(A) Orthotopic transplantation of 200–106 Tgfbr2 cKO CD34+ SCC cells into the anorectal transition zone of recipient nude mice resulted in secondary tumor formation with 100% efficiency (n = 89 in sum; 200 cells, n = 4; 1000 cells, n = 6; 5000 cells, n = 2; 10,000 cells, n = 10; 100,000 cells, n = 44; 200,000 cells, n = 7; 300,000 cells, n = 6; 500,000 cells, n = 4; 750,000 cells, n = 2; 1,000,000 cells, n = 4). Data represent the mean number of days after transplantation before palpable tumor formation ± standard deviation. (B) CD34+ SCC cells were enriched for tumor forming efficiency, compared to CD34− SCC cells or YFP+α6-β1- SCC cells, when transplanted orthotopically into a tertiary mouse. (C–E) H and E staining revealed that orthotopic transplant of cultured Tgfbr2 cKO CD34+ SCC cells into a secondary recipient (C) or orthotopic transplant of Tgfbr2 cKO CD34− SCC cells (D) or orthotopic transplant of Tgfbr2 cKO CD34+ SCC cells directly into a tertiary recipient (E) results in SCC formation which recapitulate the morphology of the Tgfbr2 deficient tumor of origin (n = 1/13 CD34−, n = 8/13 CD34+). Scale bars: 100 µm (C–E), 50 µm (C’–E’). (F–H) Using the same FACS strategy as employed for the primary Tgfbr2 cKO anorectal SCC, the secondary and tertiary anorectal tumors were sorted and distinct CD34+ and CD34− epithelial populations were isolated, maintaining the tumor hierarchy of the Tgfbr2 cKO tumor of origin. Of the one tertiary mouse that developed a tumor from transplant of CD34− cells, CD34 was re-expressed within the tumor environment (G).