Insight into pericytes in glioblastoma angiogenesis:In vivo tracking by two-photon microscopy and proteomic profiling

Qinghong Wang; Chengyan Ma; Xinpei Wang; Mengyuan Li; Xingjiu Yang; Ran Gao

doi:10.1002/ame2.70073

Abstract：Background: Glioblastoma (GBM) is a highly aggressive brain tumor characterized by aberrant angiogenesis and an immunosuppressive microenvironment. Pericytes are aberrantly recruited but their spatiotemporal roles and molecular changes remain un-clear. This study investigated platelet-derived growth factor receptor beta-positive (Pdgfrb+) pericyte dynamics and reprogramming in GBM vasculature. Methods: We generated GL261-Luc and GL261-CFP glioblastoma cells via lentivi-ral transduction and established two transgenic models. (1) For pericyte labeling, Ai14 reporter mice was crossed with PDGFRβ-P2A-CreER T2 mice for tdTomato-specific lineage tracing (PT mice). (2) For conditional ablation, we generated inducible Pdgfrb-expressing cell ablation models (PT mice was crossed with ROSA-DTA mice). An intravital imaging platform (FITC-dextran/CFP/tdTomato+two-photon microscopy) tracked pericytes, vessels, and tumor cells, while FACS-sorted Pdgfrb+ cells from GBM and normal brain were analyzed by LC-MS/MS proteomics. Results: Cre-mediated ablation of Pdgfrb-expressing cells revealed stage-dependent effects on GBM growth: early ablation inhibited progression while late ablation pro-moted it. Pericytes undergo dual spatial reorganization in GBM: regional enrichment with pre-sprouting accumulation at the tumor-brain interface, and focal positioning with preferential localization at vascular branch points. Concurrently, GBM vascula-ture displayed simplified branching, dilation, and pericyte remodeling (shorter pro-cesses, higher density). Proteomics revealed 1426 altered proteins, with upregulated proliferation pathways (e.g., matrix metallopeptidase 14 [Mmp14], lysyl oxidase like 2 [Loxl2]) and downregulated homeostasis functions (e.g., transforming growth factor beta 1 [Tgfb1]), validated by scRNA-seq in human GBM. Conclusions: This study demonstrates that during early GBM progression, peri-cytes actively drive tumor angiogenesis through molecular reprogramming toward proliferative and pro-angiogenic phenotypes, with the integrated imaging-proteomics framework revealing potential therapeutic targets for disrupting pericyte-mediated vascular remodeling.