Abstract
BACKGROUND: Glioblastoma (GBM) represents the most aggressive form of primary brain tumors, typically resulting in a median survival of less than two years. Immunosuppression characterized by macrophage polarization into a protumorigenic phenotype and T cell dysfunction belong to key features of glioblastoma microenvironment. Recently, mesenchymal cells were identified in GBM, but their immunomodulatory effect has not been elucidated yet. This study aimed to establish a 3D GBM spheroid model to investigate the influence of mesenchymal cells derived from GBM patients on the phenotype of human T cells. MATERIAL AND METHODS: Glioma stem-like cells (GSCs) and paired mesenchymal cell cultures were derived from 3 human GBMs. CD4+ T-lymphocytes were isolated from buffy coats of healthy donors using Ficoll Paque gradient separation, followed by negative magnetic isolation. T-lymphocytes were activated using anti-CD2, anti-CD3, and anti-CD28 antibodies. 3D spheroids were generated by encapsulating the GSC and T-lymphocytes in extracellular matrix (Geltrex) with or without mesenchymal cells. After co-culture for 5-days, the spheroids were dissociated, and the expression of T-cell activation markers CD69 and CD25, along with immune checkpoint molecules LAG3, TIM-3, and PD-1 was analyzed by flow cytometry. Immunohistochemistry was performed on frozen sections of the spheroids to investigate the spatial distribution of the cell populations. RESULTS: In spheroids with mesenchymal cells, CD4+ T-lymphocytes exhibited elevated expression of the activation marker CD69, whereas no significant changes were observed in CD25 expression. PD-1 expression was higher in spheroids containing mesenchymal cells, whereas no difference was noted in TIM3 and LAG3 expression levels. Uniform distribution of glioblastoma-derived cells was revealed in spheroids containing glioma stem-like cells with and without mesenchymal cells and T-lymphocytes did not exhibit any specific spatial distribution pattern. CONCLUSION: We have successfully established a 3D spheroid model to investigate the impact of mesenchymal cells on the GBM microenvironment. Our findings suggest that mesenchymal cells can influence the phenotype of CD4+ T-lymphocytes in glioblastoma. This research was funded by project National Institute for Cancer Research (Programme EXCELES, ID Project No. LX22NPO5102) by the European Union - Next Generation EU, and Charles University Cooperatio Program,,Oncology and Haematology“.