Yumm5

YUMM1.7 (SCC227) and YUMMER (SCC243) lines from Millipore Sigma (Burlington, MA, USA); YUMM1.G1 (CRL-3363), YUMM3.3 (CRL-3365), YUMM4.1 (CRL-3366), YUMM5.2 (CRL-3367) from ATCC (Manassas, VA, USA) were maintained in DMEM-F12 medium (ATCC (Manassas, VA, USA), 30-2006) supplemented with 10% FBS (Gibco (Waltham, MA, USA) #10437-028), 1% NEAA (Gibco, 11140-50), and 1% pen-Strep (ThermoFisher (Waltham, MA, USA), 15140122). For generating Vem–resistant lines, cells were seeded at ~20% confluence and allowed to adhere overnight, then cultured in medium containing 5 µM Vem (AmBeed (Arlington Heights, IL, USA), A116840) and refreshed every 2–3 days. Stably Vem–resistant cells reached a confluence after two months. Generated Vem–resistant cells were named YUMM1.7_R and YUMMER_R to be distinguished from their isogenic parentals, YUMM1.7_P and YUMMER_P, respectively.

Murine B16-F10, YUMM1.7, YUMM1.G1, YUMM3.3, YUMM4.1, and YUMM5.2 melanoma cells were newly purchased from ATCC (Gaithersburg, MD), and YUMMER1.7D4 from Sigma (St. Louis, MO). All melanoma lines used were at low passage, < 70% confluency, and maintained in RPMI-1640 medium (Life Technologies, Carlsbad, CA) supplemented with 10% (v/v) fetal bovine serum (FBS, Sigma) and 1% (v/v) penicillin/streptomycin (Life Technologies) in standard culture flasks for 2D expansion or in 6-well ultra-low attachment plates (Corning, Glendale, AZ) for 3D tumor spheroid culture, as described^{5 (link)}. PD-1 (Pdcd1) OE and vector control B16-F10 melanoma cells were generated previously^{5 (link)} and cultured in the presence of 1 μg/mL puromycin (Life Technologies) and 500 μg/mL neomycin (G418 sulfate, Life Technologies). PD-1^−/− knockout (KO) B16-F10 melanoma cells were generated and validated as described below. Cells grown in 2D were harvested using 0.1% (v/v) versene solution (Life Technologies), as described^{5 (link)}, and 3D tumor spheroids were dissociated into single cell suspension after 5 days in culture for subsequent flow cytometric analysis using enzyme-free dissociation buffer (Thermo Fisher, Waltham, MA), per the manufacturer’s instructions.