SanHuang decoction may suppress breast cancer by regulating M1 macrophage polarization via NF-κB signaling pathway: in vitro and in vivo studies

Introduction

Breast cancer (BC) is one of the most prevalent malignancies in women, accounting for more than 24% of new female cancer cases and about 15% of cancer-related death in the world (1). BC is a leading cause of cancer-related mortality in women globally, posing a significant threat to their health and well-being (2). SanHuang decoction (SHD) is a Chinese herbal formula designed by of Jiangsu Provincial Hospital of Chinese Medicine for the treatment of BC and has been widely prescribed in the clinical treatment of patients with BC. SHD is composed of three herbal medicines: Astragalus membranaceus (30 g), Rheum palmatum (10 g), and turmeric (10 g). Previous studies have indicated that SHD can alleviate preoperative clinical symptoms in patients with BC (3); however, the mechanism by which this occurs has not yet been clarified.

Tumor-associated macrophages (TAMs) are one of the major cellular components in the tumor microenvironment (TME) and play a critical role in tumor pathogenesis (4). TAMs can be broadly classified as antitumorigenic (M1 polarized) or protumorigenic (M2 polarized) based on their ability to either promote or suppress tumor growth (5). The M1 phenotype typically produces tumor necrosis factor (TNF), interleukin (IL)-12 and interferon gamma (IFN-γ), inducible chemokines C-X-C motif chemokine ligand (CXCL) 9, and CXCL10 to exert proinflammatory and antitumor effects (6). Studies have reported that M1 macrophages can significantly promote BC cell apoptosis and decrease invasion (7,8), supporting their potential benefits in patients with BC.

Nuclear factor kappa B (NF-κB) is a crucial transcription factor in cells. Recent research has demonstrated that NF-κB signaling pathway is essential for governing the inflammatory response in cancers and that activating NF-κB pathway is crucial for the activation of M1 TAMs. Thus, NF-κB pathway is considered to be a hallmark of cancer progression and a potential therapeutic target (9). This study focused on evaluating the effects of SHD on BC in vitro and in vivo with the aim of identifying the possible antitumor activities and elucidating the underlying molecular mechanisms of its effect. We present this article in accordance with the MDAR reporting checklist (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1975/rc).

Methods

Chemicals and reagents

Polymethyl methacrylate (PMA) and lipopolysaccharide (LPS) were purchased from MedChemExpress (MCE; Monmouth Junction, NJ, USA). Cell counting kit-8 (CCK-8), bicinchoninic acid (BCA) protein content detection kit, and rabbit anti-GAPDH were obtained from KeyGEN BioTECH (Nanjing, China). IFN-γ, IL-4, and IL-13 were purchased from Novoprotein (Suzhou, China). The primary antibodies anti-IL-6, anti-BCL-2-associated X (BAX), anti-BCL-2, anti-Cas3, anti-IκB, anti-p-P65, anti-P65, were purchased from Abcam (Cambridge, UK). Anti-IκB, anti-TNF-α, anti-IκB, and anti-IL-1β were purchased from Proteintech Group (Rosemont, IL, USA). Transwell chambers were purchased from Corning Incorporated (Corning, NY, USA).

SHD preparation

Radix astragali Preparata (300 g), turmeric (100 g), and cooked rhubarb (100 g) were added with 8 times the amount of distilled water, and the solution was filtered and concentrated with a rotary evaporator until it contained 2 kg·L^-1 of raw medicine. The concentrated solution was freeze-dried to obtain 23 g of freeze-dried powder (raw medicine amount 10 g·g^-1) by the Teaching and Research Office of Traditional Chinese Medicine Processing at Nanjing University of Chinese Medicine (Nanjing, China).

Cell culture and macrophage polarization

MDA-MB-231 cells, a triple-negative BC cell line, were obtained from the National Collection of Authenticated Cell Cultures of China (Shanghai, China) and were cultured in 90% L-15 medium supplemented with 10% fetal bovine serum (FBS). THP-1 cells were purchased from Sunncell (cat. no. SNL-044; Wuhan, China). THP-1 monocytes were cultured in 90% RPMI-1640 medium supplemented with 10% FBS. The THP-1 cells were differentiated into M0 macrophages via incubation in 5 ng/mL of PMA for 48 hours, and then the cells were treated with 100 ng/mL of LPS and 2.5 ng/mL of IFN-γ for 48 hours to obtain M1 macrophages.

