Single-cell RNA-seq and CITE-seq of autochthonous KP and KPN mouse tumors

Colorectal cancers are complex ecosystems, with interactions between the cancer epithelium and the tumor microenvironment (TME) shaping disease progression. Cancer-associated fibroblasts are key TME components, influencing epithelial plasticity and immune cell infiltration. In this study, we collected autochthonous tumours from KP (villinCreER KrasG12D/+ Trp53fl/fl) and KPN (villinCreER KrasG12D/+ Trp53fl/fl Rosa26N1icd/+) mice, colorectal cancer models that develop invasive tumours and metastasise to the liver with high penetrance and short latency. We aimed to characterise the cellular composition of these tumours, focusing on shifts in different cell compartments, particularly fibroblast phenotypes. Dissociated single cells were enriched for fibroblasts by fluorescence-activated cell sorting based on negative expression of Epcam and Cd45 (Double negative, DN). Single-cell RNA sequencing and CITE-seq were performed. CITE-seq staining used the TotalSeq™-C Mouse Universal Cocktail V1.0 (BioLegend), and cell hashing was performed using TotalSeq™-C0308 anti-mouse Hashtag 8 (Barcode: TATAGAACGCCAGGC) and TotalSeq™-C0314 anti-mouse Hashtag 14 (Barcode: CTTTCGCCAACTCTG).