Spatial and vascular determinants of tumor-infiltrating lymphocyte expansion in glioblastoma 2260218

Abstract Introduction Adoptive transfer of tumor-infiltrating lymphocytes (TILs) has shown promise in solid tumors but remains limited in glioblastoma (GBM), where immune dysfunction and spatial exclusion constrain TIL outgrowth. We profiled GBM tumors that did or did not yield TIL cultures to identify microenvironmental features predictive of successful expansion. Methods TIL expansion was attempted in 40 tumors; 24 (60%) achieved successful TIL expansion following optimized rapid expansion protocol (REP) culture. Expanded TILs were profiled by spectral flow cytometry and TCR sequencing. A subset of TIL+ (n = 5) and TIL⁻ (n = 6) tumors underwent single-cell RNA sequencing, CODEX multiplex proteomics, and Xenium spatial transcriptomics to characterize immune states, distict niches, receptor—ligand interactions, and vascular topology. Results Expanded TILs proliferated 100—1000-fold from pre-REP to REP, were CD4+-enriched, and dominated by effector-memory phenotypes in both CD4+ and CD8+ compartments with minimal Tregs or naïve subsets. Overall T-cell infiltration was low (< 3% of total cells), yet TIL+ tumors contained more stem-like TCF7+ CD8+ cells and lower HAVCR2 and TOX expression. Spatial analysis revealed 2.3-fold higher CD31+ endothelial density and shorter T cell—endothelium distances (p < 0.05) in TIL+ tumors, suggesting greater vascular accessibility. TIL⁻ tumors showed 1.6-fold stronger inhibitory TAM—microglia signaling, whereas TIL+ regions demonstrated enriched T cell—endothelial and TAM—macrophage interactions, indicating more permissive immune niches. Conclusion These findings identify vascular architecture and immune topology as key determinants of TIL expansion potential. Spatial-immune profiling may guide patient selection and inform strategies to recondition the GBM microenvironment for improved TIL and combination immunotherapies. Funding Source n/a Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)