Pericytes, essential components of the tumor microenvironment, undergo phenotypic alterations that influence cancer progression, yet the molecular mechanisms governing these changes remain poorly understood. Here, we investigate the role of Notch3 signaling in pericyte phenotype and functions in colorectal cancer (CRC). Using lineage tracing approaches, we show that murine tumor pericytes originate from normal tissue-resident pericytes, which proliferate inside tumors. In vivo genetic manipulation reveals that Notch3 pathway activation promotes pericyte proliferation, while suppressing contractile protein expression, and leads to increased endothelial cell proliferation and reduced blood vessel integrity. In contrast, Notch3 deletion leads to decreased endothelial proliferation, blood vessel normalization, and a significant reduction in tumorigenesis in an advanced orthotopic mouse model. Single-cell RNA sequencing analysis uncovers significant pericyte heterogeneity in both mouse colitis-associated cancer and human CRC. It specifically identifies distinct subpopulations characterized by differential Notch3 activity, which is enriched in a synthetic subset and absent in a contractile subset, further supporting our in vivo findings. Our results establish Notch3 as a key regulator of pericyte phenotypic plasticity in CRC and suggest that targeting this pathway could represent a promising strategy for improving therapeutic outcomes through vascular normalization.
© 2026. The Author(s).