Tumor-associated neutrophils drive liver-specific metastatic progression in breast cancer through methionine metabolism mediated by methionine adenosyltransferase II alpha

Introduction

Breast cancer (BC) remains one of the most commonly diagnosed malignancies among women worldwide, accounting for approximately 2.3 million new cases annually (1). Although significant advances have been achieved in prevention, early detection, and adjuvant therapies, a substantial proportion of patients develop metastatic disease, with approximately 5% presenting with metastasis at initial diagnosis and nearly 30% progressing to metastatic disease during the course of treatment (2). Currently, metastatic BC remains largely incurable, and for most patients, standard chemotherapy confers primarily palliative benefits without durable disease control (3).

This clinical challenge is further compounded by the organ-specific nature of BC metastasis. Accumulating evidence indicates that BC exhibits a predilection for metastasizing to specific distant organs, including the bone, lungs, liver, and brain, a phenomenon referred to as metastatic organotropism (4). This organ-specific dissemination contributes to the heterogeneity of therapeutic responses and significantly impacts patient prognosis. Although the molecular mechanisms governing BC metastasis to the lungs and bones have been extensively elucidated (5-8), the mechanistic basis underlying hepatic colonization remains poorly defined, representing a critical gap in our understanding of metastatic progression.

Neutrophils have emerged as key regulators of the tumor microenvironment (TME), exerting diverse roles in cancer progression, immune modulation, and metastasis (9,10). Recent studies have highlighted that neutrophils can be co-opted by various stimuli to promote metastasis through multiple mechanisms. For instance, chemotherapy can induce neutrophil extracellular trap (NET) formation, which awakens dormant disseminated tumor cells and facilitates metastatic relapse (11). Beyond releasing NETs, neutrophils can also physically interact with tumor cells to form a signaling niche that enhances tumor cell aggressiveness (12). Furthermore, in the context of ovarian cancer, NETs have been shown to establish a pre-metastatic niche in the omentum by expanding immunosuppressive innate-like B cells (13). Conversely, innovative therapeutic strategies such as neutrophil-mimicking nanomedicine have been designed to eliminate intracellular bacteria and enhance chemotherapeutic efficacy, highlighting the potential of targeting neutrophil-related pathways (14). Additionally, chronic stress has been identified as a potent inducer of metastasis by glucocorticoid-mediated alterations in neutrophil circadian rhythm and NET formation (15). These findings underscore the functional plasticity of tumor-associated neutrophils (TANs), which can exhibit various roles depending on the context within the TME (16,17). With substantial evidence highlighting the diverse roles of TANs in cancer progression and metastasis, their role in driving hepatic metastasis in BC remains poorly defined. In this study, we identified a neutrophil-enriched TME as a defining feature of BC liver metastasis (BCLM). Using transcriptomic data from the Gene Expression Omnibus (GEO) database, we observed a distinct accumulation of TANs in liver metastasis compared to other metastatic sites. Functional assays revealed that TANs promoted cancer stemness and chemoresistance in BC cells. Mechanistically, TANs were found to upregulate methionine adenosyltransferase II alpha (MAT2A), a key enzyme in the methionine cycle, thereby enhancing methionine-dependent metabolic reprogramming. Pharmacological inhibition of MAT2A using FIDAS-5 (a selective MAT2A inhibitor) effectively reversed the TANs-induced stem-like phenotypes. Metabolomic profiling of clinical samples further confirmed activation of methionine metabolism in liver-metastatic tumors. Collectively, our findings uncover a previously unrecognized TAN-MAT2A-methionine axis that drives liver-specific metastatic progression in BC and highlight methionine metabolism as a potential therapeutic vulnerability in BCLM. We present this article in accordance with the MDAR reporting checklist (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-aw-2278/rc).

