Breast cancer (BC) has emerged as the most prevalent malignancy among women worldwide, with a mortality rate that ranks second only to lung cancer. It is estimated that there will be approximately 43,170 deaths (6.6%) in the United States in 2023 (Siegel et al., 2023). From 2008 to 2022, the global incidence of BC experienced a significant increase of 20%, while the mortality rate rose by 14% (Sher et al., 2022). This alarming trend is reflected in the estimated global burden of BC, which surged to 2.26 million new cases in 2020, a substantial increase from the nearly 1.7 million cases recorded in 2012 (Torre et al., 2017; Sung et al., 2021).

A growing body of research highlights the critical role of the tumor microenvironment (TME) in the development and progression of BC (Denkert et al., 2018; Pruneri et al., 2018). The TME is a complex ecosystem composed of tumor cells, stromal cells, immune cells, metabolites, and secreted proteins. Recent advances in TME research have revealed that the remodeling of metabolism within TME also plays a pivotal role in suppressing tumor immunity. For instance, the excessive production of metabolic by-products can disrupt the metabolic rewiring of T cells, thereby impairing their anti-tumor function (Park et al., 2023). Therefore, investigating the immune cells and metabolites that causal influence the risk of BC is of paramount importance. Such exploration is essential for elucidating the mechanisms underlying the immune microenvironment in the progression of BC.

Traditionally, meticulously planned randomized controlled trials (RCTs) have been considered as the gold standard for establishing causal relationships between exposure factors and outcomes. However, their implementation often requires ethical approval and extensive follow-up, making them complex and resource-intensive (Smylie et al., 2024). To efficiently identify potential disease-exposure relationships during the exploratory stage, Mendelian randomization (MR) analysis has emerged as a powerful epidemiological tool. MR is commonly employed to assess the causal relationship between exposure factors and outcomes by utilizing genetic variants, known as single nucleotide polymorphisms (SNPs), as instrumental variables (IVs) (Birney, 2022). These IVs act as effective proxies for observed exposures, providing more robust estimates of causality compared to traditional observational studies (Fang et al., 2024). Recent MR studies have demonstrated that SNPs related to immune cells can serve as exposure factors influencing tumor development (Bouras et al., 2022; Yu et al., 2022; Aru et al., 2023; Yin et al., 2023). However, the relationship between immune cells, metabolites, and BC has not been explored using MR.

In this study, MR was employed to identify immune cells significantly impacting BC incidence. Subsequently, reverse MR was applied to determine that BC did not affect the levels of these immune cells. Next, the SNPs related to metabolites were selected as intermediaries. Finally, through mediation analysis, we discovered that IgD- CD38⁺ B cells could elevate the risk of BC by influencing Succinoyltaurine and Glycerate.

With the increasing focus on tumor immunity, researchers are paying growing attention to the role of the Tumor Microenvironment (TME) in tumorigenesis. The alterations in TME extend beyond changes in infiltrated immune cells to include variations in various metabolite levels resulting from the metabolic reprogramming of cells within the TME. Metabolic reprogramming occurs not only in tumor cells but also in stromal cells, including immune cells, within primary tumor tissues. This metabolic reprogramming leads to a TME characterized by increased acidity, nutrient deficiencies, and hypoxia. These conditions not only exacerbate metabolic reprogramming in tumor cells and immune cells but also contribute to the creation of an immunosuppressive microenvironment. This, in turn, regulates immune cells differentiation of and promotes tumor progression (Zheng et al., 2009; Lian et al., 2022). Consequently, further exploration is warranted to better understand the role of metabolic reprogramming in immune cells during the processes of tumorigenesis and tumor progression.

MR relies on three key assumptions. Genetic variants must 1) strongly associate with the exposure factor; 2) not associate with any confounding factors; 3) influence the outcome only through the exposure factor and not through any direct causal pathway. If the assumption 1) is not met, MR analysis cannot be performed; If the assumptions 2) or/and 3) are not satisfied, it may lead to false positive results (Larsson et al., 2023). In this research, we employed a similar approach to the previous studies on SNPs of immune cells and metabolites to obtain IVs for MR analysis and ensure the validity of these three assumptions (Figure 1) (Wang et al., 2024; Wang and Zhang, 2024). In this investigation, we initially identified a causal relationship between IgD- CD38⁺ B cells and an increased risk of BC, whereas no significant causal link was observed between the risk of BC and IgD- CD38⁺ B cells. Subsequently, through an exploration of the mediating effects of metabolites, we uncovered a negative causal association between IgD- CD38⁺ B cells and Succinoyltaurine levels. Simultaneously, there was a negative causal relationship between Succinoyltaurine levels and the risk of BC. Conversely, a positive causal relationship was noted between the levels of IgD- CD38⁺ B cells and Glycerate, and likewise, a positive causal link existed between Glycerate levels and the risk of BC. In summary, we inferred that IgD- CD38⁺ B cells could contribute to an increased risk of BC through both positive and negative mediating effects involving Glycerate and Succinoyltaurine.

