In colorectal cancer, oncogenic mutations transform a hierarchically organized and homeostatic epithelium into invasive cancer tissue lacking visible organization. We sought to define transcriptional states of colorectal cancer cells and signals controlling their development by performing single‐cell transcriptome analysis of tumors and matched non‐cancerous tissues of twelve colorectal cancer patients. We defined patient‐overarching colorectal cancer cell clusters characterized by differential activities of oncogenic signaling pathways such as mitogen‐activated protein kinase and oncogenic traits such as replication stress. RNA metabolic labeling and assessment of RNA velocity in patient‐derived organoids revealed developmental trajectories of colorectal cancer cells organized along a mitogen‐activated protein kinase activity gradient. This was in contrast to normal colon organoid cells developing along graded Wnt activity. Experimental targeting of EGFR‐BRAF‐MEK in cancer organoids affected signaling and gene expression contingent on predictive KRAS/BRAF mutations and induced cell plasticity overriding default developmental trajectories. Our results highlight directional cancer cell development as a driver of non‐genetic cancer cell heterogeneity and re‐routing of trajectories as a response to targeted therapy.

In colorectal cancer, oncogenic mutations transform a hierarchically organized and homeostatic epithelium into invasive cancer tissue. To define differences in cellular composition between the normal colon and colorectal cancer, and to map potential cellular interactions between tumor cells and their microenvironment, we profiled transcriptomes of >50,000 single cells from tumors and matched normal tissues of eight colorectal cancer patients. We find that tumor formation is accompanied by changes in epithelial, immune and stromal cell compartments in all patients. In the epithelium, we identify a continuum of five tumor-specific stem cell and progenitor-like populations, and persistent multilineage differentiation. We find multiple stromal and immune cell types to be consistently expanded in tumor compared to the normal colon, including cancer-associated fibroblasts, pericytes, monocytes, macrophages and a subset of T cells. We identify epithelial tumor cells and cancer-associated fibroblasts as relevant for assigning colorectal cancer consensus molecular subtypes. Our survey of growth factors in the tumor microenvironment identifies cell types responsible for increased paracrine EGFR, MET and TGF-β signaling in tumor tissue compared to the normal colon.We show that matched colorectal cancer organoids retain cell type heterogeneity, allowing to define a distinct differentiation trajectory encompassing stem and progenitor-like tumor cells. In summary, our single-cell analyses provide insights into cell types and signals shaping colorectal cancer cell plasticity.