Molecular mechanisms underlying breast cancer lymph node metastasis remain unclear. Using single-cell sequencing, we investigated the transcriptome profile of 96,796 single cells from 15 paired samples of primary tumors and axillary lymph nodes. We identified nine cancer cell subclusters including CD44 + / ALDH2 + /ALDH6A1 + breast cancer stem cells (BCSCs), which had a copy-number variants profile similar to that of normal breast tissue. Importantly, BCSCs existed only in primary tumors and evolved into metastatic clusters infiltrating into lymph nodes. Furthermore, transcriptome data suggested that NECTIN2-TIGIT-mediated interactions between metastatic breast cancer cells and tumor microenvironment (TME) cells, which promoted immune escape and lymph node metastasis. This study is the first to delineate the transcriptome profile of breast cancer lymph node metastasis using single-cell RNA sequencing. Our findings offer novel insights into the mechanisms underlying breast cancer metastasis and have implications in developing novel therapies to inhibit the initiation of breast cancer metastasis.

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The potentially different genetics and epigenetics in the primary tumors and metastases affect the efficacy of treatment in breast cancer patients. Nevertheless, the cellular and molecular mechanisms of breast cancer lymph node metastasis still remain elusive. Here, we employed single-cell RNA sequencing (scRNA-seq) to acquire the transcriptomic profiles of individual cells from primary tumours, negative, and positive lymph nodes. We also performed a single-cell assay for transposase-accessible chromatin (ATAC) sequencing (scATAC-seq) of the positive and negative lymph node samples to get the chromatin accessibility profile. We identified a novel cell subpopulation with an abnormally high expression level of CXCL14 in the positive lymph node of breast cancer patients. Cell trajectory analysis also revealed that CXCL14 was increased expressed in the late pseudo-time. Moreover. Based on a tissue microarray of 55 patients and the Oncomine database, We validated that CXCL14 expression was significantly higher in breast cancer patients with lymph node metastasis. Furthermore, scATAC-seq identified several transcription factors (TFs) that may be potential regulation factors for the lymph node metastasis of breast cancer. Thus, our findings will improve our current understanding of the mechanism for lymph node metastasis, and are potentially valuable in providing novel prognosis markers for lymphatic metastasis of breast cancer.