A Single-cell Perturbation Landscape of Colonic Stem Cell Polarisation

Abstract

Cancer cells are regulated by oncogenic mutations and microenvironmental signals, yet these processes are often studied separately. To functionally map how cell-intrinsic and cell-extrinsic cues co-regulate cell-fate in colorectal cancer (CRC), we performed a systematic single-cell analysis of 1,071 colonic organoid cultures regulated by 1) CRC oncogenic mutations, 2) microenvironmental fibroblasts and macrophages, 3) stromal ligands, and 4) signalling inhibitors. Multiplexed single-cell analysis revealed a stepwise epithelial differentiation landscape dictated by combinations of oncogenes and stromal ligands, spanning from fibroblast-induced Clusterin (CLU)+ revival colonic stem cells (revCSC) to oncogene-driven LRIG1+ hyper-proliferative CSC (proCSC). The transition from revCSC to proCSC is regulated by decreasing WNT3A and TGF-{beta}-driven YAP signalling and increasing KRASG12D or stromal EGF/Epiregulin-activated MAPK/PI3K flux. We find APC-loss and KRASG12D collaboratively limit access to revCSC and disrupt stromal-epithelial communication - trapping epithelia in the proCSC fate. These results reveal that oncogenic mutations dominate homeostatic differentiation by obstructing cell-extrinsic regulation of cell-fate plasticity. HighlightsO_LI1,071-condition single-cell transition map of colonic stem cell polarisation regulated by oncogenic and mircoenvironmental cues. C_LIO_LIFibroblasts polarise WT colonic epithelia towards Clu+ revCSC via TGF-{beta}1 and YAP signalling. C_LIO_LIAPC-loss and KRASG12D drive a Birc5+, Lrig1+, and Ephb2+ proCSC fate via MAPK and PI3K. C_LIO_LIOncogenic mutations disrupt stromal regulation of epithelial plasticity, trapping cells in the proCSC fate. C_LI