Tumor cells evade anti-tumor immunity by reprogramming tumor microenvironment (TME). Using multiplexed single-cell proteomics to analyze 50 TME-associated proteins across treatment-naive triple-negative breast cancer (TNBC) specimens, we discovered that cancer stem cells (CSCs) drive differentiation and expansion of regulatory T cells (Tregs) via extracellular vesicle (EV)-mediated paracrine signaling. TSPAN8, an integral membrane protein on CSC-derived EVs, interacts with CD103 (integrin αEβ7) on T cells, triggering the formation of LKB1-STRAD-MO25 complex and sequential phosphorylation of LKB1 and AMPKα. This cascade enhances FOXP3 expression, which transactivates CD103, creating a positive feedback loop that drives clonal expansion of immunosuppressive CD103+FOXP3+ Tregs and their associated niche. This EV membrane topology-based mechanism operates independently of canonical EV cargo internalization. Neutralizing EVs-TSPAN8+ with a monoclonal antibody synergized with anti-PD-1 therapy in preclinical models, suggesting a potential approach targeting both CSCs and TME immunosuppression, particularly in TNBC subpopulation with high TSPAN8+ CSCs.

Background and purpose: Breast cancer is one of the leading malignancies affecting women's health, with high incidence. Nucleotide-binding oligomerization domain containing 1 (NOD1), a member of the pattern recognition receptor family, is involved in regulating the immune microenvironment in various cancers. However, the specific regulatory functions and mechanisms of NOD1 in the breast cancer immune microenvironment remain unclear. This study aimed to investigate the effects of NOD1 on tumor-associated macrophages (TAM) within the breast cancer immune microenvironment and to explore the underlying mechanisms involved. Methods: The public databases and multiplex immunohistochemistry (mIHC) were utilized to analyze the correlation between NOD1 expression and immune infiltration in human breast cancer. Multicolor flow cytometry was performed to assess the immune cell composition and changes in NOD1-overexpressing breast tumor allografts. In vitro, chemotaxis assays and quantitative real-time polymerase chain reaction (qRT-PCR) were used to evaluate the effect of NOD1-overexpressing breast cancer cells on macrophage chemotaxis and polarization. Additionally, the effect of NOD1 on macrophage recruitment and polarization in breast tumor allografts was assessed by flow cytometry. Fluorescence-activated cell sorting (FACS) was used to isolate macrophages pre-conditioned by NOD1-overexpressing breast cancer stem cells, which were then cocultured with parental breast cancer cells. Flow cytometry was used to assess the impact of NOD1-conditioned macrophages on the stemness of breast cancer cells. Results: The database analysis and multicolor flow cytometry results demonstrated significant correlations between NOD1 expression and TAM infiltration (P＜0.01). In vitro functional assays of macrophages demonstrated that NOD1-overexpressing breast cancer cells enhanced macrophage chemotaxis (P＜0.001) and upregulated the expression of M2 macrophage markers. Compared with control groups, NOD1-overexpressing breast tumor allografts showed increased macrophage infiltration and polarization (P＜0.01). NOD1-educated macrophages promoted malignant progression of breast cancer by increasing the proportion of breast cancer stem cells (P＜0.05). Conclusion: In breast cancer, NOD1 remodels the immune microenvironment by recruiting macrophages and inducing their polarization towards the M2 phenotype. Furthermore, NOD1-educated macrophages enhance breast cancer cell stemness, thereby accelerating tumor progression. NOD1 represents a potential therapeutic target in breast cancer.

Research on the intratumoral microbiota is shifting from descriptive analyses of presence and abundance toward understanding its spatial heterogeneity and local function. Here we propose the concept of the microbiota-residing spatial niche (MRSN), defined as a functional unit formed by interactions between microbiota and neighboring tumor, immune, and stromal cells within a defined spatial context, and characterized by four key features: spatial discernibility, functional consistency, microbial dependency, and clinical relevance. In this review, we discuss the limitations of single-omics approaches and present a framework centered on spatial multi-omics. This framework integrates spatial transcriptomics, multiplex immunoimaging, spatial metabolomics, and graph neural networks, enabling a stepwise analysis that first localizes microbiota, then characterizes their surrounding cellular neighborhoods, and finally validates their functional roles. We classify MRSNs into immunosuppressive, immunostimulatory, protumorigenic, and antitumorigenic types, and demonstrate how they modulate therapy responses through local metabolic remodeling and immune regulation. We also contrast the systemic immunomodulatory effects of gut microbiota with the localized influence of intratumoral microbiota. Finally, we discuss challenges in technical validation, temporal dynamics, and model translation, and propose strategies for precision interventions targeting niche vulnerabilities and clinical stratification. This framework provides a unified conceptual basis for understanding intratumoral microbiota functions and informs next-generation spatially guided therapeutic strategies.

