Breast cancer (BC) is the most frequently diagnosed neoplasm and the leading cause of cancer mortality among women [1]. In 2022, there will be estimated 290,560 new cases and 42,780 cancer deaths due to BC in the USA [2]. BC represents the most important cancer-related cause of disease burden worldwide, especially in developed countries [3]. Despite the continuous improvement of BC treatments in recent years, the survival of advanced BC is still not ideal. Lymph node metastasis is one of the main pathways of tumor metastasis, especially for breast tissue with abundant lymphatic vessels and lymphatic network [4]. The metastatic cancer cells in the lymph nodes can directly enter other lymphatic vessels and then spread widely in the body [5]. Lymph node metastasis is of great significance to BC’s prognosis, indicating the worsened prognosis [6]. Lymph node metastases is a complex biological process affected by a complex gene regulatory network and various growth factors, involving tumor movement, vascular invasion, and clonogenicity in the microenvironment [7]. In the process of lymph node metastases, lymph nodes provide a supportive environment for tumor cells of a specific genetic background, supporting their clonal growth and further distant metastasis [8].

The process of lymphatic metastasis in breast cancer has been found to be associated with a specific type of macrophages expressing podoplanin [9]. Macrophages display two different phenotypes in response to different environmental stimuli: M1 and M2 macrophages. M2 macrophages can produce large amounts of cytokines that cause Th2-type immune responses, suppressing immune function in the tumor microenvironment, inducing angiogenesis, and supporting tumor growth and metastasis [10]. In the tumor microenvironment, the majority of tumor-associated macrophages (TAMs) are of the M2 phenotype [11]. Increased numbers of M2 macrophages have been significantly correlated with lymph node metastasis, larger tumor size, poor differentiation of cancer cells, and an elevated risk of recurrence in BC [12]. The experimental data of Watari et al. showed that M2 macrophages correlated with lymph node metastasis in highly metastatic cancer [13]. Therefore, it is crucial to further investigate the biological characteristics of M2 macrophages in the context of breast cancer lymph node metastasis. Understanding their role and mechanisms in promoting metastasis could potentially lead to the development of targeted therapies.

With the development of sequencing technology, single-cell RNA sequencing (scRNA-seq) has brought new data at the cellular level to researchers, enriching the tools for single-cell analysis of tumors. The scRNA-seq enables researchers to explore the different biological properties of individual cells in complex tissues and understand the response of cell subpopulations to environmental elements. It has provided new solutions to various omics-related problems in the life sciences, and the related research has become increasingly popular. In this study, we collected a set of single-cell sequencing data (GSE158399) to investigate the role of M2 phenotype of tumor-associated macrophages and mine the key M2 macrophages-related genes for lymph node metastasis in breast cancer.

Lymph node metastasis is a common problem in breast cancer, which seriously affects the survival and prognosis of patients. Therefore, it has become increasingly urgent to understand its pathogenesis and find a specific and sensitive treatment method. The mobility and invasiveness of cells are the keys to tumor metastasis, and the movement and migration of tumor cells play a leading role in the whole process of tumor metastasis. The study conducted by Watari et al. [18] stated that inflammatory stimuli could help to establish the tumor microenvironment, and the growth, invasion, and matastases could be induced through the activation of macrophages. In our study, we observed a fundamental distinction between samples obtained from primary tumors and lymph nodes. The single-cell analysis of lymph node samples revealed a predominant presence of immune leukocytes, whereas immune leukocytes constituted only a small portion of the primary tumor samples. Further classification of cell types based on single-cell data demonstrated that the majority of cells in the primary tumors were macrophages expressing the CD68 marker, particularly M2 macrophages expressing FCGR2B. Additionally, Mahmoud et al. [19] discovered that higher numbers of CD68+ macrophages were associated with lower overall survival rates in breast cancer patients. This finding underscores the potential significance of M2 macrophages in lymph node metastasis and its clinical implications.

