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. 2026 Jul 3;20:544583. doi: 10.2147/DDDT.S544583

Metformin Sensitizes HR+/HER2- Breast Cancer Cells to CDK4/6 Inhibitor via Suppressing of PI3K/AKT/mTOR Signaling Pathway

Lin Zhu 1, Jiawen Yang 1, Fengmei Li 2, Dandan Wang 1, Wenda Zhang 1, Jingmin Zhang 1, Zhenfeng Duan 3, Youhong Hu 1, Yubing Zhou 1,✉
PMCID: PMC13340342  PMID: 42415939

Abstract

Purpose

CDK4/6 (Cyclin-dependent kinase 4/6) inhibitors are recommended as the first-line treatment for Hormone receptor-positive/human epidermal growth receptor 2-negative (HR+/HER2-) breast cancer, but their efficacy is limited. Metformin, a widely used antidiabetic drug, has demonstrated antitumor potential and can enhance the efficacy of various cancer therapies. Nevertheless, its specific role in sensitizing HR+/HER2- breast cancer to CDK4/6 inhibition and the underlying mechanisms remain poorly defined.

Methods

In this study, the combined anti-HR+/HER2- breast cancer effects of metformin with CDK4/6 inhibitor palbociclib and its potential mechanisms were investigated using human HR+/HER2- breast cancer cell lines in vitro and transplanted tumor mouse models in vivo.

Results

Our study demonstrated that the combination of metformin and palbociclib synergistically inhibited proliferation and migration, and promoted apoptosis in HR+/HER2- breast cancer cells. This synergistic anticancer effect was linked to suppression of the PI3K/AKT/mTOR pathway. Mechanistically, metformin downregulated CDK4 protein expression, and its combination with palbociclib led to a more profound suppression of Cyclin D1 and E2F, along with enhanced inhibition of Rb phosphorylation. Moreover, the metformin-palbociclib combination synergistically suppressed tumor growth in vivo.

Conclusion

Our findings highlighted that metformin could serve as an adjunctive therapy alongside CDK4/6 inhibitors, offering a promising synergistic treatment strategy for HR+/HER2- breast cancer.

Keywords: metformin, CDK4/6 inhibitor, palbociclib, HR+/HER2- breast cancer, PI3K/AKT/mTOR pathway

Graphical Abstract

Metformin and CDK4/6 inhibitors affect PI3K-AKT-mTOR and Cyclin D1-CDK4-Rb-E2F pathways. A cell signaling diagram labeled extracellular space, cytoplasm and nucleus, with a legend showing arrows for producing or promoting and inhibiting. Metformin is shown entering through a membrane channel and inhibiting PI3K. PI3K connects by a downward arrow to AKT and AKT connects by a downward arrow to mTOR. mTOR connects by a rightward arrow toward the nucleus and the label cell proliferation. AKT also connects by a rightward arrow to Cyclin D1. Cyclin D1 is linked to CDK4 and CDK4 connects by a rightward arrow to Rb in the nucleus. Rb connects by a downward arrow to E2F. E2F connects by a leftward arrow toward the label cell proliferation. CDK4/6 inhibitor is shown inhibiting Cyclin D1 and inhibiting a separate mitochondrial oval containing BCL-2 and MCL-1. Metformin is also shown inhibiting CDK4. The mitochondrial oval connects by a rightward arrow toward the label cell apoptosis.

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Introduction

HR+/HER2- breast cancer is the most prevalent type of breast cancer.1 Investigations have shown that a significant proportion of patients with HR+/HER2- breast cancer present at advanced stages.2 In 2015, the approval of CDK4/6 inhibitors added an additional option for the systemic treatment of patients with advanced breast cancer.3 Currently, the US Food and Drug Administration (FDA) has approved three selective CDK4/6 inhibitors, including palbociclib, ribociclib, and abemaciclib, for the treatment of HR+/HER2- metastatic breast cancer.4 However, with several years of clinical experience, CDK4/6 inhibitors have increasingly revealed certain limitations. The use of CDK4/6 inhibitors leads to a high frequency and incidence of myelosuppression, leukopenia and neutropenia.5 Additionally, resistance to CDK4/6 inhibitors is a growing concern, probably due to mutation-activated epigenetic changes in the tumor cells.6 Over time, side effects and resistance to CDK4/6 inhibitors have progressively diminished their clinical efficacy. Therefore, it is of great significance to search for some new effective and low-toxic treatments or drugs to enhance the efficacy of CDK4/6 inhibitors and treat HR+/HER2-breast cancer.

Metformin (N, N-dimethylbiguanide) is the classic antihyperglycemic agent for the treatment of type 2 diabetes. Metformin has evolved over the years to become an effective and safe drug. A large number of studies in recent years have found that metformin has anti-tumor effects.7 The main anti-tumor mechanism of metformin is related to the activation of adenosine monophosphate-activated protein kinase (AMPK) and inhibition of insulin receptor (IR) and insulin-like growth factor receptor 1 (IGF-R1).8,9 Currently, several clinical trials on Metformin have been conducted for various types of cancers such as breast cancer, ovarian cancer, esophageal squamous cell carcinoma (ESCC).10–12 These studies reflect a growing interest in the potential of metformin as a cancer therapeutic. The majority of these trials are concentrated on evaluating metformin as an adjuvant therapy.13 The anticancer effect of metformin was found to be strongly correlated with dose and duration of administration.14,15 Low doses of metformin may not be sufficient to achieve strong tumor-killing ability. Metformin is increasingly recognized for its valuable role as an adjunctive therapy in combination with antitumor drugs.16 The potential of metformin to enhance the efficacy of chemotherapy, radiotherapy and targeted therapy has been demonstrated in recent preclinical studies and clinical trials.17–20 Therefore, the emerging role of metformin in oncology is underscored by its ability to complement existing cancer treatments.

A large-scale prospective study has revealed a significant association between metformin use and a reduced risk of breast cancer development.21 While metformin has shown promise as an anti-cancer adjunct, a critical unanswered question is whether and how it can be effectively leveraged to improve the efficacy of CDK4/6 inhibitors in HR+/HER2- breast cancer. Previous studies have not systematically investigated this specific combination nor delineated the mechanistic basis for any potential synergy. Crucially, it remains unknown if metformin’s action intersects with the known pathways to CDK4/6 inhibition, such as the CDK4/6 axis. In this study, we investigated the synergistic antitumor effects and potential mechanisms of metformin in combination with the CDK4/6 inhibitor palbociclib on HR+/HER2- breast cancer cell lines in vitro and transplanted tumor mice models in vivo.

