A Single-cell Perturbation Landscape of Colonic Stem Cell Polarisation
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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 1,107 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.
肿瘤细胞受致癌突变与微环境信号共同调控，但过往研究往往将这两类过程分开探究。为系统性解析结直肠癌（colorectal cancer, CRC）中细胞内在与细胞外在信号如何协同调控细胞命运，我们对1107例结肠类器官培养物开展了系统单细胞分析，这些培养物分别受四类因素调控：1）结直肠癌致癌突变；2）微环境成纤维细胞与巨噬细胞；3）基质配体；4）信号通路抑制剂。多重单细胞分析揭示了由致癌基因与基质配体组合调控的渐进式上皮分化图谱，其覆盖范围从成纤维细胞诱导的簇集蛋白（Clusterin, CLU）阳性复苏结肠干细胞（revCSC），延伸至致癌基因驱动的LRIG1阳性高增殖性肿瘤干细胞（proCSC）。从复苏结肠干细胞（revCSC）向高增殖性肿瘤干细胞（proCSC）的转变，受WNT3A水平下调、TGF-β介导的YAP信号通路活性降低，以及KRASG12D突变或基质EGF/表皮调节素（Epiregulin）激活的MAPK/PI3K信号通量上调共同调控。我们发现APC缺失与KRASG12D突变可协同限制细胞获取复苏结肠干细胞（revCSC）表型，并破坏基质-上皮细胞间的信号交流，将上皮细胞固定于高增殖性肿瘤干细胞（proCSC）的命运状态。本研究结果表明，致癌突变通过阻断细胞外在信号对细胞命运可塑性的调控，主导了结直肠癌的稳态分化进程。
