Gene expression of Cancer-associated fibroblasts from orthotopically engrafted murine colon tumors on the single cell level (Sort-Seq)

The EMT-transcription factor ZEB1 is heterogeneously expressed in tumor cells and in cancer-associated fibroblasts (CAFs) in colorectal cancer (CRC). While ZEB1 in tumor cells regulates metastasis and therapy resistance, its role in CAFs is largely unknown. Combining fibroblast-specific Zeb1 deletion with immunocompetent mouse models of CRC, we observe that inflammation-driven tumorigenesis is accelerated, whereas invasion and metastasis in sporadic cancers is reduced upon fibroblast-specific loss of Zeb1. Single-cell transcriptomics, histological characterization and in vitro modelling reveal a crucial role in CAF polarization, promoting myofibroblastic features by restricting inflammatory activation. Zeb1 deficiency impairs collagen deposition and CAF barrier function but increasesd NFκB-mediated cytokine production, jointly promoting lymphocyte recruitment and immune checkpoint activation. We used single cell RNA sequencing (scRNA-seq) via SORT-Seq to analyze the transcriptome of orthotopically engrafted murine colon cancer organoids. Cell types within whole tumors were isolated by FACS (following colon tumor digestion) using marker combinations (Epcam, CD45 and CD31).