Methods

Data collection and processing

The transcriptome data generated by microarray profiling of 29 metastatic BC patients were obtained from the GEO database GSE54323 (https://www.ncbi.nlm.nih.gov/geo/), which is the only GEO database meeting three essential criteria: (I) matched primary and metastatic tumor samples; (II) inclusion of both locoregional (lymph node) and distant (bone, brain, visceral) metastases; and (III) standardized RNA-sequencing (RNA-seq) profiling. Strict exclusion criteria were applied to further refine the selection of appropriate GEO datasets, as follows: absence of metastasis information; more than 10% of the transcriptome data lacking gene annotation; and insufficient input data for Cell-type Identification By Estimating Relative Subsets Of RNA Transcripts (CIBERSORT) analysis. A total of 14 metastatic BC cases were included in the final analysis: 5 with liver metastasis, 2 with local metastasis, 2 with axillary lymph node involvement, and 5 with bone metastasis.

An additional cohort of 12 BC patients with liver metastasis was enrolled at The First Affiliated Hospital with Nanjing Medical University. Clinical data and TANs infiltration levels were collected and independently evaluated by at least two certified pathologists.

Cell lines and cell culture

The human BC cell lines MCF-7 (RRID: CVCL_0031), MDA-MB-231 (RRID: CVCL_0062), and SK-BR-3 (RRID: CVCL_0033) were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The non-tumorigenic human mammary epithelial cell line MCF-10A (RRID: CVCL_0598) was also obtained from the same repository. All cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 20% fetal bovine serum (FBS), 100 µg/mL penicillin G, and 100 U/mL streptomycin, and maintained in a humidified incubator at 37 ℃ with 5% CO₂.

Quantitative real-time polymerase chain reaction and western blot (qRT-PCR)

Primary human BC cells (sex of origin: female; species: Homo sapiens; genetic modification status: unmodified) collected at The First Affiliated Hospital with Nanjing Medical University were used for qRT-PCR and western blot analysis. Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and reverse-transcribed into complementary DNA (cDNA) using the cDNA Synthesis Kit (Thermo Scientific, Waltham, MA, USA). qRT-PCR was conducted on the CFX96 Real-Time PCR System (Bio-Rad, Hercules, CA, USA) with SYBR Green PCR Master Mix (Applied Biosystems, Waltham, MA, USA). Protein extracts from primary BC and BC cell lines were analyzed by western blotting following the manufacturer’s standard protocol using the following antibodies: anti-SOX2 (Cat# 23064, RRID: AB_2714146, Cell Signaling Technology, Danvers, MA, USA), anti-OCT4 (cat. #2750, RRID: AB_823583, Cell Signaling Technology), anti-β-catenin (cat. #9562, RRID: AB_331149, Cell Signaling Technology), and anti-β-actin (cat. #A1978, RRID: AB_476692, Sigma-Aldrich, St Louis, MO, USA).

Single-cell RNA-seq analysis

To examine the immune cell landscape in the liver microenvironment of BC, we analyzed publicly available single-cell RNA-seq data from mouse models via the Single Cell Portal. Neutrophils were identified based on canonical marker genes, and their abundance in the liver microenvironment was assessed using standard clustering and visualization methods.

Co-culture of TANs and BC cells

TANs were isolated from fresh BC tissues collected in sterile RPMI-1640 medium with 1% penicillin-streptomycin. Tissues were mechanically dissected into 2 mm³ fragments and enzymatically digested using 2 mg/mL collagenase type IV (Sigma) and 0.1 mg/mL DNase I (Sigma) in RPMI-1640 at 37 ℃ for 60 min with agitation. After termination with RPMI-1640 with 10% FBS, single-cell suspensions were obtained by sequential filtration (100 to 40 µm) and red blood cell (RBC) lysis ACK buffer (Beyotime, Shanghai, China). CD66b⁺ TANs were isolated by magnetic sorting, with purity (>85% CD66b⁺) confirmed by flow cytometry. All procedures were completed within 4 hours post-resection. TANs and BC cells were co-cultured at a 2:1 ratio in transwell systems (0.4-µm pores) for 48 hours. After co-culture, cancer cells were collected for stemness marker analysis by qRT-PCR and western blot and spheroid formation assays. Data shown are from three independent biological replicates (n=3), each representing TANs isolated from a distinct patient tumor sample. All measurements within each biological replicate were performed in technical triplicates to ensure analytical precision.

