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Characterization of Immune Cell Subsets of Tumor Infiltrating Lymphocytes in Brain Metastases

Authors: Croft, Priyakshi Kalita-de; Chittoory, Haarika; Nguyen, Tam H.; Saunus, Jodi M.; Kim, Woo Gyeong; Reed, Amy E. McCart; Lim, Malcolm; De Luca, Xavier M.; Ferguson, Kaltin; Niland, Colleen; Mazzieri, Roberta; Dolcetti, Riccardo; Simpson, Peter T.; Lakhani, Sunil R.


Issue: Biology (Basel). 2021 May 11;10(5):425. doi


The heterogeneity of tumor infiltrating lymphocytes (TILs) is not well characterized in brain metastasis. To address this, we performed a targeted analysis of immune-cell subsets in brain metastasis tissues to test immunosuppressive routes involved in brain metastasis. We performed multiplex immunofluorescence (mIF), using commercially available validated antibodies on formalin-fixed paraffin embedded whole sections. We quantitated the subsets of immune-cells utilizing a targeted panel of proteins including PanCK, CD8, CD4, VISTA and IBA-1, and analyzed an average of 15,000 cells per sample. Classifying tumors as either high (>30%) or low (<30%) TILs, we found that increased TILs density correlated with survival. Phenotyping these TILs we found tumors with low TILs had significantly higher expression of the immune-checkpoint molecule VISTA in tumor cells (p < 0.01) as well as in their microenvironment (p < 0.001). Contrastingly, the tumors with high TILs displayed higher levels of microglia, as measured by IBA-1 expression. Low TILs-tumors displayed CD8+ T-cells that co-express VISTA (p < 0.01) significantly more compared to high TILs group, where CD8+cells significantly co-express IBA-11 (p < 0.05). These results were supported by RNA analysis of a publicly available, independent cohort. Our work contributes to a growing understanding of the immune surveillance escape routes active in brain metastasis.