Cancer stem cells promote lymph nodes metastasis of breast cancer by reprogramming tumor microenvironment

Abstract

Breast cancer progression and metastasis are governed by a complex interplay within the tumor immune microenvironment (TIME), involving numerous cell types. Lymph node metastasis (LNM) is a key prognostic marker associated with distant organ metastasis and reduced patient survival, but the mechanisms underlying its promotion by breast cancer stem cells (CSCs) remain unclear. Our study sought to unravel how CSCs reprogram TIME to facilitate LNM. Utilizing single-cell RNA sequencing, we profiled TIME in primary cancer and corresponding metastatic lymph node samples from patients at our institution. To verify the derived data, we cultured CSCs and performed validation assays employing flow cytometry and CyTOF. Our analysis revealed distinct differences in cellular infiltration patterns between tumor and LNM samples. Importantly, RAC2 and PTTG1 double-positive CSCs, which exhibit the highest stem-like attributes, were markedly enriched in metastatic lymph nodes. These CSCs are hypothesized to foster metastasis via activation of specific metastasis-related transcription factors and signaling pathways. Additionally, our data suggest that CSCs might modulate adaptive and innate immune cell evolution, thereby further contributing to metastasis. In summary, this study illuminates a critical role of CSCs in modifying TIME to facilitate LNM. The enrichment of highly stem-like CSCs in metastatic lymph nodes offers novel therapeutic targeting opportunities and deepens our understanding of breast cancer metastasis.

Keywords: Breast cancer 1; Cancer stem cells (CSCs) 2; Lymph node metastasis 5; Single-cell RNA sequencing (scRNA-seq) 4; Tumor immune microenvironment (TIME) 3.

Graphical abstract

Fig. 1

Global cellular landscape of primary breast cancer and paired lymph node metastases. (A) Single cell map mapping cluster. (B) Single cell map mapping PTPRC. (C) Single cell map shows malignant cells-stromal cells-immune cells. (D) Single cell map maps cell cluster CellType. (E) Bubble chart shows CellMarker of cell type. (F) The bar chart shows the abundance proportion of each cell type in each patient. (G) Multi-group volcano map shows differential genes in primary and paired lymph node metastases of different cell types of breast cancer. (H) Single cell map shows Cluster of malignant cells. (I) Single cell map maps the characteristic marker genes ACTA2 of malignant cell subgroups in patients.

Fig. 2

Cloning and evolution of breast CSCs in primary breast cancer and lymph node metastases. (A) Bubble chart shows the biological function of significant activation of malignant cell subgroups. (B) Single cell map shows tumor stemness score of malignant cells. The violin map shows the difference of the stemness score of malignant cell tumor between primary tumor and lymph node metastasis. (C) The single cell map shows the pseudotime of the clonal evolution of malignant cells. (D) Single cell map-pie chart mapping cell subgroups show the track of clonal evolution of malignant cells. (E) Heatmap-Motif-TFs-cell cluster shows the GRN module of malignant subclones. (F) The ring network diagram shows the cellular communication events of breast cancer stem cells to malignant subclones.

Fig. 3

Reprogramming of adaptive immune cells by breast cancer stem cells in primary breast cancer and lymph node metastases. (A) Single cell map shows CD8+ T cell Cluster. (B) Bubble chart shows the biological function of significant activation of CD8+ T cell subgroups. (C) Display of B cell Cluster by single cell map. (D) The ring network diagram shows the cellular communication events between breast cancer stem cells and B cell subgroups.

Fig. 4

CSCs promote the formation of innate immune cells in primary breast cancer and paired lymph node metastasis. (A) Single cell map shows Macrophage Cluster. (B) Heatmap-Motif-TFs- cell cluster shows the gene regulatory network (GRN) module of Mac cell subgroups. (C) Single cell map-pie chart mapping MDDC cell subgroups show the track of phenotypic transformation of MDDC cell subgroups. (D) The ring network diagram shows the cellular communication events between breast CSCs and MDDC subgroups.

Fig. 5

CSCs cultured in MDA-MB-231 and MDA-MB-468 cell lines showed strong stemness performance. (A) Two groups of human breast cancer cell lines, MDA-MB-231 and MDA-MB-468, were selected to culture CSCs after cell resuscitation and cell passage. (B) The expressions of CD133, CD44 and CD24 in the two groups of cell lines were detected, respectively. (c) The expressions of SOX2, OCT4, EPCAM, ALDH1, NANOG and VIMENTIN in the two groups of cell lines were detected, respectively.

Fig. 6

Transfer-related proteins were highly expressed in CSC group of MDA-MB-231 cell line. (A) t-SNE showed the distribution of all cells in the MDA-MB-231 cell line of control group and the CSC group, and all cells were divided into 10 clusters. (B) Data were repeated three times after integration. (C) Data analysis showed the distribution of each subcluster in the control group and the CSC group. (D) Scatter plots show the enrichment of each subcluster in control group and CSC group. (E) The heatmap shows the expression of 34 markers in 10 subclusters. (F) In cluster 3, the expressions of PSTAT3, CXCR3, TIM3, VIMENTIN, SOX2, CD44 and ALDH1 were increased. In cluster 6, CXCR3 and TIM3 expression levels were increased.