Cell coculture with transwell

Coculture was performed with transwell chambers with 0.4 µm of polyvinylidene fluoride (PVDF) membrane. The upper chamber was seeded with 1×10⁵ of MDA-MB-231 cells, while 1×10⁴ of M0 macrophages and M1-polarized macrophages were seeded in the lower chamber. Forty-eight hours later, MDA-MB-231 cells were collected and used for subsequent experiments.

CCK-8 assay of the cell proliferation inhibition rate

After coculture for 48 hours, MDA-MB-231 cells were collected, and the cell proliferation inhibition rate was measured by CCK-8. MDA-MB-231 cells were seeded in 96-well plates at a density of 6×10⁴ cells/ mL and then treated with different concentrations of SHD (0, 0.1, 0.5, 1, 5, 10, 20, and 40 mg/mL) for 48 and 72 hours. After three washes with phosphate-buffered saline (PBS), cells were incubated with 5 µL of CCK-8 solution for 2 hours, the optical density value at 450 nm was measured with a microplate reader, and the inhibition rate was calculated.

Inhibitionrate=negativecontrolgroup−experimentalgroupnegativecontrolgroup×100%[1]

Wound-healing assay

After coculture for 48 hours, cells were treated with 10 mg/mL of SHD for 72 hours. MDA-MB-231 cells from the cocultures were collected and seeded in six-well plates. When MDA-MB-231 cell monolayer cultures reached approximately 80% confluency, scratches were made across the plates with a pipette tip. After plates were washed with PBS, medium was added, MDA-MB-231 cells were grown for 0 and 24 hours, and photographs (×100) were obtained at different time points.

Transwell assay

After coculture for 48 hours, cells were treated with 10 mg/mL of SHD for 72 hours, and then MDA-MB-231 cells from the cocultures were collected and cultured for 24 hours. The upper side of the basement membrane of the transwell chambers was coated with 30 µL of Matrigel dilution and dried at 37 ℃ for 120 min. After a conventional detachment process, the cells were collected and counted, with the cell density being adjusted to 1×10⁵ cells/mL. Subsequently, 100 µL of cell suspension was added into the apical transwell chamber, while 500 µL of culture medium with 20% FBS was added to the basolateral chambers. After a conventional culture for 24 hours, the transwell chambers were subsequently removed, and crystal violet staining was applied at 37 ℃ for 30 min. Three visual fields were selected on a random basis under an inverted microscope (×200), and images were obtained. The number of cells that migrated through the membrane was counted. Three replicate wells were set for each group, and the average value was calculated.

Immunofluorescence

MDA-MB-231 cells from the cocultures were collected and counted. Cells were blocked for 10 min with 3% H₂O₂ methanol solution at room temperature and then incubated with anti-CD68 (1:500), anti-CD86 (1:100), and anti-CD206 (1:100) overnight at 4 ℃. Subsequently, 50 µL of TRITC-labeled sheep anti-mouse polymer and FITC-labeled sheep anti-rabbit polymer were added for 1 hour at 37 ℃. After this, 100 µL of Hoechst stain was added for color development of hematoxylin counterstaining for 5 min and followed by three washes with PBS. The protein expression was observed under a light microscope, and three areas with high expression were identified and photographed (×200).

Western blot assay

After coculture for 48 hours, MDA-MB-231 cells from the coculture were collected, and proteins were extracted and collected via RIPA lysis buffer, mixed with 10 µL of phosphatase inhibitor, 1 µL of protease inhibitor, and 5 µL of 100-mM phenylmethylsulfonyl fluoride. The protein concentrations of each group were determined with a BCA kit. Subsequently, electrophoresis was conducted, blocking was performed with 10% milk powder for 2 hours, and incubation with primary antibodies anti-BAX (diluted to 1:5,000), anti-BCL-2 (diluted to 1:2,000), anti-cleaved caspase 3 (diluted to 1:2,000), anti-P65 (diluted to 1:2,000), anti-p-P65 (diluted to 1:5,000), anti-IκB (diluted to 1:1,0000), anti-p-IκB (diluted to 1:500), and anti-IL-6 (diluted to 1:1,000) was completed. After another wash, secondary antibodies were added and incubated for 2 hours at room temperature. ImageJ software (US National Institutes of Health, Bethesda, MD, USA) was used to analyze the gray scale.

