SEX AND SPATIOTEMPORAL DIFFERENCES OF MYELOID CELL DYNAMICS IN GLIOBLASTOMA MOUSE MODEL

Glioblastoma (GBM) is the most lethal primary brain tumor in adults, with male showing both higher incidence and poorer outcomes than females. The limited efficacy of current treatment strategies, the failure of immunotherapy-based clinical trials, and the high recurrence rate are largely attributed to the profoundly immunosuppressive tumor microenvironment, which is predominantly constituted by myeloid cells. Although GBM exhibits a documented sex dimorphism, patient sex only recently has been recognized as a biological variable in GBM research. Particularly, the spatial, temporal, and sex-specific dynamics of tumor-infiltrating myeloid populations remain poorly defined. Elucidating how myeloid cells differ along tumor progression and in a sex-dependent manner may provide critical insights into sex-specific vulnerabilities and inform the development of more effective, tailored therapeutic strategies. To address these questions, we first orthotopically implanted the murine SB28 GBM cell line into male and female C57Bl/6J mice, and monitored tumor progression in vivo using bioluminescence imaging. To comprehensively characterize the immune populations composing tumor microenvironment, particularly lymphoid and myeloid cells, we employed at early and late stages high-dimensional flow cytometry and immunofluorescence. To evaluate the translational relevance of our findings, we investigated immune-infiltrative patterns in human GBM exploiting publicly available patient datasets. In vitro, SB28 cells exhibited markers consistent with mesenchymal-like transcriptional profile, and an immune-evasive phenotype characterized by low MHC-I expression. In vivo, SB28 cells resulted in robust engraftment, reproducible growth kinetics, highly infiltrative tumors and myeloid- dominated microenvironment with limited lymphoid infiltration, collectively recapitulating the defining biological and immunological features of human GBM. Longitudinal analyses of the spatiotemporal myeloid dynamics revealed a progressive shift in myeloid composition, from early-stage microglia (MG) predominance to late-stage accumulation of infiltrating monocyte-derived macrophages (MDM), accompanied by a significant variation of activation-markers. These changes delineated a specific GBM-associated myeloid phenotype characterized by features of monocyte-differentiation, activation, and a mixed pro-inflammatory and anti-inflammatory profile, ultimately converging toward a GBM-specific immunosuppressive state. Subsequently, sex-stratified analyses highlighted that male mice developed significantly larger tumors than females, suggesting that early events, yet unidentified, drive sex-specific tumor growth trajectories. Deeper characterization demonstrated that male tumors displayed earlier and more pronounced myeloid infiltration, particularly MG infiltration, while female tumors were predominantly infiltrated by circulating peripheral myeloid cells, primarily monocytes. Consistent with these findings, interrogation of human GBM datasets revealed enrichment of MG-associated gene signatures in male. Moreover, early-stage MG in male mice displayed weaker polarization in response to GBM compared to females. Overall, this work establishes the SB28 model as a robust and reproducible platform for studying glioblastoma, enabling the identification of temporal and sex-specific remodeling of the myeloid compartment during disease progression. By integrating preclinical and human data, the study reveals how the myeloid microenvironment evolves across time and sex, providing a mechanistic basis for sex-dimorphic outcomes in GBM and underscoring the importance of incorporating sex as a biological variable in immune-oncology research. These findings highlight the potential of sex-informed, myeloid-targeted therapeutic strategies to advance personalized treatment approaches for GBM.