Tumorsphere formation assay

Single-cell suspensions of BC cells (1,000 cells per well) were seeded into 12-well ultra-low attachment culture plates (Corning, New York, NY, USA) and cultured for 7 days in serum-free Dulbecco’s modified Eagle medium (DMEM)/F12 medium. The medium was supplemented with 20 ng/mL epidermal growth factor (EGF) (Invitrogen), 10 ng/mL basic fibroblast growth factor (bFGF) (Invitrogen), B27 supplement (1:50, Invitrogen), N2 supplement (1:100, Invitrogen), 1% sodium pyruvate, 100 µg/mL penicillin G, and 100 U/mL streptomycin. Spheroids with diameters ≥50 µm were counted under an inverted microscope (Leica, Wetzlar, Germany). The data presented are derived from three independent biological replicates (n=3), each performed with technical triplicates, ensuring statistical robustness and reproducibility.

Chemoresistance assay

Cell viability was assessed using the Cell Counting kit-8 (CCK-8; Dojindo, Kumamoto, Japan) following the manufacturer’s instructions. In brief, BC cells were seeded in 96-well plates at a density of 1×10⁴ cells per well and allowed to adhere overnight. The next day, cells were treated with 5 µg/mL of cisplatin (CDDP; Sigma). Each experiment was performed in triplicate and independently repeated at least 3 times to ensure reproducibility.

Methionine cycle inhibition assay

Primary BC cells were co-cultured with TANs, followed by treatment with FIDAS-5 (5 µM final concentration; Merck Millipore, Burlington, MA, USA). The messenger RNA (mRNA) levels of pluripotency-associated transcription factors were then analyzed by qRT-PCR. Our selected concentrations of CDDP and FIDAS-5 were based on their established therapeutic windows from our previous studies (18,19), and have further been validated in BC models through our comprehensive dose-response analyses.

Clinical sample preparation and metabolomic analysis

Three BC tissue samples with confirmed liver metastasis and three paired BC tissue samples without liver metastasis were collected from female patients (age range, 45–68 years) at The First Affiliated Hospital with Nanjing Medical University. The reporting of metabolomics data and methodologies in this study adheres to the relevant guidelines and minimum information requirements as outlined by the Metabolomics Standards Initiative (MSI), which falls under the broader Minimum Information for Biological and Biomedical Investigations (MIBBI) framework. All specimens were subjected to metabolomics analysis following the standardized protocols detailed below.

Following removal of the culture medium, adherent cells were gently rinsed with 150 mM sodium chloride solution and detached from the plate surface using a sterile cell scraper. The collected cells were then quenched with 4 volumes of 150 mM sodium chloride solution, vortexed for 1 minute, and centrifuged at 3,000 ×g for 5 minutes at 4 ℃, after which the supernatant was discarded. Polar metabolites (aqueous phase) and lipids (organic phase) were subsequently isolated from the cell pellets using a two-phase liquid-liquid extraction method as previously reported (19-21). Metabolomic analysis was carried out using an ultra-performance liquid chromatography (UPLC) system (Waters Corporation, Milford, MA, USA) in conjunction with a mass spectrometer (Thermo Fisher Scientific).

Ethics consideration

This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Ethics Committee of The First Affiliated Hospital with Nanjing Medical University (No. 2023-SRFA-408; date of approval: March 3rd, 2023). Written informed consent was provided by each participant.

Statistical analysis

The statistical analysis was carried out using SPSS 23.0 (IBM Corp., Armonk, NY, USA) and GraphPad Prism 6 (GraphPad Software, San Diego, CA, USA). The Student’s t-test was used to compare the difference of clinicopathologic features between two groups. All statistical tests were two-sided, and a P value of <0.05 (*), <0.01 (**), or <0.001 (***) was considered significant.

