A Single-cell Perturbation Landscape of Colonic Stem Cell Polarisation

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-β-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. Highlights ### Competing Interest Statement The authors have declared no competing interest.