. Upon sacrifice, tumors were excised and the skin tissue was dissected away. Scissors were used to mince the tumor into chunks before mixing with 3 to 4 mL of PBS within a Falcon tube on ice strained through a 40 µm nylon mesh cell strainer (Thermo Fisher Scientific, Waltham, MA; cat. no. 08-771-1). The tumor chunks were collected and placed into a Falcon tube with freshly prepared 4 mL of RPMI with 5% FBS with 1 mg/mL collagenase A (Sigma-Aldrich, St. Louis, MO; cat. no. C9891) and 100 µL of 1 mg/mL DNase I (Roche, Basel, Switzerland; cat. no. 10104159001). This mixture was incubated for 30 minutes at 37°C. The Falcon tube was gently inverted every 5 minutes. After incubation, the solution was filtered through a 40 µm nylon mesh cell strainer and centrifuged for 4 minutes at 1000 rpm. The pellet was resuspended in 2.5 mL of PBS. 2.5 mL of ACK Lysing Buffer (Quality Biological, Gaithersburg, MD; cat. no. 118-156-101) for 30 seconds and incubated at room temperature for 30 seconds to remove red blood cells. After centrifugation, the supernatant was removed, and the pellet was resuspended in PBS. Trypan Blue was used to check cell viability and cell count before proceeding with Dead Cell Removal Kit (Miltenyi Biotec, Bergisch Gladbach, Germany; cat. no. 130-090-101) per manufacturer's instructions. Cell preparations with under 70% cell viability were excluded from analysis Library preparation and sequencing were performed by the University of Chicago Genomics Core (RRID:SCR_019196). Chromium Next GEM Single Cell 3′ Reagent Kits v3.1 (10x Genomics, Pleasanton, CA) were used for library generation and NovaSeq X-10B-100 (Illumina, San Diego, CA) for sequencing.