Tumor-associated macrophages (TAMs) play a key role in the growth of breast cancer. The role of TAMs in cancer pathogenesis depends on their phenotypic and functional polarization [20]. Tumor cells can disguise as normal cells to deceive macrophages and inhibit the phagocytosis of tumor cells by macrophages [21]. Studies have shown that the high infiltration of TAMs is essential in promoting breast cancer cell metastasis. The infiltration density of TAMs in primary breast tumor tissue is significantly higher than in adjacent tissue. At the same time, the infiltration of TAMs in primary breast tumor tissue increases with tumor stage and size [22]. Previous studies have shown that infiltration of macrophages in the tumor microenvironment supported tumor growth, angiogenesis, metastasis/invasion, inflammation, and immunosuppression by secreting factors that promote tumor progression [23]. TAMs are heterogeneous populations with different subsets performing different functions [24]. TAMs primarily exhibit a phenotype resembling alternatively activated M2 macrophages, which possess anti-inflammatory properties and promote tumor growth. M2-type macrophages largely contribute to the aggressiveness of malignant tumors [18]. TAMs located in primary tumors are mostly associated with poor prognosis in cancer patients. In this study, we found that M2 macrophages mainly aggregated in breast cancer lymph node metastasis-positive samples, and M2 macrophages in orthotopic tumor tissue had stronger migration and activation ability. This phenomenon suggests to us that M2 macrophages play an important role in promoting lymph node metastasis in patients. Depletion or phenotypic reversal (M2 to M1) of TAMs has been shown to halt tumor progression in mouse models of breast cancer [25]. In order to reveal the biological role of M2 macrophages in the process of lymph node metastasis, we analyzed the differences of M2 macrophages from three samples and annotated their functions. The results showed that M2 macrophages may help tumor cells complete metastasis through phagosomes, and M2 macrophages may also have immune function defects. New clinical research suggested that an immunodeficiency of macrophages 22 may cause Crohn’s disease (CD). The reason for granulomas is the reduced ability to clear invading bacteria induced by a weakened attraction of granulocytes to the intestinal wall. So far, there are few reports on whether M2 macrophages promote tumor cell metastasis through phagosomes and immunodeficiency.

Based on the above analysis, we analyzed the difference between the gene sets of “gobp_macrophage_activation_ involvedin_immune_response” and “gobp_macrophage_ migration” in M2 macrophages, respectively. The previous study analyzed the data in GSE158399 and found the elevated expression level of CXCL14 in PLs, which might be valuable in predicting the prognosis of breast cancer with lymph node metastasis [23]. In this study, we found that SUCNR1, C3aR1, C5aR1, MMP14, THBS 1, TSP-1, and TREM2, which are abnormally expressed in tumor tissue in situ, may be potentially associated with lymph node metastasis.

Keiran et al. [26] discovered that the activation of SUCNR1 plays a crucial role in promoting the anti-inflammatory phenotype of macrophages, thus contributing to the anti-inflammatory response. It is demonstrated that Succinate and its receptor SUCNR1 can suppress immune responses. Moreover, the receptor SUCNR1 can also suppresse immune responses, and SUCNR1 deficiency in macrophages could lead to enhanced inflammatory responses [27]. C3AR1 was down-regulated in osteosarcoma tissues and cells, and its overexpression inhibited the proliferation, migration, and invasion of osteosarcoma cells and induced apoptosis [28]. These findings may appear contradictory to our conclusions, but they emphasize that the interaction between C3AR1 and cell migration can have divergent roles in different types of cancer. In addition, C5α can also promote the malignant development of HBc-positive hepatocellular carcinoma through C5AR1 [29]. Matrix metalloproteinases (MMPs) are key factors in extracellular matrix remodeling and cell migration during tumor metastasis. In particular, MMP-14, a membrane-anchored MMP, is closely involved in these processes. The study found that Bladers I and IV were associated with cell migration. Bladers IV is required for MMP-14 homodimerization. The interaction between MMP-14 and CD44 leads to phosphorylation of the EGF receptor and downstream activation of MAPK and PI3K signaling pathways involved in cell migration [30]. Reports provide evidence [31] that THBS1 derived from oral squamous cell carcinoma (OSCC) exosomes is involved in the polarization of macrophages towards an M1-like phenotype.

In contrast, conditioned medium from exosomes induced M1-like TAMs and significantly promoted the malignant migration of OSCCs. Perturbation of macrophage migration inhibitory factor expression in mouse melanoma suppresses tumor formation by up-regulating Thrombospondin-1 (TSP-1) [32]. TSP-1 is a secreted protein that inhibits angiogenesis, modulates anti-tumor immunity, stimulates tumor cell migration, and modulates extracellular proteases and growth factors in the tumor microenvironment. Furthermore, in polyomavirus middle T antigen (Pyt) transgenic mice, TSP-1 in the mammary tumor microenvironment inhibits angiogenesis and tumor growth, yet promotes lung metastasis in Pyt transgenic mice [33]. Research has shown that targeting TREM2 on tumor-associated macrophages enhances immunotherapy [34].

However, there are potential limitations in this study. Firstly, the primary breast cancer tissues, PLs, and NLs in the GSE158399 dataset were from one patient with Luminal B subtype breast cancer, which may restrict the generalizability of the findings. Secondly, the current mainstream tissue sequencing methods may not effectively detect the key genes associated with M2 macrophages. Therefore, there could be additional genes involved in the M2 macrophage phenotype that were not identified in this study. Lastly, further investigations are needed to validate and expand upon the interesting findings presented here. In conclusion, this study found that M2 macrophages play an important role in promoting lymph node metastasis in breast cancer patients, possibly through immune activation and phagosomes to help tumor cells complete metastasis, thus exerting their biological function. In addition, we also discovered key genes that M2 macrophages significantly up-regulated in immune response and cell migration to discover key molecular targets regulating breast cancer metastasis and providing new strategies for the prevention and treatment of breast cancer metastasis. These results may have important implications for understanding the mechanism of lymph node metastasis in breast cancer.