Modeling ITH using fluorescently labeled DMBA/TPA-induced squamous cell skin carcinoma cell lines. (A–C) Flow cytometric analysis of T cell infiltration in subcutaneous tumors derived from injection of CIT6-YFP and CIT9-RFP cell lines into mice carrying an unactivated Confetti cassette. n = 9. All data are a representation of at least three independent experiments. (D) IF staining of CD3⁺ T cells in 8-µm cryosections of subcutaneous CIT6-YFP and CIT9-RFP tumors, with DAPI staining cell nuclei, and quantification of T cell count per field. Scale bar = 100 µm. T cells were counted in 52 (CIT6-YFP) and 39 (CIT9-RFP) tiles in each arm. Data are a representation of two independent experiments. (E) Schematic of mixed-population tumor generation. A 1:1 mixture of CIT6-YFP and CIT9-RFP cell lines was subcutaneously injected into the right hind flank of mice carrying an unactivated Confetti cassette, and the resulting tumors were harvested for downstream analyses when they reached 1 cm in diameter at 21–28 days after injection. (F and G) Whole-tumor images by fluorescent dissecting microscope (F) and a representative image of a tumor cross-section (8-µm cryosection) (G) of tumors that were a 1:1 mixture of CIT6-YFP and CIT9-RFP cell lines. Scale bars in F and G are 2 and 1 mm, respectively. Images are representation of >100 tumors we have analyzed throughout the experiments in the paper. Statistical significance was determined by Student’s t test (A–D). *P < 0.05, **P < 0.01, and ***P < 0.001.