM/F12 supplemented with 50 ng/mL human epidermal growth factor (Peprotech), 12.5 ng/mL human FGF (fibroblast growth factor) 10 (Peprotech), 10 mM nicotinamide (Sigma-Aldrich), 0.2% bovine serum albumin (Wako), insulin-transferrin-selenium supplement (Thermo Fisher) and 4i cocktail for KYK models.