3D heterotypic models of glioblastoma reveal the impact of microglia on cellular organization and the production of a distinct secretome.

Glioblastoma (GBM) is a deadly brain tumor with a very poor prognosis. Development of new therapeutics is hindered by the lack of appropriate preclinical models that reflect the complexity of the tumor microenvironment, especially the crucial role of microglia. In this study, we investigated the impact of microglia on GBM models using humanized 3D spheroids. Homotypic and heterotypic spheroids were created out of a GBM-derived cell line (DKMG) or patient-derived glioma stem cells (GB22-13), along with a microglia cell line (HMC3). Heterotypic glioma-HMC3 spheroids exhibited increased proliferation and greater drug resistance to chemotherapy drug Temozolomide compared with homotypic spheroids. Heterotypic spheroids also grew larger, developed multinucleated structures within 7 days, and had a greater invasive potential. Additionally, a distinct core-shell structure emerged in the heterotypic spheroids, with glioma cells concentrated in the core and a surrounding layer of microglia forming a protective shell that appeared to hinder drug penetration to the tumor core. Further, heterotypic cells were able to induce migration and polarization of peripheral blood monocytes (THP-1) towards M2 phenotypes, increasing immune evasion. These findings highlight the critical role of microglia in GBM development and progression, demonstrating their contribution to both reduced drug diffusion and increased tumor growth.