Characterization and Functional Analysis of Macrophages in Tumour-Draining Lymph Nodes

Abstract

Metastasis to lymph nodes (LNs) represents a critical point in the progression of the disease, promoting spread to distant organs and systemic immunosuppression. Consequently, metastatic LNs have emerged as potential targets for enhancing the efficacy of immunotherapies. The role of macrophages in metastatic LNs remains largely uncharacterized, presenting a significant gap in our understanding of cancer metastasis and immune evasion. In primary tumours, tumour-associated macrophages (TAM) are predominantly associated with worsening clinical outcomes. Hence, understanding macrophage subsets and their roles in metastatic LNs could provide new insights into their roles in cancer progression and immune response to cancer. We leveraged spatial transcriptomics which revealed an abnormal accumulation of SPP1+ macrophages surrounding tumorous areas in breast cancer sentinel LNs. Single-cell RNA-sequencing of myeloid cells in sentinel LNs revealed five macrophage subsets including SPP1+ macrophages. Immunofluorescence on sentinel LN sections validated these findings on a protein level. In vitro co-culture models of cancer cells and macrophages suggested that direct cell-cell communication influences SPP1 expression in macrophages. Additionally, recombinant SPP1 enhanced T-cell glycolysis and oxidative phosphorylation, and downregulated CD44 expression in activated T cells, potentially impacting T cell metabolism and function within metastatic LNs. Our studies characterized macrophage heterogeneity within sentinel LNs at a single-cell level and revealed SPP1+ macrophages adjacent to tumour regions. We conclude that SPP1 from TAMs may modulate T cell metabolism in metastatic LNs, thereby fine-tuning the immune response against cancer. Further functional investigations into LN TAMs are crucial for elucidating their roles in cancer progression, metastasis, and immune evasion.