Materials and Methods

Cell Culture and Reagents

The HR+/HER2- breast cancer human-derived cell lines MCF7 and T47D, and the mice-derived breast cancer cell line EMT6 were selected for this study. MCF7, T47D and EMT6 cells were purchased from Shanghai QuiCell Biotechnology Company. T47D cells was cultured in DMEM (Solarbio, China) containing 10% fetal bovine serum (FBS, Biological Industries, Israel). MCF7 and EMT6 cells were cultured in RPMI-1640 (Solarbio, China) with 10% FBS. The media all contained penicillin (100 U/mL) and streptomycin (0.1 mg/mL). Cultures were maintained at 5% CO2, 37 °C.

Metformin hydrochloride (Beyotime, China) was dissolved in phosphate buffer solution (PBS, Servicebio, China). Palbociclib (MedChemExpress, USA) was dissolved in dimethyl sulfoxide (DMSO, Solarbio, China) and diluted with medium in vitro, and dissolved in 0.5% CMC-Na in vivo. D-luciferin (Meilunbio, China) was dissolved in DMSO and diluted with medium.

Cell Proliferation Assay

MCF7 and T47D cells were seeded into 96-well plates at densities of 4000 and 12,000 cells per well, respectively, and incubated with or without treatment 48 or 72 hours. Cell Counting Kit-8 (CCK-8) was added (10 μL/well) and incubated for 2 hours before measuring absorbance at 450 nm using a microplate reader (Bio Tek, USA). The combinatorial index (CI) was calculated using CompuSyn software (ComboSyn Inc., USA).

The ability of the cells to proliferate was detected using the colony formation assay and EdU assay. MCF7 and T47D cells were plated in 6-well plates at a density of 600 and 1000 cells per well and then continued to be cultured with or without drugs for 14 days, with fluid changes and observation of cell status every 3 days. After cloning was completed, the clones were fixed by 4% paraformaldehyde and stained with crystal violet, then photographed and counted. Furthermore, the 5-ethynyl 2′-deoxyuridine (EdU) proliferation assays were conducted according to the Cell Proliferation Kit with Alexa Fluor 555 (Beyotime, China) manufacturer’s directions and photographed under a fluorescence microscope (Leica DMi8, USA).

Immunofluorescence Assay

MCF7 and T47D cells were plated in 24-well plates at a density of 15,000 and 30,000 cells per well and treated with or without drugs for 72 hours. Post-treatment, the cells were fixed with 4% paraformaldehyde and permeabilized with 0.5% TritonX-100 (Beyotime, China) and blocked in blocking buffer for immunostaining (Beyotime, China). After blocking, the cells were incubated with Ki67 (Proteintech, China) or β-Tubulin (Zenbio, China) primary antibody overnight. Then, after incubation CoraLite 594 (Red) conjugated goat anti-rabbit antibody or CoraLite 488 (Green) conjugated goat anti-mouse antibody (Proteintech, China) at room temperature and protected from light for 1h, the cells were washed and sealed by anti-fluorescent attenuation sealer with DAPI (Solarbio, China). Finally, they were then photographed by fluorescence microscope.

Cell Migration and Invasion Assays

For the wound healing assay, MCF7 and T47D cells were seeded in 6-well plates at densities of 45,000 and 60,000 cells per well, respectively, incubated overnight, and then scratched with a 200 μL pipette tip. After PBS wash, cells were incubated in 5% FBS medium ± drugs for 48 hours. The scratches were photographed under a microscope (ZEISS, Germany). Relative migration rate was calculated as: (wound width at the start time point - wound width at the end time point) / wound width at the start time point × 100%.

For transwell assay, following a 72-hour drug pretreatment in 6-well plates, MCF7 and T47D cells were seeded at densities of 4×105 and 6×105 cells per well, respectively, into the upper chambers of transwell plates. The assay was conducted with 100 μL of 2% FBS medium in the upper chamber and 600 μL of 20% FBS medium in the lower chamber. After 24 hours, cells on the underside were fixed and stained with crystal violet. For invasion, the upper chamber was precoated with matrigel (ABW Matrigengel, China), polymerized at 37 °C for 4 hours, and then cells were added. Migration and invasion were quantified using ImageJ-Fiji software (NIH, USA).

Cell Apoptosis Analysis

After treating the cells with drugs for 72 hours, apoptotic cells were detected using the PE Annexin V Apoptosis Detection Kit (BD, USA). Briefly, collected cells were resuspended in binding buffer and stained with PE Annexin V (Excitation/Emission: 488/575 nm) and 7-AAD (Excitation/Emission: 488/655 nm) for 15 minutes at room temperature in the dark. Unstained cells and single‑stained controls (Annexin V only, 7‑AAD only) were included in each experiment for accurate fluorescence compensation and quadrant setting. Flow cytometry data were acquired on a BD FACSVerse flow cytometer equipped with a 488-nm laser, using the appropriate filter configurations for PE (575/25 nm BP filter) and 7-AAD (695/40 nm BP filter). During analysis, a sequential gating strategy was applied: cell populations were first gated on FSC-A vs. SSC-A to exclude debris, followed by FSC-H vs. FSC-A gating to exclude doublets, and finally, the single-cell population was analyzed for Annexin V and 7-AAD signal to distinguish viable, early apoptotic, late apoptotic, and necrotic populations. Data were processed using FlowJo 10.8.1 software (BD, USA).

Cell Cycle Analysis

After 72 hours of drug treatment, the cell cycle was analyzed. Briefly, cells were collected, fixed in 70% ice-cold ethanol overnight at 4 °C, and then treated with RNase A. The cell nuclei were stained with Propidium Iodide (PI; Excitation/Emission: 488 nm/617 nm) using the Cell Cycle and Apoptosis Analysis Kit (Servicebio, China) according to the manufacturer’s protocol. Unstained cells were used to set the baseline fluorescence. Data were acquired on a BD FACSVerse flow cytometer equipped with a 488-nm laser and a 586/42 nm BP filter for PI detection. During analysis, a sequential gating strategy was applied: the primary cell population was first gated on FSC-A vs. SSC-A to exclude debris, followed by FSC-H vs. FSC-A gating to rigorously exclude doublets and select single cells for accurate DNA content analysis. The cell cycle distribution (G0/G1, S, and G2/M phases) was modeled from the PI-area (PI-A) histogram of the singlet population using ModFit LT 5.0 software (Verity Software House, USA).