Tumor size of patients with BC

Forty patients with BC undergoing neoadjuvant chemotherapy at Jiangsu Provincial Hospital of Chinese Medicine were enrolled. Patients were randomly divided into a control group and an observation group. The control group received neoadjuvant chemotherapy (NACT) while the observation group received NACT and SHD orally for 9 weeks. Breast magnetic resonance imaging (MRI) plain scans and enhanced scans were performed, and the length of the largest lesion of the tumor was measured to evaluate the tumor size. This study was approved by the Ethics Committee of Affiliated Hospital of Nanjing University of Chinese Medicine (Jiangsu Province Hospital of Chinese Medicine) (ethics number: 2022NL-031), and informed consent was obtained from all patients. The study conformed to the provisions of the Declaration of Helsinki and its subsequent amendments.

Immunofluorescence staining of tumor tissue

The sections of BC tissue from treated patients were first immersed in anhydrous alcohol for 5 min. They were then sequentially treated with 95%, 85%, and 75% alcohol (I and II) for 5 min each. The sections were placed in repair solution and heated to boiling (95 ℃) for 15–20 min and then subjected to high heat for 4 min and low heat for 10 min. After natural cooling for more than 20 min to room temperature, 10% bovine serum albumin (BSA) sealing solution was applied, and the sections were incubated at room temperature for 30 min. Appropriate amounts of anti-CD68 and anti-CD86 antibodies were added, and the sections were incubated overnight at 4 ℃. Horseradish peroxidase-labeled secondary antibodies were then applied and incubated at room temperature for 50 min. Tyramide signal amplification fluorescent dye reaction solution was added to the sections and allowed to react at room temperature for 1–15 min. After the sections were slightly dried, they were sealed with anti-fluorescence quencher (including DAPI). Protein expression was observed under a light microscope, and three areas with high expression were selected, photographed, and stored. All images were taken at ×400 magnification.

Statistical analysis

Statistical analysis was performed with GraphPad Prism 8 (Dotmatics, Boston, MA, USA), and the results were compared via one-way analysis of variance (ANOVA). All data are expressed as the mean ± standard deviation (SD), with a P value <0.05 being considered statistically significant.

Results

Successful establishment of M0 and M1 macrophages

CD68+ and CD86 double labels were used to detect M1 macrophage marker proteins. The immunofluorescence results showed that after stimulation with 5 ng/mL of PMA for 48 hours and further induction for 48 hours with 100 ng/mL of LPS and 2.5 ng/mL of IFN-γ, CD68-positive cells were present in M0 macrophages (M0/THP-1), while CD68- and CD86-positive cells were present in M1 macrophages (Figure 1). This indicated that THP-1 cells were successfully differentiated into M1-polarized macrophages (M1/THP-1).

Figure 1 Double staining of CD68 (red fluorescence) and CD86 (green fluorescence) in M0 and M1 macrophages (×20).

Effect of SHD on cell proliferation

After coculture with M0/THP-1 or M1/THP-1 for 48 hours, MDA-MB-231 cells from the cocultures were collected and treated with different concentrations of SHD (0, 0.1, 0.5, 1, 5, 10, 20, and 40 mg/mL) for 48 hours and 72 hours. CKK-8 assay indicated that the coculture with M1/THP-1 had a greater inhibitory effect on MDA-MB-231 cell proliferation than did coculture with M0/THP-1, and the inhibition rates of 10 mg/mL SHD and 20 mg/mL SHD for 72 hours, respectively, were significant (P<0.05). Given the toxicity of SHD at a high concentration, the subsequent experiments included 10 mg/mL of SHD intervention for 72 hours as the experimental condition (Figure 2).

Figure 2 CCK-8 detection of the inhibitory effects of the SHD at different concentrations on MDA-MB-231 cells. **, P<0.01 compared with MDA-MB-231 + M0 for 48 hours with SHD (0. 1 mg/mL); ^##, P<0.01 compared with MDA-MB-231 + M0 for 48 hours with same concentration of SHD. CCK-8, cell counting kit-8; SHD, SanHuang decoction.

