P10.01 Effect of mesenchymal cells on infiltration and phenotype of T cells in a 3D glioblastoma spheroid model

Abstract

Abstract BACKGROUND Glioblastoma (GBM), the most malignant primary brain tumor with a median survival less than two years, is characterized by aggressive invasive growth, presence of hypoxic and necrotic regions, and T-cell dysfunction. Mesenchymal cells with characteristics of cancer associated fibroblasts are present in GBMs, but it remains to be established whether they contribute to T cell sequestration in the tumor stroma and functional inactivation similarly to extracranial tumors. Murine models do not reflect several aspects of human immune system and 2D cell culture systems often fail to recapitulate complex interactions in the tumor microenvironment. 3D spheroids prepared from patient-derived cell cultures offer higher degree of complexity and biological relevance. The aim of this study was to establish a 3D GBM spheroid model to determine the effect of patient derived mesenchymal cells on human T cell infiltration and phenotype. MATERIAL AND METHODS Glioma stem-like cell and mesenchymal cell cultures were derived from human GBMs. Naïve T lymphocytes were isolated from buffy coats of healthy donors by Ficoll paque gradient separation, followed by negative magnetic isolation techniques using Naïve Pan T Cell Isolation Kit (Miltenyi Biotec). 3D spheroids were prepared by embedding the cells in 20 µl of Geltrex (Thermo Fisher Scientific). T cells activated by magnetic beads with anti-CD2, anti-CD3 and anti-CD28 antibodies (Miltenyi Biotec) and IL-2 were subsequently added to the spheroids. After 5 days of co-culture, spheroids were dissociated, and viable cells were counted using automated cell counter (Biorad). Expression of a T-cell activation marker CD69 and immune checkpoint molecules LAG3, and PD-1 in CD3+ cells was analyzed by flow cytometry. Mesenchymal, stem cell and lymphocyte markers TE-7, Nestin and CD45 respectively, were analyzed by immunohistochemistry (IHC) on frozen spheroid sections. RESULTS We prepared spheroids comprising glioma stem-like cells with and without mesenchymal cells. Glioblastoma-derived cells were evenly dispersed throughout the spheroids as demonstrated by IHC. T cells infiltrated into both types of spheroids, but their number in spheroids containing mesenchymal cells was about 40% (p=0.002) lower compared to the ones without mesenchymal cells. In both types of spheroids, the presence of activated allogeneic T cells led to more than a 50% reduction of the glioblastoma-derived cells. Ongoing studies evaluate the effect of mesenchymal cells on the phenotype of spheroid infiltrating T cells. CONCLUSION We have successfully established a 3D spheroid model to study the role of mesenchymal cells in GBM microenvironment. Our results indicate that mesenchymal cells may influence the infiltration of T cells and modulate their phenotype. SUPPORT Project was financed by the Center for Tumor Ecology (CZ.02.1.01/0.0/0.0/16_019/0000785).