Defining the perivascular tumour microenvironment after neoadjuvant chemotherapy within triple negative breast cancer and correlation to metastasis

Background: Triple negative breast cancer (TNBC) remains a formidable challenge due to its high metastatic potential, limited treatment options and poor survival rates. In mouse models of cancer, tumour associated macrophages (TAMs) have been shown to accumulate around tumour blood vessels, where they promote tumour angiogenesis, immunosuppression and metastasis, reduce the efficacy of frontline anti-cancer treatments, and drive relapse.
Methods: Multiplex immunofluorescence staining and AI-based image analysis were used to quantify TAMs (and other immune cells) in perivascular (PV) and non-PV areas of human TNBCs in both untreated and neoadjuvant chemotherapy (NAC)-treated TNBCs. This was assessed in both tumour cell islands and the tumour stroma. Follow-up data for a 3-year period after surgery enabled these immune features to be correlated with metastasis. An ex vivo microfluidics assay was also established to model the PV features observed in TNBC sections. This used human endothelial cells to create perfusable microvessels in a fibrin gel, fibroblasts, cancer cells and macrophages.
Results: In tumors from patients who developed metastases after NAC, there were significantly lower levels of stromal CD163+ TAMs (especially those expressing the
negative checkpoint regulator, T-cell immunoglobulin and mucin domain 3, ‘TIM-3’) than in those from disease-free patients. Although these cells were predominantly PV in tumours, significant differences in their density between the above 2 patient groups were only seen in non-PV areas of the stroma. CD4+ T cells (but not CD8+ T cells) preferentially located to PV areas in all TNBCs, whereas Tregs only did so only after NAC. Distinct subsets of CD4+ and CD8+ T cells formed PV clusters with CD163+ TAMs and Tregs which were retained after NAC.
Conclusions: The density of stromal TIM-3+ CD163+ TAMs may represent a new biomarker in NAC-treated TNBC to identify patients at a higher risk of relapse.