Effect of SHD on the migration of MDA-MB-231 cells

Wound-healing and transwell assays were implemented to assess the ability of SHD to inhibit the migration of MDA-MB-231 cells. After MDA-MB-231 cells were cocultured with M0/THP-1 or M1/THP-1 for 48 hours, 10 mg/mL of SHD was added for 72 hours, then MDA-MB-231 cells were collected and inoculated into a six-well plate and cultured for 24 hours, then the cells were scratched with pipette tips (shown in Figure 3). As shown in Figure 3A, compared with the migration rate in the MDA-MB-231 + M0/THP-1 group, those in the MDA-MB-231 + M0/THP-1 + SHD, MDA-MB-231 + M1/THP-1, and MDA-MB-231 + M1/THP-1 + SHD groups were significantly lower (P<0.01). Meanwhile, compared with the MDA-MB-231 + M0/THP-1 + SHD group, the MDA-MB-231 + M1/THP-1 + SHD group showed a greater inhibition in migration (P<0.01). As shown in Figure 3B, the number of migrated cells in the MDA-MB-231 + M0/THP-1 + SHD, MDA-MB-231 + M1/THP-1, and MDA-MB-231 + M1/THP-1 + SHD groups was significantly lowered compared with that in the MDA-MB-231 + M0/THP-1 group. This result confirmed that M1/THP-1 and SHD exerted a significant inhibitory effect on the migration of MDA-MB-231 cells, while SHD combined with M1/THP-1 exhibited more significant effects in this regard.

Figure 3 Effect of SHD on MDA-MB-231 migration ability. (A) Images obtained under an inverted microscope (×100) after the wound-healing assay. (B) After crystal violet staining, images were obtained under an inverted microscope (×200) and the number of cells that migrated through the membrane was counted. **, P<0.01 compared with the MDA-MB-231 + M0/THP-1 group; ^##, P<0.01 compared with the MDA-MB-231 + M0/THP-1 + SHD group. SHD, SanHuang decoction.

Effect of SHD on regulating the inflammation and apoptosis of MDA-MB-231 cells

After coculturing, the ability of SHD to regulate the inflammation and apoptosis of MDA-MB-231 cells was analyzed via Western blotting. As shown in Figure 4, compared with the MDA-MB-231 + M0/THP-1 group, the expressions of IL-6, TNF-α, IL-1β, BAX and cleaved-caspase 3 were significantly upregulated while BCL-2 was decreased in other groups. In addition, M1/THP-1 produced a stronger regulatory effect than did SHD in pro-inflammatory, confirming that M1 can regulate inflammatory responses in the TME.

Figure 4 Regulation of M1 macrophage-related secretion factor expression in the microenvironment of MDA-MB-231 cells by SHD. (A) Effect of SHD on inflammation-related factors. (B) Effect of SHD on apoptosis-related factors. **, P<0.01 compared with the MDA-MB-231 + M0/THP-1 group; ^##, P<0.01 compared with the MDA-MB-231 + M0/THP-1 + SHD group. BAX, BCL-2-associated X; BCL-2, B-cell lymphoma 2; IL-1β, interleukin-1 beta; IL-6, interleukin-6; SHD, SanHuang decoction; TNF-α, tumor necrosis factor alpha.

SHD’s regulation of the NF-κB signaling pathway in MDA-MB-231 cells

To further investigate the mechanism by which SHD inhibits the migration of MDA-MB-231 cell from the cocultures, the effect of SHD on NF-κB phosphorylation was assessed via Western blot analysis. As shown in Figure 5, the phosphorylation of P65 and IκB was significantly upregulated in the MDA-MB-231 + M0/THP-1 + SHD, MDA-MB-231 + M1/THP-1, and MDA-MB-231 + M1/THP-1 + SHD groups; in this regard, the combination of SHD and M1/THP-1 produced a significantly greater effect, which was consistent with the previously mentioned findings. Due to these results, we speculated that SHD could activate NF-κB signaling pathway, strengthen the expression of cytokines of M1/THP-1, promote an inflammatory response in the TME, and thus regulate cell apoptosis.

Figure 5 Expression of NF-κB signaling pathway in the cells of each group. **, P<0.01 compared with the MDA-MB-231 + M0/THP-1 group; ^#, P<0.05, ^##, P<0.01 compared with the MDA-MB-231 + M0/THP-1 + SHD group. SHD, SanHuang decoction.

Effect of SHD on tumor size in patients treated with NACT

Tumor size was measured via breast MRI scanning with enhancement before treatment and after 9 weeks of treatment in the NACT + SHD group and NACT group. As shown in Figure 6, the difference in tumor size between the two groups at 0 weeks was not statistically significant. After 9 weeks of treatment, the tumor size of both groups was significantly reduced (P<0.01), with the tumor size reduction being significantly greater in the NACT + SHD group than in the NACT group (P<0.05).

