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-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.
Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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