Results

TME analysis across BC metastatic sites

To investigate the TME associated with different metastatic patterns in BC, we analyzed transcriptomic data of metastatic lesions from the GEO dataset GSE54323. Notably, the immune composition of BCLM was markedly distinct from that of other metastatic sites (Figure 1A). Principal component analysis (PCA) further revealed that the TME of local metastasis differed significantly from that of distant metastasis (Figure 1B), with additional heterogeneity observed among distant metastatic sites (Figure 1C). To identify differentially represented immune cell populations, we generated a heatmap depicting the relative abundance of 22 immune cell types (Figure 1D). Together, these results indicate that the TME varies substantially across metastatic sites in BC, with TANs potentially playing a key role in liver metastasis.

Figure 1 TME analysis across BC metastatic sites. (A) Immune cell composition across different metastatic patterns in BC. (B) PCA comparing local versus distant metastases. (C) PCA comparing liver and bone metastases. (D) Heatmap showing the relative abundance of 22 immune cell types across various BC metastatic patterns (n=14). BC, breast cancer; LN, lymph node; PCA, principal component analysis; TME, tumor microenvironment.

TANs characterize BCLM

To further elucidate the involvement of TANs in BCLM, we analyzed immune cell proportions within hepatic metastatic lesions and adjacent non-tumor tissue, revealing neutrophils as the predominant immune cell type (P=0.004) (Figure 2A). Given the prominent presence of neutrophils in the liver metastatic microenvironment of BC, we further investigated their role through the Single Cell Portal. Single-cell RNA-seq revealed a marked expansion of neutrophils in the liver microenvironment of BC in mouse models, suggesting a pivotal role in metastatic progression (Figure 2B). Additionally, we employed CIBERSORT to assess immune cell composition in BC patients with liver metastasis, and the resulting correlation matrix is presented in Figure 2C.

Figure 2 TANs characterize BCLM. (A) Comparison of immune cell proportions between the tumor site (red) and paratumor site (blue) in the liver lesions of BCLM (n=14). (B) Single-cell RNA-sequencing analysis of liver microenvironment in a mouse model with breast cancer (Single Cell Portal). (C) Correlation matrix of immune cell composition within the BCLM microenvironment. BCLM, breast cancer with liver metastasis; NK, natural killer.

TANs promote stem-like properties in BC cells

To examine the impact of TANs on BC stemness, we co-cultured TANs with primary BC cells, and discovered this treatment markedly enhanced stem-like features of BC cells, as evidenced by increased expression of stemness-associated markers (Figure 3A-3C). In line with these findings, spheroid formation assays demonstrated a significant increase in the sphere-forming ability of BC cells following TANs co-culture, further supporting the role of TANs in promoting cancer cell self-renewal (Figure 3D,3E). Moreover, proteomic analysis of TAN-secreted factors identified interleukin-6 (IL-6) as a key mediator, and pharmacological inhibition of STAT3 abolished TAN-induced MAT2A upregulation in BC cells (Figure 3F,3G). We further treated BC cells directly with TAN-conditioned medium and confirmed that soluble factors released by TANs are indeed capable of significantly increasing MAT2A expression and stemness-associated markers in BC cells, as shown in Figure S1.

Figure 3 TANs promote stem-like properties in BC cells. (A) Schematic diagram of the co-culture system involving primary BC cells and TANs. (B) mRNA expression levels of stemness-associated markers in primary BC cells with or without TANs co-culture (n=3). (C) Protein expression levels of stemness-associated markers in primary BC cells with or without TANs co-culture. (D,E) Sphere formation efficiency of primary BC cells with or without TANs co-culture. (D) Quantification of sphere formation; data are presented as mean ± SD. (E) Representative images of tumor spheres (scale bar, 100 µm) (n=3). (F) Comparative profiling of secretory proteins in TANs vs. Peri-Neu (n=3). (G) mRNA levels of MAT2A in primary BC cells co-cultured with TANs or Peri-Neu, with or without STAT3 inhibitor (Stattic, 5 µM), as assessed by qRT-PCR (n=3). BC, breast cancer; MAT2A, methionine adenosyltransferase II alpha; mRNA, messenger RNA; PBS, phosphate-buffered saline; Peri-Neu, peripheral blood neutrophils; qRT-PCR, quantitative real-time polymerase chain reaction and western blot; SD, standard deviation; TANs, tumor-associated neutrophils.

