Abstract
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.
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Data availability
All in-house scRNA-seq data have been deposited in the Gene Expression Omnibus (GEO) database under the accession numbers GSE296872 and GSE296873. The numerical source data for the graphs can be found in Supplementary Data 2. All other data are available from the corresponding author (or other sources, as applicable) on reasonable request.
Code availability
All scripts used to generate the figures are available on GitHub [https://github.com/AthanasiaSt/Notch3-regulates-pericyte-phenotypic-plasticity-in-colorectal-cancer.git].
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Acknowledgements
The authors would like to thank Sofia Grammenoudi for assistance in FACS sorting experiments. The authors would also like to thank Fleming’s Animal House and Flow Cytometry facilities. This work was funded by Worldwide Cancer Research (Grant No: 22-0126, to V.K.), and the project Fib3R (Grant No: 3001, to V.K.) was funded by the Hellenic Foundation of Research and Innovation (H.F.R.I.) under the “2nd Call for H.F.R.I. Research Projects to support Faculty members and Researchers.” The authors acknowledge support of this work by the project SingleOut (HFRI-FM17C3-3780) funded by the Hellenic Foundation for Research and Innovation (H.F.R.I.) under the “1st Call for H.F.R.I. Research Projects to support Faculty members and Researchers and the procurement of high-cost research equipment”. The authors also acknowledge support by the Research Infrastructure projects InfrafrontierGR (MIS 5002802) and pMedGR (MIS 5002802) funded by the Operational Programme “Competitiveness, Entrepreneurship and Innovation” (NSRF 2014-2020) and co-financed by Greece and the European Union (European Regional Development Fund), as well as by project MIS 6004752 funded by the Regional Operational Programme “ATTICA” (NSRF 2021-2027) and co-financed by Greece and the European Union (European Regional Development Fund). The graphical abstract was created using BioRender (https://BioRender.com/kvnz47d).
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N.C. and V.K. designed the study; N.C. V.Z.A, C.P., A.M., and M.S. performed experiments; A.S. performed the bioinformatics analysis; C.N. and V.K. supervised the bioinformatics analysis; N.C., A.S., and V.K. interpreted the experimental results, wrote the manuscript, and prepared the figures; V.K. supervised the study; all authors critically revised and approved the final manuscript.
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Chalkidi, N., Stavropoulou, A., Arvaniti, VZ. et al. Notch3 regulates pericyte phenotypic plasticity in colorectal cancer. Commun Biol (2026). https://doi.org/10.1038/s42003-026-09629-4
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DOI: https://doi.org/10.1038/s42003-026-09629-4
