Quantitative analysis of immune cells within the tumor microenvironment of glioblastoma and their relevance for prognosis

Abstract

Glioblastoma (GBM) is a high malignant tumor with no effective treatment. To comprehensively characterize the landscape of immune cells in GBM and evaluate their correlation with prognosis, we developed a multispectral fluorescent imaging pipeline that included tumor-infiltrating lymphocytic markers (CD3, CD4, CD8, FOXP3, NKP46), immune checkpoint markers (PD-1, PD-L1), and markers to characterize myeloid cells (CD68, CD66b, CD163, HLA-DR), to spatially quantify 18 immune cell subsets in 21 GBM cases. We found that macrophages are the most abundant in GBM microenvironment, followed by T cells and neutrophils, while NK and NKT cells are the least. Previously unreported CD8⁺ Treg, PD-L1⁺ neutrophils, and high proportion of PD-1⁺ NK and PD-1⁺ T cells were also detected. Single high densities of PD-1⁺CD8⁺ T cells, neutrophils, and PD-L1-expressing CD68⁺ cells were associated with longer survival. Moreover, closer proximity of T cells to PD-L1⁺ macrophages or PD-L1⁺ neutrophils were associated with poor prognosis. Correlative analysis revealed circulating PMN-MDSC and e-MDSC were positively correlated with intratumoral M2 macrophages, while circulating NK cells were inversely associated with infiltrating CD4⁺ Treg cells in GBM patients. Our findings highlighted the potential roles of infiltrating immune cells in prognosis prediction and developing novel immunotherapeutic strategies for GBM patients.

Keywords: Glioblastoma; Immune cells; Multiplex fluorescent immunohistochemistry; Prognosis; Tumor microenvironment.