Single-cell analysis reveals immune modulation and metabolic switch in tumor-draining lymph nodes

Abstract

ABSTRACT Lymph-node metastasis is a prognosis factor for poor clinical outcome of breast cancer patients. Currently, how breast cancer cells establish pre-metastatic niche in the tumor-draining lymph nodes (TDLNs) is still unclear. To address this question, we isolated heterogeneous cells including immune and stromal cells from naive lymph nodes (LNs) of the FVB/NJ mice and TDLNs of the MMTV-PyMT mice. Single-cell RNA sequencing was performed to investigate the transcriptome of the cells and various bioinformatics analyses were used to identify the altered pathways. Our results revealed several significant changes between naïve LNs and TDLNs. First, according to immunologic signature and pathway analysis, CD4+ and CD8 + T cells showed upregulated angiogenesis pathway genes and higher regulatory T (Treg)-associated genes while they demonstrated downregulation of interferon response and inflammatory response gene signatures, concurrently suggesting an immunosuppressive microenvironment in the TDLNs. Second, profiling of B cells showed down-regulation of marginal zone B lymphocytes in the TDLNs, which was validated by flow cytometric analysis. Third, we found the enhancement of oxidative phosphorylation pathway in the fibroblastic reticular cells (FRCs) of the MMTV-PyMT mice and the elevation of related genes including Prdx3, Ndufa4 and Uqcrb, suggesting massive ATP consumption and TCA cycle metabolism in the FRCs. Collectively, our results reveal the reprogramming of TDLNs during breast cancer progression at single-cell level in a spontaneous breast cancer model and suggest the changes in immune modulation and metabolic switch are key alterations in the preparation of pre-metastatic niche by breast cancer cells.