An oncogenic phenoscape of colonic stem cell polarization

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, we performed a systematic single-cell analysis of 1,107 colonic organoid cultures regulated by (1) colorectal cancer (CRC) oncogenic mutations, (2) microenvironmental fibroblasts and macrophages, (3) stromal ligands, and (4) signaling inhibitors. Multiplexed single-cell analysis revealed a stepwise epithelial differentiation phenoscape dictated by combinations of oncogenes and stromal ligands, spanning from fibroblast-induced Clusterin (CLU)+ revival colonic stem cells (revCSCs) to oncogene-driven LRIG1+ hyper-proliferative CSCs (proCSCs). The transition from revCSCs to proCSCs is regulated by decreasing WNT3A and TGF-β-driven YAP signaling and increasing KRASG12D or stromal EGF/Epiregulin-activated MAPK/PI3K flux. We find that APC loss and KRASG12D collaboratively limit access to revCSCs 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. [Display omitted] •A colonic stem cell phenoscape regulated by oncogenic and microenvironmental cues•Fibroblasts polarize colonic epithelia toward revCSCs via TGF-β1 and YAP•APC loss and KRASG12D disrupt stromal regulation and trap epithelia in a proCSC fate•scRNA-seq data-driven Waddington-like landscapes of stem cell polarization Highly multiplexed single-cell analysis of colonic organoids perturbed by oncogenic mutations and microenvironmental signals reveals a continuous phenoscape of colonic stem cell polarization in colorectal cancer. Oncogenic mutations collaboratively dominate homeostatic stromal signaling, trapping epithelial cells in a cancerous state.