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

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-1CD8 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.