Alves-Pereira, C. F.; Kim, G. D.; Sherpa, N.; Colvin, K.; Khan, S. M.; Phan, K. P.; Wang, A. Z.; Dunn, I. F.; Johanns, T.; Tsitsykov, E.; Desai, R.; Dunn, G. P.; Petti, A. A.

Glioblastoma (GBM) develops within a complex tumor ecosystem whose temporal dynamics remain poorly understood. Here, we performed longitudinal single-cell RNA sequencing and spatial transcriptomics across multiple timepoints in two widely used murine GBM models - CT2A and GL261 - which differ markedly in aggressiveness and response to immune checkpoint blockade. Tumor cell transcriptomes revealed model-specific programs: CT2A cells progressively upregulated epithelial-mesenchymal transition (EMT), non-classical MHC Class I, and progressively, hypoxia response pathways, resembling the human mesenchymal GBM cell state, while GL261 cells exhibited MHC Class II expression and developmental signatures resembling oligodendrocyte progenitor and astrocytic states. Ligand-receptor interaction analyses identified thrombospondins (Thbs1, Thbs2) and osteopontin (Spp1) as CT2A-specific tumor ligands mediating tumorigenic interactions with immune cells, with downstream targets enriched for EMT and TGF-{beta} pathways. Conversely, the GL261 model presented a differential potential to engage neuronal and perivascular guidance networks, with Glutamate and L1 cell adhesion molecule (L1cam) as lead signaling partners. The CT2A immune compartment exhibited progressive microglia-to-macrophage phenotypic conversion, enhanced macrophage infiltration driven by Spp1, and elevated T cell exhaustion, while GL261 maintained a distinct adaptive immune communication hub via MHC class II-CD4 signaling. Elevated THBS1, THBS2, and SPP1 expression correlated with poor survival in human GBM datasets. Together, these findings reveal divergent tumor-immune ecosystems in CT2A and GL261 that recapitulate distinct aspects of human GBM, with implications for therapeutic targeting.