Transcriptome Sequencing

Total RNA was isolated using Trizol reagent (Thermo Fisher, USA) according to the manufacturer’s instructions. RNA quality and purity were assessed using the Bioanalyzer 2100 and RNA 6000 Nano LabChip Kit (Agilent, USA), with high-quality samples (RIN > 7.0) selected for library preparation. mRNA was then purified from 5 µg of total RNA using Dynabeads Oligo (dT) (Thermo Fisher, USA) through two rounds of purification. The mRNA was fragmented using divalent cations at 94 °C for 5–7 min. Fragmented RNA was reverse-transcribed into cDNA using SuperScript™ II Reverse Transcriptase (Invitrogen, USA), followed by second-strand synthesis with E. coli DNA polymerase I (NEB, USA), RNase H (NEB, USA), and dUTP Solution (Thermo Fisher, USA). The cDNA ends were A-tailed and ligated to dual-index adapters with T-overhangs. Size selection was performed using AMPureXP beads. U-labeled strands were treated with UDG enzyme (NEB, USA), and the library was amplified by PCR (95 °C for 3 min; 8 cycles of 98 °C for 15 sec, 60 °C for 15 sec, 72 °C for 30 sec; final extension at 72 °C for 5 min). The final cDNA library had an average insert size of 300±50 bp and was sequenced (2×150 bp PE150) on an Illumina Novaseq™ 6000 following standard protocols.

Western Blot Analysis

After 72 h of drug treatment, cells were harvested and lysed using RIPA lysis buffer (Beyotime, China) with protease (Solarbio, China) and phosphatase inhibitors (MedChemExpress, USA). Protein concentration was measured by BCA protein assay kit (Solarbio, China). Proteins separated by SDS-PAGE were transferred to PVDF (Merck Millipore, Germany) and blocked with Protein Free Rapid Blocking Buffer (Epizyme, China). Subsequently, the strips were incubated with the corresponding primary antibody (Supplementary Table 1) overnight at 4 °C. Secondary antibodies Dylight-conjugated anti-rabbit or anti-mouse IgG (Immunoway, China) were applied for 1 hour. Membranes were imaged using the LI-COR Odyssey CLx with 680 nm for mouse and 800 nm for rabbit antibodies.

Animal Experiment

All animal studies were ethically approved by Zhengzhou University Academy of Medical Sciences (2023101701). All procedures were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and complied with the 3R principles (Replacement, Reduction, Refinement) to ensure animal welfare. The EMT6 mice breast cancer cells expressing luciferase were constructed. Four-week-old female BALB/c mice were purchased from Beijing Vital River Laboratory Animal Technology Co. and raised under standard conditions. After more than a week of adaptation, mice were injected with 1×106 Luc-EMT6 mouse breast cancer cells subcutaneously (diluted in serum-free medium). When tumor reached 100 mm3, the mice were randomly divided into four groups (n=6): (i) control (PBS), (ii) metformin (100 mg/kg/day), (iii) palbociclib (25 mg/kg/day), (iv) combination (Metformin: 100 mg/kg/day, Palbociclib: 25 mg/kg/day). The dose of metformin and palbociclib was chosen based on previous preclinical studies and corresponds to a human equivalent dose within the standard clinical range, as calculated by body surface area normalization.22,23 Treatments were given orally for 14 days. Tumor size was measured every 3 days using calipers and calculated as: tumor volume (mm3) = maximum length (mm) × vertical width (mm)2/2. Tumor size was detected and photographed using a small animal vivo imaging system (IVIS Spectrum, USA). Prior to the use of IVIS Spectrum isoflurane combined with a respiratory anesthesia machine was used to anesthetize the mice. The mice were euthanized after the photography. Tumors were removed for weighing and photography.

Immunohistochemistry Analysis

The dissected mice tumors were embedded in paraffin and sectioned. The sections were then deparaffinized in xylene and hydrated in graded ethanol. After citrate-based antigen repair, the endogenous peroxidase of the sections was quenched by 3% hydrogen peroxide. After serum blocking, sections were incubated with Ki67 (proteintech, China), p-AKT (proteintech, China), or p-mTOR (proteintech, China) specific primary antibodies in a wet box overnight at 4 °C. Diaminobenzidine was used for color development. Staining intensity of IHC images was analyzed using ImageJ software. Quantitative analysis was performed using ImageJ software by measuring mean optical density across at least five random fields per sample after 8-bit grayscale conversion and uniform threshold application, with three independent biological replicates (tumors from three different mice) analyzed per group.

Statistical Analysis

Statistical analysis was performed using GraphPad Prism 9 (GraphPad Software, USA). Data are expressed as mean ± standard deviation (SD). Statistical significance between two groups was determined by the Student’s t-test, while one-way ANOVA was used for comparisons across multiple groups. A P-value < 0.05 was considered significant. The exact n-value for each experiment (representing biological replicates) is provided in the corresponding figure legend.

Results

Metformin and Palbociclib Alone or Together Inhibited HR+/HER2- Breast Cancer Cell Viability

To assess the anticancer effects of palbociclib and metformin on HR+/HER2- breast cancer cell lines MCF7 and T47D, cytotoxicity was evaluated using the CCK-8 assay. The drugs’ half maximum inhibitory concentrations (IC50) were determined after treating cells with escalating drug concentrations for 48 or 72 hours. Both palbociclib and metformin showed time- and concentration-dependent inhibition of cell viability in MCF7 and T47D cells. The 48-hour IC50 values for palbociclib were 9.71 ± 0.23 μM for MCF7 and 8.55 ± 0.22 μM for T47D, decreasing to 5.09 ± 0.37 μM and 4.70 ± 0.10 μM at 72 hours (Figure 1A and B). For metformin, the 48-hour IC50 values were 30.37 ± 1.51 mM for MCF7 and 20.21 ± 1.32 mM for T47D, which reduced to 13.80 ± 1.06 mM and 7.92 ± 0.38 mM at 72 hours (Figure 1C and D).

