Glioma angiogenesis phosphoproteome landscape and biomarker sets identified with phenome-centered multiomics toward 3P medical approaches.

Protein phosphorylation is an important molecular event in tumor angiogenesis that is a canonical hallmark in glioma. We hypothesize that the phosphoproteome and phosphorylation-mediated signaling networks are significantly different in glioma neovascular tissues compared to controls, which aimed to identify glioma angiogenesis phosphoproteomic landscape, phosphorylation-mediated signaling pathways, kinase-substrate networks, and phosphorylation biomarkers with integration of phosphoprotein data and multiomics data, for deep understanding of molecular mechanisms of glioma angiogenesis, discovery of effective antiangiogenesis therapeutic targets, and establishment of angiogenesis-related phosphorylation biomarker signature for patient stratification, early-stage diagnosis, and effective prognostic assessment, in the framework of predictive, preventive, and personalized medicine (PPPM, 3PM) approaches. This study used laser capture microdissection to isolate neovascular tissues from gliomas, followed by quantitative phosphoproteomics analysis, which identified 195 differentially phosphorylated proteins (DPPs) with 635 phosphosites and 58 hub DPPs. Pathway analysis of 195 DPPs found that cell adhesion-related pathways and HIF-1 signaling pathway were significantly regulated by phosphorylation to associate with glioma angiogenesis. Upstream kinase analysis found 321 upstream kinases to regulate the intratumoral neovascular tissue-associated phosphorylation, including 12 kinases that were differentially expressed in glioma neovascular tissues and 2 kinases (CAMK2D and MYLK) that were also DPPs, and 48 chemotherapeutic agents as kinase inhibitors such as staurosporine that had antiangiogenesis effects in glioma. Integrated analysis of DPPs and DEGs (differentially expressed genes) revealed 82 overlapped molecules; of them, SYN1, STX1A, PRKAR2B, PACSIN1, LSP1, HSPB1, and DMTN were associated with overall survival of glioma, and ANK1, L1CAM, and LSP1 were constructed as glioma prognosis signature. Immunohistochemistry confirmed hypophosphorylation at PDHA1-Ser293/300 in glioma angiogenesis. This study provided the first phosphoproteome landscape, kinase profile, phosphorylation-mediated signaling pathway network alterations in human glioma neovascular tissues, and effective tumor angiogenesis-based biomarkers for patient stratification, prognostic assessment, and targeted therapy in glioma. These findings provide concrete molecular targets for antiangiogenic therapy and establish clinically actionable biomarkers for glioma patient stratification in the 3PM framework. Supplementary Information: The online version contains supplementary material available at 10.1007/s13167-025-00428-1.