Despite the pivotal role of tumor immune microenvironment (TIME) in breast cancer (BC) progression, the functional contributions of mast cells (MCs) within the TIME remain poorly understood. Utilizing single-cell RNA sequencing on tumor (T) and adjacent para-tumor (PT) tissues from BC patients, we identified a distinct transcriptional profile in T-derived tumor-resident MCs (MCt) compared to their PT counterparts. Survival analysis revealed that MCt signature gene set significantly correlated with poor clinical outcomes. To assess functional roles, we established in vitro co-culture systems and in vivo murine allograft models, which demonstrated that MCt promoted BC cell proliferation and enriched ALDH+ breast cancer stem cells (BCSCs). Mechanistically, through molecular inhibitors, agonist, recombinant protein, and gene-knockdown cell lines, we found that MCt-derived IFNB1 activated the Type I interferon pathway in BC cells via the IFNAR1-STAT1 axis. Reciprocally, BC cells upregulated IFNB1 expression in MCs via stem cell factor (SCF)-mediated c-KIT-MAPK/ERK signaling. This bidirectional crosstalk established a self-reinforcing IFNB1/SCF feedforward loop driving BC progression and stemness, which was further validated in BC patient tissues by multiplex immunohistochemistry. Collectively, our findings characterize a pro-tumorigenic MC subset within the TIME and propose a potential novel therapeutic strategy for BC by disrupting the MCt-BC cell interplay.

Breast cancer brain metastasis (BCBrM) remains a major clinical challenge with limited therapeutic options and poor prognosis. Despite advances in systemic therapy, the incidence of BCBrM is rising due to prolonged survival of patients with advanced breast cancer, yet effective brain-targeted strategies remain scarce, underscoring a critical research gap. This review integrates recent mechanistic insights that illuminate the complex biology underpinning BCBrM and explores how these discoveries are driving therapeutic innovation. We detail the metastatic cascade from local invasion to brain colonization, and examine key signaling pathways orchestrating brain-specific metastasis. Emphasis is placed on the dynamic crosstalk between tumor cells and the brain microenvironment, including astrocytes, microglia, and neurons, as well as metabolic reprogramming and immune evasion. We critically evaluate current preclinical models and their translational relevance, highlighting recent advances in humanized and imaging-based systems. Emerging therapies, such as central nervous system-penetrant kinase inhibitors, antibody–drug conjugates, and immunotherapies, are discussed alongside persistent challenges in drug delivery and resistance. Finally, we outline future directions, calling for cross-disciplinary collaboration and innovative clinical trial designs to personalize care and improve patient outcomes. Together, this review underscores the urgent need to bridge biology and therapy to transform the management of BCBrM.

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer with limited effective therapeutic options readily available. We have previously demonstrated that lovastatin, an FDA-approved lipid-lowering drug, selectively inhibits the stemness properties of TNBC. However, the intracellular targets of lovastatin in TNBC remain largely unknown. Here, we unexpectedly uncovered ribosome biogenesis as the predominant pathway targeted by lovastatin in TNBC. Lovastatin induced the translocation of ribosome biogenesis-related proteins including nucleophosmin (NPM), nucleolar and coiled-body phosphoprotein 1 (NOLC1), and the ribosomal protein RPL3. Lovastatin also suppressed the transcript levels of rRNAs and increased the nuclear protein level and transcriptional activity of p53, a master mediator of nucleolar stress. A prognostic model generated from 10 ribosome biogenesis-related genes showed outstanding performance in predicting the survival of TNBC patients. Mitochondrial ribosomal protein S27 (MRPS27), the top-ranked risky model gene, was highly expressed and correlated with tumor stage and lymph node involvement in TNBC. Mechanistically, MRPS27 knockdown inhibited the stemness properties and the malignant phenotypes of TNBC. Overexpression of MRPS27 attenuated the stemness-inhibitory effect of lovastatin in TNBC cells. Our findings reveal that dysregulated ribosome biogenesis is a targetable vulnerability and targeting MRPS27 could be a novel therapeutic strategy for TNBC patients.

Tumor-resident microbiota in breast cancer promotes cancer initiation and malignant progression. However, targeting microbiota to improve the effects of breast cancer therapy has not been investigated in detail. Here, we evaluated the microbiota composition of breast tumors and found that enterotoxigenic Bacteroides fragilis (ETBF) was highly enriched in the tumors of patients who did not respond to taxane-based neoadjuvant chemotherapy. ETBF, albeit at low biomass, secreted the toxic protein BFT-1 to promote breast cancer cell stemness and chemoresistance. Mechanistic studies showed that BFT-1 directly bound to NOD1 and stabilized NOD1 protein. NOD1 was highly expressed on ALDH+ breast cancer stem cells (BCSCs) and cooperated with GAK to phosphorylate NUMB and promote its lysosomal degradation, thereby activating the NOTCH1-HEY1 signaling pathway to increase BCSCs. NOD1 inhibition and ETBF clearance increase the chemosensitivity of breast cancer by impairing BCSCs.