Figure 1.

8 plots: cell viability, IC50 and combo index for drug treatments. Image A: Two bar charts for MCF7 and T47D show cell viability (%) against Palbociclib (µM) at 48 h and 72 h across concentrations (0 to 25 µM). Image B: Bar chart for IC50 of Palbociclib (µM) for MCF7 and T47D at 48 h (MCF7 ~10, T47D ~9) and 72 h (both ~5). Image C: Two bar charts for MCF7 and T47D show cell viability (%) against Metformin (mM) at 48 h and 72 h across concentrations (0 to 100 mM). Image D: Bar chart for IC50 of Metformin (mM) for MCF7 at 48 h (~30) and 72 h (~14) and T47D at 48 h (~20) and 72 h (~8). Image E: Line graph of cell viability (%) against Palbociclib (µM) with control, 2.5 mM and 5 mM Metformin. Image F: Similar line graph as E. Image G: Scatter plot with legend ′Combination′ shows Fa vs. CI, with points around Fa 0.4-0.8 and CI 0.5-1.1, reference line at CI=1. Image H: Similar scatter plot as G, with points around Fa 0.4-0.9 and CI 0.4-1.1.

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Metformin and palbociclib alone or together inhibited HR+/HER2- breast cancer cell viability. (A) Viability of MCF7 and T47D cells after palbociclib treatment for 48 or 72 h. (B) Palbociclib IC50 for MCF7 and T47D cells. (C) Viability of MCF7 and T47D cells after metformin treatment for 48 or 72 h. (D) Metformin IC50 for MCF7 and T47D cells. (E and F) Viability of MCF7 (E) and T47D (F) cells treated with combined metformin and palbociclib for 72 h. (G and H) CI plots for MCF7 (G) and T47D (H) cells. CI values <1, =1, and >1 indicate synergistic, additive, and antagonistic effects. Data show mean ± SD, n=3.

To determine if metformin and palbociclib exhibit synergistic anticancer effects, MCF7 and T47D cells were exposed to IC10 or IC20 concentrations of metformin (2.5 mM or 5 mM) combined with increasing doses of palbociclib for 72 hours. The combination therapy significantly potentiated the inhibition of proliferation in HR+/HER2- breast cancer cells compared to monotherapy (Figure 1E and F). Drug combination index (CI) values were below 1, suggesting a synergistic effect of the metformin and palbociclib combination (Figure 1G and H). Based on the observed cell viability inhibition in Figure 1E and F, the optimal drug concentration combination was selected for further studies.

Metformin Synergizes with Palbociclib to Inhibit Proliferation of HR+/HER2- Breast Cancer Cells

The proliferation effects of the metformin and palbociclib combination on HR+/HER2- breast cancer cells MCF7 and T47D were assessed using colony formation, CCK-8, EdU, and immunofluorescence assays. Colony formation assays indicated that low concentrations of metformin combined with palbociclib were able to significantly inhibit the colony-forming ability of the cells, compared to the single-agent treatment (Figure 2A). CCK-8 results showed that the relative proliferation of metformin combined with palbociclib decreased significantly with time and was statistically different from palbociclib monotherapy at 96 hours of treatment (Figure 2B). EdU assays revealed a significantly reduced rate of EdU-positive cells in the combination group compared to controls and single-drug groups (Figure 2C–E). Immunofluorescence assays for Ki67, a proliferation marker, demonstrated reduced numbers and fluorescence intensity of Ki67 red dots in combination-treated cells, significantly lower than in monotherapy groups (Figure 2F and G, Supplementary Figure 1A). These results suggest that metformin synergizes with palbociclib to significantly inhibit the proliferation of HR+/HER2- breast cancer cell line in vitro, and is superior to monotherapy.

Figure 2.

Colony formation, proliferation, EdU and Ki67 assays in MCF7/T47D cells under four treatments. A scientific figure with seven panels (A-G) compares treatments on MCF7 and T47D breast cancer cells. Panel A shows colony formation images and bar graphs, with the combination treatment resulting in the fewest colonies. Panel B presents growth curves over 96 hours, where the combination treatment shows the lowest proliferation, marked with ***. Panels C and D display EdU assay micrographs, with the combination treatment showing minimal EdU signal. Panel E′s bar graphs confirm the combination treatment has the lowest percentage of EdU-positive cells, marked with ***. Panels F and G show immunofluorescence micrographs for Ki67, DAPI, Tubulin and merged channels, with the combination treatment showing significantly reduced Ki67 signal compared to control and single-drug treatments.

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Metformin synergizes with palbociclib to inhibit proliferation of HR+/HER2- breast cancer cells. (A) Colony formation in untreated, metformin (2.5 mM), palbociclib (1.56 μM), and co-treated MCF7 or T47D cells, with quantification. (B) Growth curves of MCF7 or T47D cells under metformin (5 mM), palbociclib (6.25 μM), combination, and control treatments, with asterisks indicating significant differences in proliferation rate at 96 h. (C–E) EdU assay in control, metformin (2.5 mM), palbociclib (3.125 μM), and co-treated MCF7 (C) and T47D (D) cells. Scale bars: 100 μM. (F and G) Ki67 expression in control, metformin (2.5 mM), palbociclib (3.125 μM), and co-treated MCF7 (F) and T47D (G) cells for 72h. Scale bars: 50 μM. Data show mean ± SD, n=3. *P < 0.05, **P < 0.01, ***P < 0.001.

Metformin Synergizes with Palbociclib to Inhibit Migration and Invasion of HR+/HER2- Breast Cancer Cells

The impact of the metformin and palbociclib combination on the migration and invasiveness of HR+/HER2- breast cancer cells was evaluated using wound-healing and transwell assays. As shown in Figure 3A, the ability of wound healing was inhibited in the metformin and palbociclib groups compared to the untreated group. However, the two-drug combination treatment group significantly inhibited the healing ability of both cells compared to the single-drug groups. The results of the Transwell migration assay showed that metformin combined with palbociclib almost completely inhibited the migration of both cells and was stronger than the monotherapy group (Figure 3B). Matrigel transwell assay showed that metformin inhibited cell invasion, but in combination with palbociclib was able to potentiate the inhibition of cell invasion (Figure 3C). These results suggest that metformin could synergize with palbociclib to effectively inhibit migration and invasion of HR+/HER2- breast cancer cells.

