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As an indicator of clinical prognosis, lymph node metastasis of breast cancer has drawn great attention. Many reports have revealed the characteristics of metastatic breast cancer cells, however, the effect of breast cancer cells on the microenvironment components of lymph nodes and spatial transcriptome atlas remains unclear. In this study, by integrating single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics, we investigate the transcriptional profiling of six surgically excised lymph node samples and the spatial organization of one positive lymph node. We identify the existence of osteoclast-like giant cells (OGC) which have high expressions of CD68 and CD163, the biomarkers of tumor-associated macrophages (TAMs). Through a spatially resolved transcriptomic method, we find that OGCs are scattered among metastatic breast cancer cells. In the lymph node microenvironment with breast cancer cell infiltration, TAMs are enriched in protumoral pathways including NF-κB signaling pathways and NOD-like receptor signaling pathways. Further subclustering demonstrates the potential differentiation trajectory in which macrophages develop from a state of active chemokine production to a state of active lymphocyte activation. This study is the first to integrate scRNA-seq and spatial transcriptomics in the tumor microenvironment of axillary lymph nodes, offering a systematic approach to delve into breast cancer lymph node metastasis.

Aims: This study investigates the association between circulating tumor cells (CTCs) and breast cancer metastasis. Methods: A retrospective study was conducted using patients with histologically confirmed breast cancer recruited from the First Affiliated Hospital of Nanjing Medical University during the period of August 2017–October 2020. We used adjusted logistic regression, the random forest algorithm, and sensitivity analysis to study the association between CTC enumeration and tumor metastasis. Further, we performed next-generation sequencing (NGS) on the CTCs obtained from two patients with breast cancer brain metastasis. Results: A total of 41 out of 116 enrolled patients were identified with tumor metastasis. CTC enumeration was significantly higher in patients with liver metastasis than in those without liver metastasis. Patients with CTCs ≥ 5 exhibited a higher risk of tumor metastasis than those with CTCs < 5 in the adjusted model (odds ratios (OR) = 6.25, 95% confidence interval (CI) = 2.63–15.58). The random forest model identified CTC enumeration as a significant metastasis-related variable with the highest mean decrease accuracy and mean decrease Gini score. No significant association was found between CTCs and visceral metastasis with an OR of 1.29 (95% CI = 0.98–2.05, p = 0.232). Upon further investigating organ-specific metastasis, we found that patients with high CTC levels were more likely to develop liver metastasis (OR = 4.87, 95% CI = 1.34–20.17, p = 0.021). The NGS study of CTCs identified a total of 120 indel mutations (e.g., CNGB1, NTSR1, ZG16). The enriched biological processes were mechanoreceptor differentiation and macrophage activation involved in the immune response. The enriched KEGG pathways included focal adhesion, the PI3K-Akt signaling pathway, and microRNAs involved in cancer. Conclusions: Our study revealed that CTCs ≥ 5 are a risk factor for tumor metastasis in breast cancer patients. In addition, we reported that CTCs ≥ 5 might be associated with a higher risk of liver metastasis in patients with metastatic breast cancer. We have provided the mutational profiles of CTCs based on next-generation sequencing. [ABSTRACT FROM AUTHOR]

[Display omitted] • Circulating tumor cells are pro-metastasis precursors. • Heterogeneity of circulating tumor cells associated with breast cancer progression. • Genetic properties of circulating tumor cells affect distant metastases. • Components secreted by tumor microenvironment guide circulating tumor cells to specific metastatic sites. Circulating tumor cells (CTCs) are viewed as pro-metastasis precursors shed from primary tumors or metastatic sites. The phenotypic and molecular heterogeneity of CTCs is associated with breast cancer progression and prognosis. Therefore, we divided CTCs into several subtypes according to their differences in biomarker status, epithelial/mesenchymal phenotype, aggregation status, and other factors to summarize their characteristics. Considering that the organ-specific metastasis is a hallmark of breast cancer, we adopted the "seed and soil" model to further analyze the relationship between the heterogeneity of CTCs and the organotropism of breast cancer. We speculated that CTCs might not only develop their genetic potential but communicate with surroundings, including chemokine systems, hemocytes, and extracellular matrix components, to regulate the organ-specific metastases of breast cancer. [ABSTRACT FROM AUTHOR]