31 CD44-Gal9 Interaction between IDH1-mutant Grade 4 Astrocytoma and Microglia Promotes Invasion and Proliferation of Tumor Cells

Abstract Introduction IDH1-mutant grade 4 astrocytomas exhibit pronounced interactions with the tumor microenvironment that critically influence tumor progression. Tumor-associated microglia/macrophages (TAMs) are key mediators of these interactions, yet the molecular mechanisms governing tumor-TAM crosstalk remain incompletely understood. Here, we investigated the role of CD44-Galectin-9 (Gal9) signaling in patient-derived IDH1-mutant astrocytoma models. Method scRNA-seq was performed on paired primary and recurrent high-grade astrocytomas to assess CD44 and Gal9 expression. CD44 was suppressed in tumor cells using shRNA-mediated knockdown, with or without exogenous Gal9. Conditioned media from control or CD44 knockdown tumor cells was applied to induced pluripotent stem cell-derived microglia (iMGs) to evaluate phagocytic activity. Tumor behavior was further assessed in 3D cerebral organoid co-culture models. Immunofluorescence analyses were used to examine spatial expression patterns of CD44 and Gal9. Results scRNA-seq analysis revealed marked overexpression of CD44 in tumor cells, while Gal9 expression was predominantly associated with TAMs. CD44 knockdown significantly reduced tumor cell proliferation and invasion in vitro. Exogenous Gal9 enhanced proliferative and invasive behavior in control tumor cells, whereas CD44-deficient cells were unresponsive, indicating a CD44-dependent pro-tumorigenic effect of Gal9. Conditioned media from CD44 knockdown tumor cells increased microglial phagocytic activity, suggesting altered tumor-mediated immune modulation. In 3D cerebral organoid co-cultures, CD44 knockdown tumor cells exhibited reduced invasion and proliferation. Immunofluorescence revealed peritumoral enrichment of Gal9 expression in iMGs, which was diminished in CD44 knockdown samples, with CD44 and Gal9 expression showing a strong positive correlation across models. Conclusions Together, these data identify CD44-Gal9 signaling as a clinically relevant axis promoting tumor invasiveness and immune evasion in IDH1-mutant astrocytomas. Therapeutic inhibition of this interaction may represent a promising strategy to simultaneously attenuate tumor aggressiveness and reprogram the tumor microenvironment.