Figure 3.

Wound healing, migration and invasion assays for MCF7 and T47D cells under four treatments. Three panels display microscopy images and bar graphs. Panel A: Wound healing assay for MCF7 and T47D cells at 0 and 48 hours under control, metformin, palbociclib and combination treatments. Dotted lines indicate wound edges. Bar graphs show migration percentage; combination treatment results in the lowest migration (0-5%) compared to control (50-60% for MCF7, 25-30% for T47D). Triple asterisks denote significant reduction compared to control and single agents. Panel B: Transwell migration assay images and graphs show migration up to 150%. Combination treatment nearly stops migration in both cell lines, significantly lower than control and single agents, marked by triple asterisks. Panel C: Matrigel invasion assay images and graphs show invasion up to 150%. Combination treatment results in the lowest invasion (10-20%) for both cell lines, significantly lower than control and monotherapy, indicated by triple asterisks.

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Metformin synergizes with palbociclib to inhibit migration and invasion of HR+/HER2- breast cancer cells. (A) Migration of MCF7 or T47D cells treated with metformin (2.5 mM), palbociclib (1.56 μM), combination, or control treatment for 48 h. The white dotted lines indicate the wound edges. (B and C) Migration and invasion transwell assays of MCF7 or T47D cells treated with metformin (2.5 mM), palbociclib (1.56 μM), combination, or control treatment for 72 h. Data show mean ± SD, n=5. ***P < 0.001.

Metformin Synergizes with Palbociclib to Promote Apoptosis but Not Cell Cycle Inhibition in HR+/HER2- Breast Cancer Cells

The effects of metformin and palbociclib on apoptosis were assessed by flow cytometry and Western blot assays. Flow cytometry results indicated that metformin promoted apoptosis in MCF7 and T47D cells and combined with palbociclib to enhance apoptosis (Figure 4A). The expression levels of anti-apoptotic proteins BCL-2 and MCL-1 in the cells were detected by Western blot assay. As shown in Figure 4B, the expression level of BCL-2 was not reduced in both types of palbociclib-treated cells, while the expression level of MCL-1 was significantly reduced in both. The expression of BCL-2 was suppressed in metformin-treated MCF7 cells, whereas the expression of MCL-1 was suppressed in both cells. Both the expression levels of BCL-2 and MCL-1 were suppressed in cells co-treated with metformin and palbociclib and produced statistical differences from the monotherapy group. The above results suggest that metformin synergizes with palbociclib to promote apoptosis of HR+/HER2- breast cancer cells. The synergistic anti-cancer mechanism of metformin and palbociclib is related to the inhibition of anti-apoptotic proteins BCL-2 and MCL-1 expression.

Figure 4.

Multi-panel plots of apoptosis, anti-apoptotic proteins and cell-cycle changes under four treatments. Image A presents flow-cytometry data for MCF7 and T47D cells under various treatments: Control, Metformin, Palbociclib and their Combination. Dot plots use Annexin V-PE (x-axis) and 7-AAD (y-axis). MCF7 quadrant values show significant changes, especially in Q2 and Q4 under Combination treatment. T47D also shows notable shifts, particularly in Q2 and Q3. Bar charts indicate increased apoptosis rates: MCF7 from Control to Combination (~60%) and T47D from Control to Combination (~70%). Image B displays Western blot results for BCL-2, MCL-1 and beta-Tubulin in both cell lines. BCL-2 levels decrease significantly in Combination treatment for both MCF7 and T47D. MCL-1 levels also drop notably under Combination treatment. Image C features cell-cycle histograms and stacked bar charts for MCF7 and T47D, showing reduced peaks under treatments. Stacked bar charts reveal G0/G1 phase dominance, with smaller S and G2/M phases across treatments.

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Metformin synergizes with palbociclib to promote apoptosis but not cell cycle inhibition in HR+/HER2- breast cancer cells. (A) Effects and relative quantification of metformin and palbociclib on apoptosis in MCF7 or T47D cells. (B) Effects and relative quantification of metformin and palbociclib on anti-apoptotic proteins in MCF7 or T47D cells. (C) Effects and relative quantification of metformin and palbociclib on the cell cycle of MCF7 or T47D cells. Metformin: 2.5 mM, palbociclib: 3.125 μM, treatment time: 72 h. Data show mean ± SD, n=3. *P < 0.05, **P < 0.01, ***P < 0.001.

The effects of metformin and palbociclib on the cell cycle were measured by flow cytometry. The results of flow cytometry analysis showed that there was no significant difference in cell cycle parameters between metformin treatment and control group (Figure 4C). Palbociclib significantly induced cell arrest in the G0/G1 phase (MCF7: 90.14±1.47%, T47D: 93.12±1.88%). In the combination therapy group, a lower proportion of cells accumulated in the G0/G1 phase (MCF7: 88.48±1.18%, T47D: 81.12±1.12%) compared to the group treated with palbociclib alone. These results indicate that metformin did not enhance the cell cycle arrest ability of palbociclib.

Metformin Synergizes with Palbociclib to Exert Anticancer Effects Through Inhibition of PI3K/AKT/mTOR Pathway

To further investigate the underlying mechanisms by which metformin in combination with palbociclib exerts synergistic inhibitory effects on HR+/HER2− breast cancer, we analyzed the transcriptional impact of metformin and palbociclib on cells using mRNA sequencing technology. Bioinformatics analysis focused on comparing the palbociclib and combination groups to isolate metformin’s additional transcriptional effects under CDK4/6 inhibition. As shown in Figure 5A and B, compared with the palbociclib monotherapy group, the metformin plus palbociclib combination group upregulated 1428 genes and downregulated 2154 genes.

Figure 5.