Figure 6 MRI evaluation of tumor size (n=20). **, P<0.01 compared with pretreatment; ^#, P<0.05 compared with control group in the same period. MRI, magnetic resonance imaging; NACT, neoadjuvant chemotherapy; SHD, SanHuang decoction.

Effect of SHD on the regulation of M1 macrophages in patients with BC

In order to examine SHD’s regulation of M1 macrophages, immunofluorescence staining of tumor tissue was carried out after surgery. Immunofluorescence staining (Figure 7) indicated that CD68-positive cells were present in the two groups. However, in the NACT group, CD86 expression was weak, while that in the NACT + SHD group was relatively strong. We thus concluded that M1 macrophages are highly expressed in the tumor tissues of patients with BC. Taken together, these results indicated that SHD ameliorates BC development in vivo, potentially via the regulation of M1 macrophages polarization.

Figure 7 Immunofluorescence double staining of CD68 (red fluorescence) and CD86 (green fluorescence) in the M1 macrophages of tumor tissues (×20) (n=6). NACT, neoadjuvant chemotherapy; SHD, SanHuang decoction.

Discussion

BC, due to its high mortality and incidence rates, remains a major health concern among women. Even with adjuvant chemotherapy, the 5-year survival rate for metastatic BC is less than 30% (10). Early symptoms of BC are often not obvious, making it difficult to detect. Thus, the majority of patients with BC are at advanced stage of disease when they are diagnosed. Treatment at this stage is challenging, often requiring NACT to reduce the size of tumor, followed by surgical treatment to achieve the goal of radical resection (11,12). NACT has been demonstrated to be of considerable clinical value in the treatment of locally advanced and inoperable BC (13). It works specifically by damaging the cancer cells or slowing their growth, yet patients often discontinue treatment due to the associated adverse events and drug resistance (14). Studies have shown that traditional Chinese medicine interventions have unique advantages in BC treatment (15,16). SHD is a Chinese herbal formula that is commonly used in the Breast Surgery Department of Jiangsu Province Hospital of Chinese Medicine. Previous studies have confirmed that this formula can significantly improve perioperative clinical symptoms and enhance the postoperative quality of life of patients with BC (3). In this study, we found that the tumor size was reduced in patients treated with NACT and SHD for 9 weeks, with this reduction being more pronounced in the group treated with both NACT and SHD compared with that treated with NACT alone. This suggests that SHD exerts a valuable therapeutic effect. Clarifying the mechanism of action of SHD in BC can provide experimental evidence for its application in preoperative chemotherapy.

TAMs are the most critical effector cells of innate immunity and the most abundant tumor-infiltrating immune cells. They play a key role in the clearance of apoptotic bodies, regulation of inflammation, and tissue repair in maintaining homeostasis in vivo (17). Research has demonstrated that TAMs are one of the main cellular components of the TME and play a crucial role in the pathogenesis of tumors (4). In recent years, TAMs have gained widespread attention as a potential target for cancer therapy (18,19). However, due to the related severe adverse reactions and low specificity, TAM-targeted therapies have not been widely applied in clinical practice (20). Further development in this field may require the discovery of novel molecular targets. Studies have shown that macrophages can be activated in response to certain signals in the TME and can be further divided into two main phenotypes: M1 macrophages (classically activated macrophages) and M2 macrophages (alternatively activated macrophages). M1-polarized macrophages have been described as being highly phagocytic and highly inflammatory, promoting antitumor immune responses in the TME by secreting nitric oxide, reactive oxygen species, and inflammatory cytokines including TNF-α, IL-1β, and IL-6 (21-23); meanwhile, M2 macrophages are mainly characterized by their production of IL-10 and Arg-1, which are known to inhibit inflammatory responses and promote tumor progression (24,25). In the TME, tumor cells promote the polarization of macrophages toward M2 TAMs (26). Therefore, selective depletion of TAMs and the reprogramming pf immunosuppressive M2-like TAMs into antitumor M1 phenotypes have emerged as approaches in antitumor therapy (27,28). THP-1-generated macrophages have been recognized as a reasonable model of macrophage polarization (29), and human M1 macrophages can be identified by the surface marker CD86 (30). As shown in Figure 1, we used immunofluorescence to examine the expression of CD86 in THP-1-derived M1 macrophages as a marker for M1/THP-1.