TANs enhance the chemoresistance of BC cells

Cancer stem cells (CSCs), characterized by their tumorigenic and self-renewal capacities, are key contributors to chemoresistance and tumor recurrence. Given the role of TANs in maintaining CSC properties, we next examined their influence on chemotherapy response. CCK-8 assays revealed that co-culture with TANs significantly increased resistance to CDDP in BC cell lines and primary BC cells (Figure 4).

Figure 4 TANs enhance chemoresistance in BC cells. (A-D) Cell viability of MCF-7 (A), MDA-MB-231 (B), SK-BR-3 (C), and primary BC cells (D) with or without TANs co-culture following CDDP treatment. (E,F) Cell viability of MCF-7 and primary BC cells with or without TANs co-culture following varying concentrations of CDDP. Data are presented as mean ± SD; n=4. *, P<0.05; **, P<0.01; ***, P<0.001. BC, breast cancer; CDDP, cisplatin; SD, standard deviation; TANs, tumor-associated neutrophils.

TANs promote stemness of BC in a methionine-dependent way

Methionine metabolism is increasingly recognized as a critical vulnerability of tumor-initiating cells. To investigate whether TANs enhance BC stemness through this metabolic axis, we examined methionine cycle activity in primary BC cells co-cultured with TANs. The results showed that TANs exposure significantly augmented both stem-like phenotypes and methionine cycle activity (Figure 5A). Importantly, expression of MAT2A, but not other key enzymes in methionine metabolism such as methylenetetrahydrofolate reductase (MTHFR) or methionine synthase (MTR), was markedly upregulated in TANs-treated BC cells compared to controls (Figure 5B). To dissect the functional dependency on methionine metabolism, we pharmacologically suppressed MAT2A using the selective inhibitor FIDAS-5. The inhibition of MAT2A led to a clear reversal of the TAN-induced acquisition of stem-like phenotypes in BC cells, as evidenced by a significant reduction in stem cell markers (Figure 5C,5D). Importantly, this blockade had no observable effect on normal mammary epithelial cells (Figure 5E), indicating that activation of the methionine cycle is essential for the pro-stemness effects mediated by TANs.

Figure 5 TANs promote stemness of BC in a methionine-dependent way. (A) Methionine metabolomic comparison in primary BC cells with or without TANs co-culture (n=5). (B) Correlation between TANs abundance and key enzymes expression of methionine cycle in primary BC cells. (C) Expression of pluripotency-associated transcription factors in primary BC cells co-cultured with TANs, with or without FIDAS-5 treatment, as assessed by qRT-PCR. (D) mRNA levels of Sox2 in primary BC cells co-cultured with TANs, with varying concentrations of FIDAS-5, as assessed by qRT-PCR. (E) Expression of pluripotency-associated transcription factors in normal mammary epithelial cells (MCF-10A) co-cultured with TANs, with or without FIDAS-5 treatment, as assessed by qRT-PCR. n=3. *, P<0.05; **, P<0.01; ***, P<0.001. BC, breast cancer; mRNA, messenger RNA; qRT-PCR, quantitative real-time polymerase chain reaction; SAH, S-adenosylhomocysteine; SAM, S-adenosylmethionine; TANs, tumor-associated neutrophils.

Clinical validation of TANs-associated methionine metabolism in BCLM

To corroborate our experimental findings, we analyzed tumor specimens from BC patients with liver metastasis (n=3) and without liver metastasis (n=3). Comparative analysis revealed a marked increase in methionine cycle activity in BC patients with liver metastasis (Figure 6A,6B). Further, in BC patients with liver metastasis, 84% cases were found to have a high or medium proportion of tumor-infiltrating neutrophils in liver lesions (Figure 6C). Moreover, correlation analysis demonstrated a positive association between intratumoral methionine levels, primary tumor size, and MAT2A expression (Figure 6D), further supporting the clinical relevance of TANs-driven methionine metabolism in metastatic progression.