Multiple graphs showing gene expression and pathway enrichment in Palbociclib vs Combination groups. Image A: Heatmap of top 100 genes, red for high and blue for low expression between Palbociclib and Combination groups. Image B: Volcano plot shows log2 fold change vs. negative log10 q-value, highlighting gene regulation. Image C: Bar graph displays pathways like Cell cycle and p53 signaling with negative log10 P value. Image D: Bubble plot with Rich Factor vs. pathways, bubble size for gene count, color for P value. Image E: Donut chart shows pathway proportions, including cancer and viral infections. Image F: Grouped bar chart differentiates upregulated and downregulated genes by gene count. Collectively, these graphs depict transcriptional impact of treatments, with significant enrichment in pathways like Cell cycle and p53 signaling.

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Transcriptome sequencing results of MCF7 cells treated with metformin in combination with palbociclib. (A) Heatmap: Top 100 differentially expressed genes in palbociclib-only and metformin + palbociclib groups. Red indicates high expression; blue indicates low expression. Deeper colors signify greater differences. (B) Volcano plot: Upregulated and downregulated genes in the metformin + palbociclib group compared to palbociclib-only group. Red: significantly upregulated genes; blue: significantly downregulated genes; gray: non-significant genes. (C) The top 20 biological pathways enriched by differentially expressed genes. (D) Enrichment factors and gene counts of different pathways enriched by differentially expressed genes. (E) The KEGG pathways enriched by the top 20 upregulated and downregulated genes among differentially expressed genes. (F) The number of upregulated and downregulated genes in the top 20 KEGG pathways enriched by differentially expressed genes. Metformin: 2.5 mM, palbociclib: 3.125 μM, treatment time: 72 h.

Additionally, further analysis of the KEGG pathway enrichment results revealed the top 20 biological pathways enriched by the differentially expressed genes between the palbociclib monotherapy group and the metformin plus palbociclib combination group. As shown in Figure 5C, the top three pathways were the cell cycle, the p53 signaling pathway, and the PI3K-AKT signaling pathway. The significant enrichment of these pathways suggests that they may play crucial roles in the treatment with metformin combined with palbociclib. Figure 5D illustrates the enrichment factors and gene counts of different pathways enriched by the differentially expressed genes between the palbociclib monotherapy group and the metformin plus palbociclib combination group. As shown in Figure 5D, the PI3K-AKT signaling pathway and the cytokine-cytokine receptor interaction pathway exhibited high enrichment factors and significance. This indicates that these pathways are highly representative within the gene set affected by the combination of metformin and palbociclib and are likely to play key roles in the underlying biological processes.

Figure 5E presents the KEGG pathway enrichment analysis of differentially expressed genes between the palbociclib monotherapy group and the metformin plus palbociclib combination group, highlighting the top 20 most significantly upregulated and downregulated genes. The outer and inner rings represent upregulated and downregulated genes, respectively, with the annotated percentages indicating the proportion of differentially expressed genes in each pathway relative to the top 20 genes. The results reveals that among the significantly upregulated genes, pathways related to cell cycle regulation and oncogenesis are predominant, suggesting that the combination therapy may enhance the activity of these pathways. Conversely, among the significantly downregulated genes, pathways such as PI3K-AKT signaling, oncogenic pathways, and cytokine-receptor interactions show a distinct trend, indicating that these pathways may be involved in the inhibitory effects of the combination therapy.

Figure 5F shows that there are 67 differentially expressed genes enriched in the PI3K-AKT signaling pathway. Compared with the palbociclib monotherapy group, the combination group had 28 upregulated genes and 39 downregulated genes in this pathway. This significant difference indicates that the PI3K-AKT signaling pathway plays a crucial role in the effects of metformin combined with palbociclib. Based on these findings, we speculate that the synergistic inhibitory effect of metformin and palbociclib on the proliferation of HR+/HER2− breast cancer cells is likely primarily associated with the regulation of the PI3K-AKT signaling pathway.

To elucidate the specific regulatory mechanisms of metformin combined with palbociclib on the PI3K/AKT/mTOR pathway, we detected the key proteins in PI3K/AKT/mTOR pathway. Compared with the single drug treatment group, the PI3K, p-AKT and p-mTOR protein in the combined treatment group were significantly inhibited, while the total AKT and mTOR protein remained unchanged (Figure 6A and Supplementary Figure 1B). Next, the expression levels of proteins related to the mechanism of palbociclib action were detected. Palbociclib treatment did not significantly alter the expression level of CDK4 protein, while metformin markedly reduced it. CDK4 expression was also decreased in the combined treatment group (Figure 6B and Supplementary Figure 1C). The relative protein expressions of p-Rb and E2F in the combined group were significantly inhibited, compared with the single drug groups. Compared to the palbociclib group, Cyclin D1 expression was significantly decreased in the combination treatment group. However, the total protein level of Rb has not changed.

Figure 6.

A multi-panel infographic of PI3K/AKT/mTOR signaling and cell assays in MCF7 and T47D cells. The image A showing western blots for MCF7 and T47D with Palbociclib and Metformin conditions. Rows are labeled PI3K, AKT, p-AKT, mTOR, p-mTOR and β-Tubulin. Band intensity for p-AKT and p-mTOR appears reduced in the combined plus plus condition compared with single-drug conditions, while AKT and mTOR appear similar across lanes. The image B showing western blots for MCF7 and T47D with Palbociclib and Metformin . Rows are labeled CDK4, Rb, pRb (Ser780), E2F, Cyclin D1 and β-Tubulin. pRb (Ser780) and E2F bands appear reduced in the combined plus plus condition. The image C showing western blots for MCF7 and T47D with Metformin, Palbociclib and 740Y-P. Rows are labeled AKT, p-AKT, mTOR, p-mTOR and β-Tubulin. p-AKT and p-mTOR bands appear higher with 740Y-P alone and lower with Metformin plus Palbociclib. The image D showing two bar charts titled MCF7 and T47D with y-axis label Cell viability percent and tick marks at 0, 50 and 100. Bars show lower viability for Palbociclib plus Metformin than control and higher viability when 740Y-P is added compared with Palbociclib plus Metformin. The image E showing colony formation images for MCF7 and T47D under Metformin, Palbociclib and 740Y-P minus and plus conditions. Fewer colonies appear with Palbociclib plus Metformin and more colonies appear when 740Y-P is added. The image F showing flow cytometry dot plots arranged as two rows labeled MCF7 and T47D, with treatment conditions indicated above. Axes are labeled Annexin V-PE on the x-axis and 7-AAD on the y-axis. Plots show more events in the Annexin V-PE positive region with Palbociclib plus Metformin and fewer with 740Y-P added. The image G showing EdU assay micrographs for MCF7 and T47D in columns of treatment conditions with labels EdU, Hoechst and Merge. EdU signal appears reduced with Palbociclib plus Metformin and increased with 740Y-P. The image H showing Transwell Migration and Invasion images for MCF7 and T47D with Metformin, Palbociclib and 740Y-P minus and plus conditions. Fewer migrated and invaded cells appear with Palbociclib plus Metformin and more with 740Y-P. The image I showing wound healing images for MCF7 and T47D at 0h and 48h with treatment conditions indicated above. The wound gap appears wider at 48h with Palbociclib plus Metformin and narrower when 740Y-P is present.

