Fig. 1. LOF of Fat1 promotes hybrid EMT state in mouse skin SCC, mouse lung cancer and human SCC.

a, Immunostaining for GFP, E-cadherin (E-cad), vimentin (VIM) and KRT14 (K14) in EPCAM⁺ control (wild-type (WT)), EPCAM⁺ Fat1-cKO and EPCAM⁻ Fat1-cKO DMBA/TPA-induced SCCs. Scale bars, 50 μm. b, c, FACS analysis (b) and percentage of EPCAM expression (c) in control and Fat1-cKO YFP⁺ skin SCCs. Mean ± s.e.m., two-tailed t-test. TC, tumour cell. d, Distribution of YFP⁺EPCAM⁻ tumour cells in CD106 (also known as VCAM1), CD61 (also known as ITGB3) and CD51 (also known as ITGAV) subpopulations in Fat1-cKO SCCs. Mean ± s.e.m. TN, EPCAM⁻ triple negative (CD106⁻CD51⁻CD61⁻); TP, EPCAM⁻ triple positive (CD106⁺CD51⁺CD61⁺). e, f, Co-immunostaining (e) and quantification (f) of KRT14 and vimentin in cytospin of FACS-isolated skin SCC tumour cells. Scale bars, 20 μm. n = 90 cells per condition and tumour. g, Immunostaining for GFP, KRT7, NKX2-1, KRT5, SOX2, KRT8 and KRT18 (KRT8/18), and vimentin in Fat1 wild-type and -knockout lung carcinomas. Scale bars, 50 μm. h, i, FACS analysis (h) and percentage of EPCAM expression (i) in control and Fat1-cKO YFP⁺ lung tumour cells. Mean ± s.e.m., two-tailed t-test. j, Distribution of YFP⁺EPCAM⁻ tumour cells in CD106/VCAM1, CD61/ ITGB3 and CD51/ITGAV subpopulations in Fat1-cKO lung carcinomas. Mean ± s.e.m. k, l, Co-immunostaining (k) and quantification (l) of pancytokeratin (pan KRT) and vimentin in cytospin of FACS-isolated lung carcinoma tumour cells. Scale bars, 20 μm. n = 70 cells per condition and tumour. In l, K denotes pancytokeratin. m, Immunostaining for KRT14 and vimentin, E-cadherin, SOX2, TRP63 and ZEB1 in FAT1 wild-type and FAT1-knockout (KO) A388 human skin SCC cell line. Scale bars, 50 μm. n, o, Representative images (n) and quantification of hybrid EMT score (o) (colocalization of pancytokeratin and vimentin) in wild-type and FAT1-mutated (MUT) head and neck, and lung, patient-derived xenografts. Scale bars, 50 μm, Mean ± s.e.m., two-tailed Mann–Whitney U test.