ZEB2 drives intra-tumor heterogeneity and skin squamous cell carcinoma formation with distinct EMP transition states.

Poor prognosis in cutaneous squamous cell carcinoma (cSCC) stems from poor differentiation, invasion and metastasis, all of which are linked to the process of epithelial-to-mesenchymal plasticity (EMP). Here we showed that the EMP-associated transcription factor ZEB2 drives cSCC heterogeneity which resembles biphasic carcinosarcoma-like tumors. Single cell RNA sequencing revealed different subpopulations ranging from fully epithelial (E) to intermediate (EM) to fully mesenchymal (M) which was associated with the graded loss of cell surface markers EPCAM, CDH1, ITGB4 and CD200. Mesenchymal features were associated with a higher metastatic capacity and anoikis resistance, yet this comes with a sensitivity towards TNF-induced cell death. Altogether we provide insights in cSCC heterogeneity and modes to target mesenchymal- metastasis inducing cells Krt14CreTg/+ -Rosa26-Zeb2Tg/+/Trp53Fl/Fl mice develop skin tumors that show a mixed epithelial-mesenchymal phenotype or fully mesenchymal-like tumors. These mice express Zeb2 as a bicistronic transcript with GFP. We performed single cell RNA sequencing on GFP+DAPI- sorted cells from a mixed epithelial-mesenchymal primary tumor and a fully mesenchymal primary tumor. We also performed single cell RNA sequencing on an early passage cell culture derived from a tumor with a mixed epithelial/mesenchymal phenotype.