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Metformin synergizes with palbociclib to exert anticancer effects through inhibition of PI3K/AKT/mTOR pathway. (A and B) PI3K/AKT/mTOR pathway and downstream protein expression in MCF7 and T47D cells under metformin, palbociclib, combination, or control treatment. (C) PI3K/AKT/mTOR pathway protein expression in MCF7 and T47D cells treated with 740Y-P, metformin+palbociclib, 740Y-P+metformin+palbociclib, or control. Cell viability (D), Colony formation (E), Apoptosis (F), EdU assay (G) of MCF7 and T47D cells treated with 740Y-P, metformin+palbociclib, 740Y-P+metformin+palbociclib, or control. In the above experiment, metformin: 2.5 mM, palbociclib: 3.125 μM, 740Y-P: 5 μM, treatment time: 72 h. Scale bars: 100 μM. (H) Transwell migration and invasion of MCF7 or T47D cells treated with 740Y-P (5 μM), metformin (2.5 mM) + palbociclib (1.56 μM), 740Y-P+metformin+palbociclib or control for 48 h. The white dotted lines indicate the wound edges. (I) The migration of MCF7 or T47D cells under the same treatment conditions for 72 h. Data show mean ± SD, n=3. *P < 0.05, ***P < 0.001.

740Y-P was Used to Reverse the Damage Caused by the Combination of Metformin and Palbociclib

To further validate that the synergistic anticancer effects of metformin and palbociclib were associated with the PI3K/AKT/mTOR pathway, 740Y-P was added to the combination treatment of metformin and palbociclib. 740Y-P is a potent activator of PI3K. The results indicate that 740Y-P reversed the changes in protein levels caused by metformin combined palbociclib (Figure 6C and Supplementary Figure 1D). After co-treatment of cells with 740Y-P and metformin and palbociclib, the relative expression levels of p-AKT and p-mTOR were up-regulated compared to the metformin plus palbociclib group.

Next, the effect of 740Y-P on cellular biological functions was further examined. The cells were divided into metformin+palbociclib group, 740Y-P mono-treatment group, and metformin + palbociclib + 740Y-P group. 740Y-P significantly reversed the decrease in cell viability and colony formation caused by treatment with metformin combined palbociclib (Figure 6D and E, Supplementary Figure 1E). In addition, apoptosis induced by metformin combined with palbociclib was also reversed by 740Y-P (Figure 6F and Supplementary Figure 1F). After the addition of 740Y-P, EdU positive cells treated with metformin and palbociclib increased, allowing cancer cells to resume proliferation (Figure 6G and Supplementary Figure 1G). 740Y-P reversed the inhibition of cell migration and invasion induced by metformin in combination with palbociclib (Figure 6H and I, Supplementary Figure 1H and I). These results suggest that 740Y-P reverses metformin combined with palbociclib resulting in HR+/HER2- breast cancer cell damage. In conclusion, metformin synergizes with palbociclib to exert anticancer effects through inhibition of PI3K/AKT/mTOR pathway.

Metformin Synergizes with Palbociclib to Inhibit Tumor Growth in vivo

Based on the above results, the effects of metformin combined with palbociclib in transplanted tumor mice model with EMT6 mice-derived breast cancer cells were further evaluated. The luciferase was marked into EMT6 cells and named Luc-EMT6 cells. When the tumor size reached 100 mm3, all mice were randomly divided into four groups of six mice each (Figure 7A). Mice were treated with PBS, metformin (100 mg/kg/day), palbociclib (25 mg/kg/day), and a combination of the two drugs by oral gavage for 14 days.

Figure 7.

Experimental design and results of drug effects on tumor growth in mice. The image A shows an experimental design for drug testing on mice with Luc-EMT6 cells. Mice are divided into groups: PBS, Metformin, Palbociclib and a combination, treated for 14 days. Image B shows a line graph of tumor volume over time, indicating reduced growth in the combination group. Image C displays luminescence images of mice under different treatments. Image D presents a bar graph of total flux, showing significant reduction in the combination group. Image E shows a line graph of mice weight over time, with no significant changes. Image F includes bar graphs of leukocyte and platelet counts, with some variations across groups. Image G shows a scatter plot of tumor weight, with the combination group having the lowest weight. Image H displays dissected tumors from each group. Image I shows immunohistochemical staining for Ki67, p-AKT and p-mTOR, with bar graphs indicating expression levels, showing reduced expression in the combination group.

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Metformin synergizes with palbociclib to inhibit tumor growth in vivo. (A) Experimental design protocol and technology roadmap for mice drug delivery. (B) The tumor volume change curve of mice during 14 days of treatment. n=6. (C and D) Tumor size and relative quantification of tumors in mice after 14 days of drug administration. n=6. (E) The tumor weight (g) of mice during 14 days of treatment. n=6. (F) The count of Leukocytes and platelets in mice after 14 days of treatment. n=6. (G and H) Weight statistics and representative images of dissected mice tumors after 14 days of treatment. n=6. (I) Immunohistochemical staining of mice tumor tissues for Ki67, p-AKT and p-mTOR antibodies. n=3. Scale: 100 μM. Data show mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ns, not significant (P > 0.05).

