Tumors were excised, mechanically minced, and incubated with serum-free RPMI containing 3 U/mL Collagenase I (Worthington Biochemical), 133 U/mL Collagenase IV (Worthington Biochemical), and 10 U/mL DNAse I (Worthington Biochemical) and rotated at 37 C for 30 min. Enzymatic digestion was blocked by adding RPMI-1640 containing 2mM EDTA and 2% FBS. The remaining tumor aggregates were dissociated mechanically. The single cell suspension was then passed through a 70mm strainer and red blood cells were lysed by incubation with 3 mL of ACK Lysis Buffer (15 mM NH4Cl, 10 mM KHCO3, 0.1 mM Na2EDTA) for 3 min at RT. Single cell suspensions were converted to barcoded scRNA-seq libraries using the Chromium Single Cell 3' Library, Gel Bead & Multiplex Kit, and Chip Kit (10x Genomics), aiming for 6,000 cells per library. Samples were processed using kits pertaining to V2 barcoding chemistry of 10x Genomics. Single samples were processed in a single well of a PCR plate, and all samples were processed in parallel in the same thermal cycler.