Reconstructing the glioblastoma microenvironment in heterotypic 3D spheroids: a multicellular model to study tumor-stromal crosstalk

Abstract

Background The complex interplay between tumor cells and the stromal components of the glioma microenvironment necessitates the development of sophisticated in vitro models capable of modelling key aspects of cellular interactions that occur beyond the limitations of conventional monocultures. Methodology The development and characterization of homo- and heterotypic 3D spheroid models incorporating CCF-STTG1 astrocytes, HMC3 microglia, and U87MG glioma cells was undertaken. The assessment of morphological, molecular, and functional properties was performed via flow cytometry, cytokine arrays, ECM analysis and invasion assays (Matrigel™/gelatin). Results Heterotypic spheroids have been observed to spontaneously self-assemble into a spatially polarized architecture, with microglia and glioma cells segregating into distinct compartments, a pattern suggestive of the cellular topology at the invasive front. The morphological, molecular, and functional properties of the generated 3D models recapitulated several established features associated with in vivo tumors, including growth, invasion, resistance to chemotherapy, and metabolic reprogramming alongside the expression of stemness markers, and key pro-invasive mediators (MMPs, SDF-1α, VEGF). Secretome profiling revealed a marked, non-additive upregulation of chemokines (IP-10, MIP-1α) and the emergence of novel correlations (HGF/SDF-1α, MCP-1/LIF), indicating potential modulation of paracrine networks involved in immune cell trafficking in the heterotypic setting. The initial formation of a rigid ECM matrix appears to be initiated by microglia, while the supply of fibronectin and laminin may be linked to astrocytes exhibiting some features of reactive gliosis, which could help organize invasion pathways. Conclusion These heterotypic 3D spheroid models offer a stroma-enriched, reproducible platform for the analysis of stromal contributions to glioma progression and for exploratory preclinical evaluation of therapeutic strategies.