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In glioblastoma (GBM), an aggressive primary brain tumor, astrocytes become activated and tumor-associated astrocytes (TAAs) exhibit different transcriptomic profiles, morphology, and functions supporting disease progression. Heterogeneity and specific roles of TAAs within various regions of tumors are poorly known. Advancements of single-cell and spatial transcriptomics allow to profile tumors at unprecedented resolution revealing cell phenotypes, hidden functionalities, and spatial architecture in disease-specific context. We combined spatial transcriptomics and multiple immunofluorescent staining to visualize TAAs heterogeneity and location of various subpopulations in three intracranial murine glioma models. Using distinct gene expression profiles, we identified subtypes of TAAs with distinct localization and inferred their specialized functionalities. Gene signatures associated with TAAs reflected their reprograming in the tumor microenvironment (TME), revealed their multiple roles and potential contributing factors shaping the local milieu. Using spatial correlation analysis of the spots, we inferred the interactome of\n                    <jats:italic>Slc1a2<\/jats:italic>\n                    (encoding a glutamate transporter) with the other markers of TAAs based on segregated areas of the tumor. The designer RGD peptide that blocked tumor-microglia communications, altered the spatial distribution of TAAs in GL261 gliomas providing new insights into cell-to-cell communication. Spatial transcriptomics combined with multiple staining unveiled multiple functional phenotypes of TAAs and interactions within TME. Altogether, we demonstrate distinct morphology of TAAs and different roles in various regions of the tumor. Glioma-induced heterogeneity of TAAs allows adaptation to the pharmacologically induced modification of the immunosuppressive TME.\n                  <\/jats:p>","DOI":"10.1371\/journal.pbio.3002893","type":"journal-article","created":{"date-parts":[[2025,10,13]],"date-time":"2025-10-13T17:35:38Z","timestamp":1760376938000},"page":"e3002893","update-policy":"https:\/\/doi.org\/10.1371\/journal.pbio.corrections_policy","source":"Crossref","is-referenced-by-count":17,"title":["Identification and characterization of tumor-associated astrocyte subpopulations and their interactions with the tumor microenvironment in experimental 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