To determine whether SHD can regulate BC through macrophage polarization, we performed immunofluorescence assays, which showed a highly positive expression of CD86 in the NACT + SHD group but a relatively weak expression in the NACT group. These findings indicate that SHD may promote M1 macrophage polarization in BC tissue. For the in vitro study, MDA-MB-231 cells were cocultured and treated with SHD, and cell proliferation was observed. We found that SHD and M1/THP-1 could both inhibit MDA-MB-231cell proliferation, but SHD showed a more significant effect. Moreover, coculture with M1/THP-1 significantly increased the inhibitory effect of SHD, which suggests that the mechanism of SHD in regulating the proliferation of BC cells may lie in the polarization of macrophages toward M1 TAMs.

To further clarify the mechanism by which SHD regulates macrophage polarization, we detected the expression of IL-6, IL-1β, and TNF-α via Western blotting. The results indicated that the protein expression of inflammatory factors increased significantly after the intervention of SHD or M1/THP-1. M1-polarized macrophages exerted a greater effect on the expression of inflammatory factors than SHD, which is consistent with the proinflammatory effect demonstrated by M1/THP-1. In another study, it was found that M1 macrophages could promote the apoptosis of mouse bladder cancer cells (23), but their effect on BC cells is unclear. To elucidate the mechanism of apoptosis in BC cells, we further examined the expression of apoptosis-related proteins. Apoptosis is mainly effectuated by a family of caspases, among which caspase 3 is particularly prominent (31). The BCL-2 family proteins are key regulators of apoptosis, and antiapoptotic proteins (BCL-2 and BCL-xL) can block apoptosis by inhibiting their proapoptotic counterparts; moreover, proapoptotic proteins (BAX and BAD) can facilitate this process by promoting the mitochondrial release of cytochrome c, ultimately resulting in the cleavage of critical cellular proteins (32). In our study, SHD and M1/THP-1 also promoted the expression of proapoptotic proteins including BAX and caspase 3 while inhibiting the expression of antiapoptotic proteins including BCL-2, thus promoting the apoptosis of BC cells.

NF-κB is a crucial transcription factor in cells, playing a significant role in tumor-associated inflammation and malignant progression. Studies on mouse models of cancer have shown that the activation of NF-κB is critical in driving cancer-associated inflammation. The mammalian NF-κB family consists of five transcription factors—p65 (also known as RelA), RelB, c-Rel, p105/p50 (NF-κB1), and p100/p52 (NF-κB2) (33)—with the NF-κBp65 dimer being its most common form. IκB proteins can suppress the activation of NF-κB dimers by interacting with the nuclear localization signal (NLS) of the highly conserved Rel homology domain (RHD) (16). The canonical pathway is rapidly triggered by proinflammatory stimuli; these include the cytokines TNF-α and IL-1β, the bacterial component LPS, and antigens, which stimulate a cascade of receptor-proximal signaling events leading to the activation of an IκB kinase (IKK) complex composed of IKKα, IKKβ, and NF-κB essential modulator (NEMO; also referred to as IKKγ) (34). NF-κB can transcribe apoptotic genes and synthesize apoptosis-related proteins, regulating cellular apoptosis. In our study, after intervention with SHD and M1/THP-1, the activation levels of NF-κB pathway indicator-related factors in MDA-MB-231 cells significantly increased in each group, suggesting that the regulatory effect of SHD on the coculture system of MDA-MB-231 cells and M1-polarized macrophages is mediated by the NF-κB signaling pathway.

Conclusions

SHD can enhance the expression of M1-polarized macrophage-related cytokines, promote the inflammatory response in the TME, and thus inhibit the proliferation of MDA-MB-231 cells. This regulation of BC’s pathological process is likely accomplished through activation of NF-κB signaling pathway. It is hoped that the findings from this study can contribute to the improvement of NACT in patients with BC.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the MDAR reporting checklist. Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1975/rc

Data Sharing Statement: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1975/dss

Peer Review File: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1975/prf

Funding: This research was funded by the Nantong Municipal Health Commission General Project (MS2024098), Nanjing University of Chinese Medicine Natural Science Foundation (XZR2024230), and Jiangsu Provincial Hospital of Traditional Chinese Medicine Hospital-Level Research Project (Y2024033).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1975/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. The study was approved by the Ethics Committee of Affiliated Hospital of Nanjing University of Chinese Medicine (Jiangsu Province Hospital of Chinese Medicine, ethics number: 2022NL-031) and informed consent was taken from all the patients. The study conformed to the provisions of the Declaration of Helsinki and its subsequent amendments.

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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(English Language Editor: J. Gray)