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

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 unclear. This study investigated platelet-derived growth factor receptor beta-positive (Pdgfrb+) pericyte dynamics and reprogramming in GBM vasculature. We generated GL261-Luc and GL261-CFP glioblastoma cells via lentiviral transduction and established two transgenic models. (1) For pericyte labeling, Ai14 reporter mice was crossed with PDGFRβ-P2A-CreERT2 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. Cre-mediated ablation of Pdgfrb-expressing cells revealed stage-dependent effects on GBM growth: early ablation inhibited progression while late ablation promoted 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 vasculature displayed simplified branching, dilation, and pericyte remodeling (shorter processes, 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. This study demonstrates that during early GBM progression, pericytes 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.