Protocols: A 1 cm3 tumor piece was harvested per tumor, chopped with a razor blade to generate a smooth paste and shaken in 7 mL dissociation solution (10%FCS, 0.2% collagenase (Roche), 0.125u/mL dispase II (Roche) in DMEM) for 1h at 37°C. The generated cell suspensions were filtered through a 100 μm filter and neutralized. The neutralized suspension was centrifuged and the cell pellet was resuspended in 10 mL 30% Percoll (Cytiva) solution. After centrifugation (400 × g, 5min), red blood cells were lysed by redissolving the cell pellet in 2 mL of ACK lysis buffer (Lonza) and incubated for 3 minutes at room temperature. ACK lysis was stopped by adding PBS till 10 mL. After a centrifugation step, the supernatant was aspirated and the cell pellet was resuspended in PBS. To isolate tumor cells, single-cell suspensions were stained with a viability dye (DAPI) and a lineage marker cocktail to exclude hematopoietic (CD45), endothelial (CD31), and erythroid (TER119) cells. The following antibodies were used: anti-CD45 (BD Biosciences, 552772), anti CD31 (eBioscience, 25-0311-82), and anti-TER119 (eBioscience, 25-5921-82). Using a fluorescence-activated cell sorter, 40,000 live, single, lineage-negative cells (DAPI⁻CD45⁻CD31⁻TER119⁻) were sorted. Cells were collected into PBS supplemented with 0.04% BSA to a final concentration of approximately 1,000 cells/µL. Cells were loaded on a GemCode Single-Cell Instrument (10x Genomics, Pleasanton, CA, USA) to generate single-cell Gel Bead-in-EMulsion (GEMs). Single-cell RNA-Seq libraries were prepared using GemCode Single-Cell 3ʹGel Bead and Library Kit (10x Genomics) according to the manufacturer's instructions.