Neurotransmitters are increasingly recognized to play important roles in limiting anti-tumor immunity. N-acetyl-aspartyl-glutamate (NAAG) has been extensively studied in neurological disorders; however, its potential role in restricting anti-tumor immunity has not been investigated. Here, we demonstrated that NAAG or its synthetase RimK-like family member B (RIMKLB) significantly disrupted anti-tumor immunity by rewiring the myeloid progenitor differentiation of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs), which in turn promoted breast cancer growth and metastasis. Mechanistically, NAAG sustained the tumor immunosuppressive microenvironment by activating an NR2B-containing NMDA receptor (NR2B-NMDAR)-dependent p38-NOTCH1 axis, and subsequently stimulating tumor cell migration and invasion, as well as inducing PMN-MDSC differentiation and expansion. In mouse models, RIMKLB ablation or NMDAR inhibition enhanced the efficacy of anti-PD-1 therapy and suppressed tumor progression. An analysis of clinical samples revealed that high levels of NAAG and NR2B-NMDAR predicted a poor prognosis in TNBC patients. Collectively, our findings have uncovered a signaling role for tumor-derived NAAG beyond its classic function as a neurotransmitter that can be targeted pharmacologically to enhance immunotherapy against breast cancer.

Despite an increasingly detailed understanding of cancer hallmarks at molecular or atomic resolution, most studies, however, fall short of investigating the systemic interactions of cancer with the human body. We propose to investigate the hallmarks of cancer from an organ-wide macroscopic view, discuss the challenges in preclinical and clinical research to study the cross-organ regulation of cancer together with potential directions to overcome these challenges, and foresee how this holistic view may be translated into more effective therapies.

The class I phosphatidylinositol 3-kinase (PI3K)-AKT signaling pathway is a key regulator of cell survival, growth, and proliferation and is among the most frequently mutated pathways in cancer. However, where and how PI3K-AKT signaling is spatially activated and organized in mammalian cells remains poorly understood. Here, we identify focal adhesions (FAs) as subcellular signaling hubs organizing the activation of PI3K-PI(3,4,5)P3-AKT signaling in human cancer cells containing p110α mutations under basal conditions. We find that class IA PI3Ks are preferentially recruited to FAs for activation, resulting in localized production of PI(3,4,5)P3 around FAs. As the effector protein of PI(3,4,5)P3, AKT1 molecules are dynamically recruited around FAs for activation. The spatial recruitment/activation of the PI3K-PI(3,4,5)P3-AKT cascade is regulated by activated FA kinase (FAK). Furthermore, combined inhibition of p110α and FAK results in a more potent inhibitory effect on cancer cells. Thus, our results unveil a growth-factor independent, compartmentalized organization mechanism for PI3K-PI(3,4,5)P3-AKT signaling.

As the main component of lipids, fatty acids are essential in supporting life activities regarding energy supply, cell composition, and signaling molecules. Enhancement of fatty acid (FA) synthesis, storage, and catabolism is observed in various cancer cells. In addition, it has become clear that tumor cells exhibit plasticity in fatty acid metabolism to facilitate aggression and treatment resistance. Here, we describe cellular fatty acid metabolism changes associated with tumor development and therapeutic resistance. Potential inhibitors of fatty acid metabolism are also discussed for tumor therapy. Therefore, exploring the targets of FA metabolism in cancer to improve the efficiency of cancer therapy is of great interest.

Breast cancer metastasis is responsible for most breast cancer-related deaths and is influenced by many factors within the tumor ecosystem, including tumor cells and microenvironment. Breast cancer stem cells (BCSCs) constitute a small population of cancer cells with unique characteristics, including their capacity for self-renewal and differentiation. Studies have shown that BCSCs not only drive tumorigenesis but also play a crucial role in promoting metastasis in breast cancer. The tumor microenvironment (TME), composed of stromal cells, immune cells, blood vessel cells, fibroblasts, and microbes in proximity to cancer cells, is increasingly recognized for its crosstalk with BCSCs and role in BCSC survival, growth, and dissemination, thereby influencing metastatic ability. Hence, a thorough understanding of BCSCs and the TME is critical for unraveling the mechanisms underlying breast cancer metastasis. In this review, we summarize current knowledge on the roles of BCSCs and the TME in breast cancer metastasis, as well as the underlying regulatory mechanisms. Furthermore, we provide an overview of relevant mouse models used to study breast cancer metastasis, as well as treatment strategies and clinical trials addressing BCSC-TME interactions during metastasis. Overall, this study provides valuable insights for the development of effective therapeutic strategies to reduce breast cancer metastasis.

The different compartments of the mammary stem cell hierarchy develop into distinct breast cancer subtypes as a result of specific genetic lesions. A recent study identifies aberrant ERBB3 low luminal progenitors with altered proteostasis and translation as the cell of origin of BRCA2 -mutant breast cancer.