Endothelial Cells Potentially Participate in the Metastasis of Triple-Negative Breast Cancer

Inhibition of triple-negative breast cancer metastasis has long been a challenge, mainly due to the difficulty in identifying factors that contribute to this process. In this study, freshly isolated triple-negative breast cancer biopsied cells obtained from consenting patients were subjected to flow cytometry and bioinformatic analysis to identify three endothelial cell subclusters: EC (ATP1B3), EC (HSPA1B), and EC (KRT7) in the tumor microenvironment. These endothelial cell subclusters exhibited distinguishing biological features. Based on differentially expressed genes derived from the subclusters, gene set enrichment analysis showed that EC (ATP1B3) and EC (HSPA1B) contribute to the process of metastasis, for example, in fibrosarcoma and anaplastic carcinoma. In this study, we identified the heterogeneity of endothelial cells in the human breast cancer and have provided insights into its role in metastasis.


Introduction
Triple-negative breast cancer (TNBC) is characterized by low or no expression of the progesterone, oestrogen, and human epidermal growth factor 2 receptors [1]. Approximately 20% of the breast cancer patients are diagnosed with TNBC [2,3]. Current therapies against TNBC include surgery, chemotherapy, and/or immune therapy, and although this has led to decreased mortality, numerous patients still undergo cancer metastasis to other organs, such as the brain, lung, and bone [2][3][4], resulting in a drastic loss of therapeutic efficiency and quality of life. The processes underlying TNBC metastasis remain unclear due, in part, to the complexity of this process [3].
The tumor microenvironment (TME) participates in tumor cell proliferation, apoptosis, and migration [5], all of which are closely related to metastasis. The cellular components in the TME include monocytes, macrophages, neutrophils, B cells, and endothelial cells [6][7][8]. Monocytes facilitate the breast tumor metastasis by CCL2-induced recruitments [9], and macrophages bridge the tumor cellextracellular matrix towards metastasis by secreting SPARC [10]. Neutrophils promote metastasis by forming extracellular traps [11,12] and B-lineage cells contribute to metastasis by upregulating STX16 and ATIC [7]. Although endothelial cells have long been considered a key cell type in tumor initiation and progression [13], their role in TNBC metastasis has yet to be fully elucidated.
The endothelial cells are heterogenous in both healthy and pathological conditions [14][15][16][17].Exploring the heterogeneity of endothelial cells in TNBC would help to understand their role in metastasis. In this study, three endothelial cell subpopulations were identified from TNBC biopsies, two of which could contribute to metastasis to fibrosarcoma and anaplastic carcinoma.

Materials and Methods
2.1. Human Biopsies. Biopsies were isolated from TNBC patients underwent surgery. After pathological examination, the residual biopsies were processed either for flow cytometry analysis or immunofluorescent staining. This study was approved by the Ethics Committee of Youjiang Medical University for Nationalities.

Identification of Endothelial Cells in the Human
Breast and TNBC Biopsied Tissues. In the freshly isolated malignant cells from patients with TNBC, a significant proportion of endothelial cells (CD31 + ) were detected (Figure 1(a)). To detect whether they could be divided into subpopulations, the expression of ATP1B [19], HSPA1B [20], and KRT7 [21] were evaluated. Interestingly, TNBC endothelial cells could be further divided into three subpopulations: EC (ATP1B3), EC (HSPA1B), and EC (KRT7) (Figure 1(a)). To compare their genetic profiles, the scRNA-Seq data of normal human breast tissues and TNBC tissues were retrieved [18]. Overall, nine cell types were identified in normal tissues and 10 in TNBC tissues (Figure 1(b)). Three markers, CD93, CDH5 (coding), and PECAM1, were used to isolate endothelial cells (Figure 1(c), Supplementary  Figure 1) [22]. The fifth population in normal breast tissue and the ninth population in TNBC tissues were endothelial cells (Figures 1(b) and 1(c)). The lack of expression of PTPRC (encoding CD45) further suggested that these two populations did not belong to leukocytes (Figure 1(c), Supplementary Figure 1). Flow cytometry analysis showed that TNBC endothelial cells constitutively expressed CDH5 and CD93 (Figure 1(d)).

The Human Breast and TNBC Endothelial Cells Are
Heterogeneous. To compare the biological features of endothelial cell subpopulations, isolated endothelial cells from the scRNAN-seq experiment were evaluated. The 2 Journal of Immunology Research  3 Journal of Immunology Research integration of the human breast and TNBC endothelial cells revealed three subclusters (Figure 2(a)). Interestingly, ATP1B3, HSPA1B, and KRT7 were preferentially expressed in three endothelial cell subpopulations (Figure 2(b)), which is consistent with the flow cytometry analysis (Figure 1(a)).
To determine distribution pattern of these 3 endothelial cell subpopulations, we performed immunofluorescent staining. Obviously, these 3 subpopulations preferentially exist in different regions of TNBC biopsies, which could be indicated by DAPI staining (Figure 2(c)). In total, 292 genes were expressed at higher levels in EC (ATP1B3), 505 were higher in EC (HSAP1B), and 118 genes were expressed at higher levels in EC (KRT7), compared among these three subpopulations (Supplementary Figure 2). The heat map shows a brief view of the similarity/disparity of the top 20 genes from the three subpopulations (Figure 2 [23][24][25]; EC (HSPA1B) could promote in antigen presentation and MAPK signalling and participates in mediating rheumatoid arthritis [26] and toxoplasmosis [27] in the breast cancer patients; and EC (KRT7) could be more important for mediating leukocyte transendothelial migration and thyroid hormone signalling. Interestingly, EC (ATP1B3) exhibited higher expression of phagocytosis-related genes in TNBC patients, RAB5A and EEA1 (Supplementary Figure 4), indicating these patients could be more susceptible to infection.

Endothelial Cell Subpopulations Contribute to TNBC
Metastasis. In comparison to the normal human breast endothelial cell subpopulations, integrated scRNA-seq data revealed that the distribution and frequencies of these subpopulations were different (Figures 3(a) and 3(b)). The proportion of subpopulation EC (ATP1B3) increased significantly (Figure 3(b)    Journal of Immunology Research ( Figure 3(c)). Unfortunately, the DEGs from EC (KRT7) could not be calculated because of their extremely low numbers. GSEA analysis revealed that most of the pathways were downregulated in TNBC endothelial cell subpopulations compared to the normal human breast tissues, such as IL-17 signalling (including IL17RA, TRAF3IP2, GSK3B, and NFKB1), TNF signalling (including TNFR1, TRADD, TAB2, and RIP1), and NOD-like receptor signalling (including NFR1, NFR5, and NIN) (Figure 3(d)), all of which are highly related to the development of cancer [28,29]. Moreover, EC (HSPA1B) in the TNBC tissues exhibited altered fluid shear stress compared to the normal human breast tissues ( Figure 3(d)), which participates in tumor metastasis [30]. Gene concept network analysis of the DEGs abstracted from the above two endothelial cell subpopulations revealed that in TNBC, EC (ATP1B3) could contribute to TNBC metastasis to fibrosarcoma, hereditary diffuse gastric cancer, and anaplastic carcinoma (Figure 4(a)), while EC (HSPA1B) could participate in TMBC metastasis of fibrosarcoma and anaplastic carcinoma (Figure 4(b)).
However, the potential role of EC (KRT7) in TNBC metastasis has not been studied because of extreme cell events. Further studies should be conducted to bridge this gap. Moreover, TNBC metastasis should be evaluated with relevant genetic modified animal models in further studies.