Figure 6 Clinical validation of TANs-associated methionine metabolism in BCLM. (A) Schematic illustration of key metabolites and enzymes involved in the methionine cycle. (B) Metabolomics analysis of the primary site of BC with or without liver metastasis (n=3) (C) Quantification of TANs infiltration in liver lesions from 12 BC patients with liver metastasis. (D) Three-dimensional correlation analysis of methionine levels, primary tumor size, and MAT2A expression in 12 BCLM patients. AA, amino acid; ADP, adenosine diphosphate; AMP, adenosine monophosphate; BC, breast cancer; BCLM, breast cancer liver metastasis; cAMP, cyclic adenosine monophosphate; CDP, cytidine diphosphate; CMP, cytidine monophosphate; F1, 6P, Fructose-1, 6-Bisphosphate; FAD, flavin adenine dinucleotide; GD3P, D-Glyceraldehyde-3-Phosphate; GDP, guanosine diphosphate; GLDC, glycine decarboxylase; Gly, glycine; Hcy, homocysteine; LM, liver metastasis; MAT2A, methionine adenosyltransferase II alpha; Met, methionine; MTHFR, methylenetetrahydrofolate reductase; MTR, methionine synthase; NAD, nicotinamide adenine dinucleotide; NADH, nicotinamide adenine dinucleotide reduced form; NADP, nicotinamide adenine dinucleotide phosphate; SAH, S-adenosylhomocysteine; SAHH, S-adenosylhomocysteine hydrolase; SAM, S-adenosylmethionine; Ser, serine; SHMT2, serine hydroxymethyltransferase 2; TANs, tumor-associated neutrophils; TCA, tricarboxylic acid; THF, tetrahydrofolate; UDP, uridine diphosphate; UMP, uridine monophosphate.

Discussion

BC remains the most common cause of cancer-related death in women globally (22). Although the prognosis for early-stage disease is generally favorable, the emergence of distant metastasis drastically reduces survival, with a 5-year survival rate dropping to approximately 23% (23). Among common metastatic sites, the liver is the third most frequent target, and BCLM is associated with particularly poor outcomes, with a median survival of only 4–8 months if left untreated (24). Although considerable progress has been made in understanding metastasis to bone and lung, the mechanisms driving liver-specific metastasis remain insufficiently characterized (25-27). The liver’s unique microenvironment—characterized by its immune cells, vasculature, and metabolic processes—appears to influence the behavior of TANs in ways that are specific to liver metastasis. Given the liver’s unique immune and vascular microenvironment, further investigation into the molecular basis of BCLM is crucial for identifying novel therapeutic targets and improving clinical outcomes. In this study, we analyzed the TME across different metastatic sites using the GEO database and identified a unique immune landscape in liver metastasis, characterized by prominent neutrophil infiltration. The liver’s distinct immune environment, including the presence of hepatic resident macrophages and specialized endothelial cells, may promote the role of TANs in liver-specific metastasis. Although TANs are implicated in metastasis across multiple organs, their enrichment and metabolic reprogramming appear particularly critical in liver metastases. The unique interaction between the liver microenvironment and TANs supports the idea that liver-specific metastasis may depend on the ability of the liver to modify the functions of infiltrating neutrophils in ways that facilitate tumor progression. Unlike lung or bone metastases, where neutrophils primarily facilitate vascular remodeling or osteolysis (7,26), TANs in the liver uniquely exploit methionine metabolism to fuel cancer stemness. The liver’s immune cell landscape is more abundant and complex compared to other organs, with a greater degree of metabolic regulation. We hypothesize that this unique immune and metabolic environment may be a key factor contributing to the distinctive nature of liver metastasis. Such context-dependent behavior underscores the liver’s distinct metabolic microenvironment as a driver of TAN-mediated chemoresistance. In addition, this neutrophil-enriched environment appears to play a critical role in promoting cancer stemness and chemoresistance, highlighting the potential of neutrophil-targeted interventions in BCLM treatment.

