Preliminary characterisation of the spatial immune and vascular environment in triple negative basal breast carcinomas using multiplex fluorescent immunohistochemistry

Triple negative breast cancers often contain higher numbers of tumour-infiltrating lymphocytes compared with other breast cancer subtypes, with their number correlating with prolonged survival. Since little is known about tumour-infiltrating lymphocyte trafficking in triple negative breast cancers, we investigated the relationship between tumour-infiltrating lymphocytes and the vascular compartment to better understand the immune tumour microenvironment in this aggressive cancer type. We aimed to identify mechanisms and signaling pathways responsible for immune cell trafficking in triple negative breast cancers, specifically of basal type, that could potentially be manipulated to change such tumours from immune "cold" to "hot" thereby increasing the likelihood of successful immunotherapy in this challenging patient population. We characterised the spatial immune environment in 10 basal breast cancers showing a range of tumour-infiltrating lymphocytes using multiplex fluorescent immunohistochemistry and quantitative digital analysis of CD3+ T cells. We examined their relationship to blood vessels and their activation status as defined by VCAM-1, ICAM-1 and PD-L1. Confirmation of the relationship between tumour-infiltrating lymphocytes and endothelial activation was performed through in silico analysis on TCGA BRCA RNA-seq data (N = 808). Significantly higher CD3+ T cell densities were observed in the stromal compartment compared with the neoplastic cell compartment (P = 0.003). ICAM-1 activated blood vessels were spatially associated with higher CD3+ T cell densities only within 30 microns of blood vessels compared with more distal activated and non-activated blood vessels (P = 0.041). In silico analysis confirmed higher numbers of tumour-infiltrating lymphocytes in basal breast cancers and that higher numbers were significantly associated with endothelial cell activation molecules, co-clustering with upregulated ICAM-1 and VCAM-1 amongst others. PD-L1 was also identified in a subset of blood vessels, suggesting an additional immune regulatory mechanism in endothelial cells. Regulating the activation status of tumour-associated vascular endothelial cells may improve T cell trafficking into basal breast tumours and enhance immunotherapeutic response.