* These authors contributed equally to this work. High-grade gliomas (HGGs) are central nervous system (CNS) tumors characterized by an immunosuppressive tumor microenvironment (TME) that drives invasion and therapy resistance. Crosstalk between CNS-resident cells (e.g., astrocytes, microglia) and HGG cells fosters extracellular matrix (ECM) remodeling and tumor progression. Understanding HGG immunosuppression and invasion mechanisms is critical for improving therapeutic strategies. However, interpatient heterogeneity in invasion and immune modulation remains poorly captured by existing models. This work aims to develop a human cell model that recapitulates key cellular and molecular interactions within HGG TMEs.

We employed a CNS-HGG 3D co-culture approach using stirred-tank culture systems. As the CNS component, we resourced to neurospheroids (iNSoids) derived from human induced pluripotent stem cells, composed of functional neurons, astrocytes, and oligodendrocytes within their native ECM. As HGG component, we employed enhanced green fluorescent protein (eGFP)-expressing adult and pediatric HGG cell lines (A172, JX6, SF7761).

Co-cultured CNS and HGG cells remained viable and retained their identity at the phenotypic and gene expression levels. Confocal microscopy imaging revealed that eGFP+ HGG cells infiltrated iNSoids with distinct, cell line-dependent invasion dynamics. Invasion depths exceeded 30 μm, with HGG cells exhibiting diverse morphological adaptations, ranging from rounded amoeboid-like migration to elongated mesenchymal-like processes. Gene expression analysis indicated that each cell line differentially regulated ECM remodeling, with upregulation of matrix metalloproteinases (e.g., MMP2, MMP14) and hyaluronan-associated genes (e.g., HYAL2, HAS2), suggesting distinct invasion mechanisms. Beyond invasion, HGG cells also influenced the surrounding immune microenvironment. Upregulation of key immunosuppression-associated genes (e.g., TGFB1, CSF-1) suggests the establishment of an anti-inflammatory TME.

Ongoing work is focused on the incorporation of microglia and patient-derived primary HGG cells to dissect onco-neuroimmune interactions at the single-cell level, increase the clinical translatability of the model, and depict HGG patient cell response to invasion and immunosuppression modulators. Ultimately, our model will provide a platform for preclinical testing that captures the heterogeneity of HGG behavior and better predicts individualized clinical response. We acknowledge funding from FCT/MCTES (PT): LA/P/0087/2020, UIDB/04462/2020 & UIDP/04462/2020, 2022.02117.PTDC, 2024.02085.BD to I.S; UI/BD/151253/2021 to C.G, iBETXplore (PI-752).