To contextualize our findings within the established field, it is important to note the recognized roles of neutrophils in tumor metastasis. Seminal studies have demonstrated that neutrophils can directly “escort” circulating tumor cells to enhance their survival and proliferation, or can shape an immunosuppressive pre-metastatic niche in distant organs such as the lung to facilitate colonization (26,28). These foundational works primarily focused on the physical interactions or secretory functions of neutrophils. In contrast, our study uncovers a distinct, metabolism-dependent mechanism through which TANs drive BC metastasis specifically to the liver. We found that the hepatic microenvironment uniquely upregulates MAT2A in TANs, reprogramming their methionine metabolism flux. Thus, our work extends the understanding of neutrophil-mediated metastasis into the novel dimension of cellular metabolism and provides a fresh mechanistic explanation for the hepatic tropism of BC metastasis and reveals a potential target for tissue-specific therapeutic intervention.

Recent advances in cancer immunology have underscored the significance of immune-related biomarkers in predicting clinical outcomes and guiding therapeutic decision-making (29-31). Among various immune components, neutrophils have emerged as a critical player due to their dual presence in both peripheral circulation and the TME (10,16,32). Numerous studies have demonstrated that elevated neutrophil counts or an increased neutrophil-to-lymphocyte ratio (NLR) are associated with poor prognosis across multiple malignancies, highlighting their potential as accessible and cost-effective prognostic indicators (33-35). Beyond systemic measures, TANs have garnered considerable interest owing to their frequent infiltration across a wide spectrum of solid tumors. This has led to a growing body of literature exploring their intratumoral dynamics and prognostic relevance, suggesting that both circulating and tumor-infiltrating neutrophils may contribute to cancer progression and immune evasion through distinct yet complementary mechanisms. Through single-cell transcriptomic analysis, our team identified neutrophils as the most dynamically altered immune cell population within the liver metastatic microenvironment. Functionally, TANs were shown to promote CSC-like properties in BC cells, including upregulation of stemness markers, impaired differentiation capacity, and increased resistance to chemotherapeutic treatment. These results highlight the potential of TANs as both biomarker and functional driver of BC progression, particularly in the context of liver metastasis.

The metabolic reprogramming of CSCs represents an emerging frontier in oncology research, offering significant therapeutic potential yet remaining underexplored (36,37). Growing evidence suggests that dysregulated metabolic pathways in CSCs play a pivotal role in tumor progression, therapeutic resistance, and poor clinical outcomes (20,38,39). Targeting CSC-specific metabolic vulnerabilities, particularly in BCLM, may thus provide a novel strategy for cancer treatment. Recent studies have highlighted the critical role of methionine metabolism in maintaining CSC properties. For instance, Wang et al. reported that CSCs exhibit unique methionine cycle activity, and its inhibition, even transiently, disrupts CSC self-renewal capacity (20). Complementary work by Gao et al. further demonstrated that dietary methionine restriction enhances therapeutic efficacy in chemoresistant models (40), underscoring the clinical relevance of this pathway. In line with these findings, our study reveals distinct methionine metabolic features in liver metastasis of BC and identifies TANs that promote BC stemness in a MAT2A dependent manner. Although we identified MAT2A as the dominant methionine cycle enzyme linked to TANs, MTR was modestly elevated in BCLM samples. However, its weaker association with stemness phenotypes and TAN infiltration suggested that MAT2A was the primary metabolic mediator in this context. MTHFR, by contrast, showed no significant alterations, highlighting the specificity of the TAN-MAT2A axis. Moreover, in our study, we observed significantly higher IL-6 secretion in TANs compared to peripheral blood neutrophils (Figure 3F), and STAT3 inhibitor (Stattic, 5 µM) reversed TAN-induced MAT2A upregulation in BC cells (Figure 3G), supporting the probability that TANs promote stemness through IL-6/STAT3-mediated MAT2A upregulation. Further studies are warranted to elucidate the additional regulators and detailed molecular mechanisms.