BC is infiltrated by various immune cells, typically found in the extracellular matrix or in direct contact with cancer cells (Salgado et al., 2015). Among these immune cells, adaptive immunity mediated by lymphocytes plays a crucial role in eliciting an effective anti-tumor response (Galon et al., 2013). While T lymphocytes primarily fulfill an anti-tumor immune function, recent research has highlighted the increasingly recognized role of infiltrating B lymphocytes in BC, with CD20⁺ B lymphocytes serving as a notable representative (Hussein and Hassan, 2006). CD20⁺ B lymphocytes contribute to an anti-tumor response by generating antibodies against BC antigens, releasing immunogenic cytokines and chemokines, and presenting antigens to T cells (Brown et al., 2014; Sarvaria et al., 2017). Nonetheless, a subset of B cells exists that fosters tumor growth by promoting inflammation and immunosuppression. This is achieved through the secretion of anti-inflammatory and angiogenic mediators, as well as interactions involving immune complexes and complement activation (Yuen et al., 2016). The IgD- CD38⁺ B cells identified in our study belong to a subtype of inhibitory B cells that can elevate the risk of BC through mediating effects (with metabolites as mediators). It has been observed that the heightened infiltration levels of IgD- CD38⁺ B cells were associated with increased inflammation (Wang et al., 2022). In regulatory B cells (Bregs) with IL10+ IgD- CD38⁺, PPARδ was significantly upregulated. However, the immunosuppressive function of IL10+ IgD- CD38⁺ Bregs could be blocked, and cancer immunotherapy enhanced, by using PPARδ inhibitors (Chen et al., 2023b). Overall, research on IgD- CD38⁺ B cells is still limited at present, and further in-depth study is required to fully understand its function and role in tumorigenesis and tumor immunity.

Single nucleotide polymorphisms (SNPs) play a pivotal role in BC. Existing research indicated a close association between the polymorphism at SNPs and the risk of BC (Jupe et al., 2001; Arancibia et al., 2021). These variations may influence gene expression, thereby impacting crucial biological processes such as cell proliferation, differentiation, and apoptosis (Morales-Pison et al., 2021). SNPs variations could lead to alterations in protein structure or function, consequently disrupting the normal regulation of cellular signaling pathways and promoting the occurrence and progression of BC (Noguchi et al., 2009). Moreover, SNPs may influence the response of patients with BC to treatment. Some SNPs could affect the activity of drug-metabolizing enzymes, thus impacting drug concentrations and efficacy in vivo, ultimately affecting treatment effects and prognosis for patients with BC (Noguchi et al., 2009; Duong et al., 2023; Oliva et al., 2023). These genes as mentioned, which could be influenced by SNPs and had an impact on the pathogenesis and treatment of BC, include non-coding genes and genes encoding protein, such as those in our research. In summary, SNPs play a significant role in the occurrence, development, and treatment of BC. Further in-depth research into the mechanisms of action of these SNPs will contribute to a better understanding of the pathophysiological processes of BC, providing a theoretical basis and clinical guidance for personalized treatment approaches.

Although we endeavored to enhance the objectivity and credibility of our research during the design stage, we acknowledge certain limitations and shortcomings. Despite utilizing genetic statistics from the open GWAS database, it is important to note that these data might not encompass all ethnic groups and populations, as our focus was primarily on the European population. Since the current GWAS datasets of immune cells and metabolites were only from European population, to ensure the same genetic background, we chose breast cancer GWAS data from European population. We also expect more GWAS datasets of other people (such as Asians, Africans, etc.) to verify our results in the future. MR analysis provides robust evidence of causality, but it relies on pivotal assumptions, including the absence of genetic confounding factors. Any violation of these assumptions may impact the accuracy of the results. Moreover, our research assumes a linear relationship between exposures and outcomes. However, we cannot assess potential nonlinear relationships. Although our analysis identified Succinoyltaurine and Glycerate as mediating factors, the regulation and influence of metabolic levels were intricate, implying the possible existence of other unconsidered mediating factors. While our study revealed a causal relationship between IgD- CD38⁺ B cells and the risk of BC, these results should be cautiously interpreted in the clinical context. Further research is needed to validate our findings and determine the clinical implications. Lastly, our study aims to generate new hypotheses rather than definitive conclusions, and there is a risk of false positives if multiple tests are not corrected. Therefore, our results should be viewed as preliminary and require further validation in future research.

This MR study provides novel genetic evidence supporting a causal relationship between IgD- CD38⁺ B cells and the risk of BC. Through mediation analysis, we identified that IgD- CD38⁺ B cells contribute to an increased risk of BC through both positive and negative mediation effects involving Glycerate and Succinoyltaurine. Overall, these findings suggest the potential for clinicians to assess the risk of BC in patients by measuring the levels of blood IgD- CD38⁺ B cells. Nevertheless, further clinical research and experiments are warranted to validate these findings in the future.