Title : Sex-dependent glial architecture in the prefrontal cortex and its relevance to glioblastoma
Abstract:
Background: Female glioblastoma (GBM) patients consistently outlive males by 3 to 5 months even after controlling for age, surgical extent, and MGMT methylation status. The biological mechanism driving this survival gap remains unresolved. Nearly all existing research examines glial biology after tumor formation, leaving pre-tumor sex differences in glial architecture unstudied. This study addressed that gap through two complementary analyses.
Methods: Study 1 quantified glial cell density across three subregions of the rat medial prefrontal cortex (mPFC) — the anterior cingulate area (ACA), prelimbic area (PL), and infralimbic area (ILA) — using DAPI nuclear morphology and automated ImageJ particle analysis (n = 3 per sex). A dorsal-to-ventral gradient analysis compared ACA density to pooled ventral mPFC density by sex using two-way ANOVA with Fisher’s LSD post-hoc. Study 2 analyzed GFAP and AIF1 gene expression in 37 human GBM tumor blocks from the Ivy Glioblastoma Atlas Project (Ivy GAP; 19 male, 18 female). A two-way ANOVA examined the effect of sex and EGFR amplification status on the log?(GFAP/AIF1) expression ratio in 28 blocks with available EGFR data.
Results: In Study 1, male rats showed a statistically significant dorsal-to-ventral glial density gradient (ACA: 78.3 vs. pooled ventral: 175.7 objects/100,000 px²; p = 0.047). Female rats showed a numerically comparable gradient that narrowly missed significance (p = 0.068), with large effect sizes in the ACA (Hedges’ g = 1.91) and ILA (g = 1.18), consistent with insufficient power at n = 3. In Study 2, neither GFAP nor AIF1 differed by sex in isolation. However, when patients were stratified by EGFR amplification status, amplification was significantly associated with an elevated GFAP/AIF1 ratio in male patients (amplified: 1.420 vs. non-amplified: 0.319; p = 0.010) but not in female patients (0.876 vs. 0.658; p = 0.552).
Conclusions: These findings suggest that sex-dependent glial differences are expressed through organizational responsiveness to perturbation rather than absolute cell quantity. The female tumor microenvironment appears more resistant to EGFR-driven oncogenic disruption of the astrocyte-to-microglial balance, which may partly explain the persistent female survival advantage in GBM. EGFR-amplified male patients may represent a biologically distinct subgroup with an astrocyte-dominant microenvironment that suppresses cytotoxic T cell infiltration and warrants sex-stratified analysis in future checkpoint inhibitor trials.
