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

SIMPLE SUMMARY: Brain metastases arising from breast cancers, occur in about 20% of women with a poor-survival outcome. Unfortunately, most patients survive only up to eighteen months from diagnosis. Therefore, there is an urgent need to understand how these cancers survive in the brain. It is thought that the immune cells in the brain, together with brain resident cells, may provide a favorable environment for cancer growth. However, this is not very well understood at this point. We aimed to profile the cells found in these tumors, focusing on five different cell types based on the marker expressed by them. Our results indicate that certain molecules contained within the cancer and the surrounding environment are associated with poor survival. This suggests that these molecules might be important in brain metastasis. This finding is a step towards our understanding of how some patients with brain metastasis survive longer than others. ABSTRACT: 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.