In the evolving landscape of MAT2A-targeted therapies, FIDAS-5, also known as AG-270 (NCT03435250), emerged as the first clinical candidate but was discontinued in 2023, potentially due to efficacy or toxicity limitations, despite showing preliminary activity in MTAP-deficient malignancies including bile duct, pancreatic, and non-small cell lung cancers (NSCLCs) (41). This experience has informed the development of next-generation inhibitors, with current clinical efforts focusing on IDE397 (NCT04794699) for MTAP-deficient NSCLC and bladder cancers, and S095035 (NCT06188702) which entered phase I trials in 2024 with undisclosed indications. These ongoing studies aim to build upon the foundational work of AG-270 while addressing its potential limitations through improved therapeutic windows and biomarker-driven patient selection strategies. Notably, MAT2A has been implicated in modulating CSC chemosensitivity, suggesting that our results align with established mechanisms of metabolic regulation in CSCs. These insights not only reinforce the importance of methionine metabolism in CSC biology but also propose actionable targets for future therapeutic development. Mechanistic exploration in our study revealed that the pro-stemness effects are dependent on methionine metabolism, as co-culture with TANs enhanced methionine cycle activity and induced MAT2A expression in tumor cells. Importantly, pharmacological inhibition of MAT2A significantly attenuated TANs-mediated CSC maintenance. Although MAT2A is indeed ubiquitously expressed as a core enzyme in methionine metabolism, its therapeutic targeting leverages a critical vulnerability that is unique to cancer cells. The uncontrolled proliferation of cancer cells creates an exceptional dependency on MAT2A for survival and growth, while normal cells only require baseline MAT2A activity to maintain basic functions and do not show significant activation of the methionine cycle under similar treatments (Figure 5E). This biological disparity supports tumor-selective targeting, much like the difference between a casual walker and a marathon runner in terms of water consumption. In this analogy, MAT2A inhibitors act as precision tools, selectively disrupting the ’supply lines’ critical for cancer cell survival, while sparing normal cells due to their lower dependency on MAT2A. This targeted approach represents a novel paradigm in cancer treatment by capitalizing on the metabolic addiction of malignant cells. These findings uncover a novel mechanism by which TANs contribute to BC progression and drug resistance through metabolic reprogramming, and highlight methionine metabolism as a potential therapeutic target in BCLM.

Limitation

There are several limitations in this study: (I) the small clinical cohort may constrain the generalizability of our findings, highlighting the need for validation in larger populations; (II) although clinical samples were analyzed to support key observations, in vivo functional validation remains necessary to confirm the mechanistic roles proposed; (III) although we focused on the MAT2A-dependent methionine pathway, additional metabolic and immune pathways may also contribute and warrant further investigation.

Conclusions

We identified a distinct immune microenvironment in BCLM, characterized by enriched neutrophil infiltration. TANs were further shown to promote cancer stemness and chemoresistance by activating methionine metabolism, primarily through upregulation of MAT2A. Pharmacological inhibition of MAT2A mitigated these effects, highlighting methionine metabolism as a potential therapeutic target. These findings emphasize the critical role of TANs and metabolic reprogramming in the progression of BCLM.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the MDAR reporting checklist. Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-aw-2278/rc

Data Sharing Statement: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-aw-2278/dss

Peer Review File: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-aw-2278/prf

Funding: This work was supported by the National Natural Science Foundation of China (No. 82372930 to L.H.); the Innovation and Entrepreneurship Talent Plan of Jiangsu Province (to L.H.); the Young Elite Talent Program of The First Affiliated Hospital with Nanjing Medical University (No. YNRCQN0309 to L.H.); Shanghai Pujiang Program (No. 22PJD042 to D.X.); and Shanghai “Rising Stars of Medical Talents” Youth Development Program.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-aw-2278/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Ethics Committee of The First Affiliated Hospital with Nanjing Medical University (No. 2023-SRFA-408; Date of Approval: March 3rd, 2023). Written informed consent was provided by each participant.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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