Expression and Membranous Localization of CD44 Define Distinct Cellular and Spatial Phenotypes Associated with Glioma Aggressiveness

Adult-type diffuse gliomas comprise, amongst others, biologically distinct isocitrate dehydrogenase (IDH)-mutant (IDH-MUT) astrocytomas and IDH-wildtype (IDH-WT) glioblastoma (GB). Although IDH-MUT astrocytomas generally have a more favorable prognosis, they frequently recur and progress. Despite extensive molecular characterization, key drivers of glioma progression remain elusive. CD44, a hyaluronic acid receptor, has been implicated in glioma invasion and progression. We investigated CD44 expression, localization, and spatial organization across IDH-WT GB and IDH-MUT astrocytomas and their association with tumor progression and cellular states. CD44 expression and localization were assessed in IDH-WT (n = 15) and IDH-MUT (n = 17) diffuse gliomas across WHO grades by immunohistochemistry (IHC). Multiplex IHC combined with AI-assisted digital pathology (HALO) evaluated associations between CD44, GFAP-positive astrocytic, and NeuN/RBFOX3-positive neuronal regions. Findings were validated using independent snRNA-seq data. IDH-WT GB exhibited significantly higher CD44 expression with prominent membranous localization. Lower-grade IDH-MUT astrocytomas showed reduced, diffuse staining, whereas higher-grade tumors acquired a membranous CD44 localization, resembling IDH-WT GB. CD44 was strongly associated with GFAP-positive astrocytic regions and inversely correlated with NeuN/RBFOX3-positive neuronal areas in IDH-MUT tumors. AI-assisted spatial analyses confirmed distinct CD44-positive astrocytic and NeuN-positive neuronal niches, supported by snRNA-seq demonstrating positive CD44-GFAP correlations. This association was strongest in astrocytic (AC-like) metaprograms of WHO grade 2, while in higher-grade tumors it was more pronounced in mesenchymal (MES-like) states. CD44 exhibits IDH- and grade-dependent spatial patterns marking glioma progression and astrocytic/mesenchymal tumor states. Membranous CD44 acquisition accompanies progression to higher grades in IDH-MUT astrocytomas, suggesting CD44 as a marker of glioma aggressiveness.