After 14 days of treatment, the mice were photographed with live imaging of small animals to observe the size of the tumors in the body. The tumor growth curve showed that metformin combined with palbociclib significantly reduced the tumor growth rate compared with the monotherapy group (Figure 7B). As shown in Figure 7C and D, the drug combination showed a more significant inhibitory effect on tumor growth than monotherapy. During the administration of the drug in mice, no deaths or adverse effects were detected in mice in the combination group, indicating that the combination was safe and effective (Figure 7E). To assess the safety profile of the drug combination, a complete blood count was performed on mice after 14 days of treatment. The results showed that the platelet count in the combination group was significantly higher than that in the palbociclib monotherapy group, while no significant difference was observed in white blood cell counts (Figure 7F). The mice were then sacrificed with the size and weight of the dissected tumors measured. Figure 7G and H show that the tumor size and weight of the mice in the combination group were reduced and were smaller than those in the monotherapy group. Metformin combined with palbociclib has a better therapeutic effect, which is consistent with the result of cells in vitro. In addition, the expressions of Ki67, p-AKT and p-mTOR in mice tumor tissues were detected by immunohistochemistry. As shown in Figure 7I, the intensity of Ki67, p-akt and p-mtor staining was decreased in tumor tissues of the metformin and palbociclib treatment groups, and was weakest in the combination group. These results indicate that metformin combined with palbociclib significantly inhibits tumor growth in HR+/HER2- breast cancer mice in vivo.

Discussion

In this study, we first observed that metformin can combine with CDK4/6 inhibitor palbociclib to exhibit a synergistic inhibitory effect on HR+/HER2- breast cancer cells, which was significantly stronger than that of monotherapy.

When mitosis occurs in breast cancer cells, initiating signals such as the upstream ER, PI3K/AKT, and mTOR/S6K signaling pathways are required.24 These signaling pathways lead to the binding of Cyclin D family to CDK family, which in turn phosphorylates Rb.25–27 Inactivation of Rb releases a number of translocation factors like E2F, a key regulator that initiates the G1 to S phase progression.28,29 CDK4/6 inhibitors such as palbociclib intervene in this process by disrupting Cyclin D-CDK binding and inhibiting Rb phosphorylation, thereby inducing cell cycle arrest.30 Our study reveals that metformin profoundly enhances this effect through a coordinated, multi-layered mechanism. Interestingly, this synergy did not manifest as a further increase in the proportion of G1-arrested cells beyond the profound level achieved by palbociclib alone. Instead, we observed a shift in cellular fate from cytostasis to apoptosis. This can be explained by the fact that the cell cycle arrest machinery was already maximally engaged by palbociclib, leaving limited room for enhancement. Our findings are consistent with previous work by Ma et al31 who also reported metformin’s ability to cooperate with CDK inhibitors to trigger apoptosis without reinforcing G1 arrest in other cancer models.

Transcriptome sequencing identified the PI3K-AKT signaling pathway as a central hub for the synergy, a finding of high significance given the extensive crosstalk and feedback loops between the PI3K/AKT/mTOR and CDK4/6-Rb-E2F axes that often limit targeted therapy efficacy.32 Notably, we demonstrate that metformin counteracts the compensatory PI3K/AKT activation known to arise from CDK4/6 inhibition, thereby quenching a key resistance mechanism.33 Beyond this, metformin uniquely downregulates CDK4 protein expression—an effect not shared by palbociclib, which primarily inhibits CDK4 kinase activity.34 Thus, the combination launches a dual assault: metformin reduces the target (CDK4) level while palbociclib inhibits the activity of the remaining target. This is further reinforced by metformin-induced suppression of Cyclin D1. Consequently, the combination achieves a collapse of the CDK4/6-Rb-E2F axis, evidenced by the enhanced suppression of Rb phosphorylation, Cyclin D1, and E2F. The reversal of these molecular and functional effects by the PI3K activator 740Y-P provides definitive genetic evidence that suppression of the PI3K/AKT/mTOR axis is the initiating event in this synergistic cascade. However, the observation that 740Y-P did not completely abolish the synergy suggests the involvement of additional, complementary mechanisms. Given the pleiotropic nature of metformin, it is plausible that other reported targets, such as AMPK or IGF-R1, contribute to a multi-faceted cellular stress that cooperates with PI3K/AKT/mTOR inhibition to enact the full therapeutic response.35 Therefore, we conclude that metformin sensitizes HR+/HER2- breast cancer cells to palbociclib primarily by orchestrating a coordinated shutdown from upstream PI3K/AKT/mTOR suppression to downstream cell cycle engine disruption, within a context of potential contributions from other pathways, thereby shifting the pharmacological effect from cytostasis to potent cytotoxicity.

Repurposing metformin for oncology requires defining a therapeutic window that maximizes efficacy while minimizing side effects. While high doses are often used in trials for refractory tumors, our study explores a lower, clinically tractable dose to synergize with CDK4/6 inhibition—a strategy yet to be clinically evaluated. This gap underscores the need for foundational preclinical rationale, which is supported by emerging evidence across cancers. For example, metformin enhances CDK4/6 inhibitor efficacy by reprogramming the senescence-associated secretory phenotype in head and neck squamous cell carcinoma, and by suppressing lysosomal trapping of the drug in non-small cell lung cancer.36,37 In line with these findings, our study demonstrates that low-dose metformin synergizes with palbociclib to significantly inhibit tumor growth in a mouse model, without exacerbating toxicity. The metformin dose of 100 mg/kg used in our mouse model corresponds to a human equivalent dose of approximately 486 mg for a 60 kg adult based on body surface area normalization. This dose falls at the lower end of the standard clinical dosing range for metformin (500–2550 mg/day).38,39 These collective insights, together with our mechanistic data, position metformin as a versatile adjunct that can target context-specific pathways to improve the therapeutic index of CDK4/6 inhibitor-based regimens.

It is important to acknowledge limitations of our study. First, the concentrations of metformin required to observe robust synergistic effects in our in vitro models fall within the millimolar range, which exceeds typical human plasma levels. This is a common challenge in preclinical metformin research, where high concentrations are often necessary to elicit a measurable phenotype within a short treatment window.40–43 Second, we utilized the mouse-derived EMT6 cell line in immunocompetent BALB/c mice, a model which, despite expressing estrogen receptor (ER), is often characterized as estrogen-independent and may not fully recapitulate the signaling dependencies of human HR+ breast cancer.44 This choice was made strategically to preserve a functional immune system, allowing for the investigation of potential immunomodulatory effects of the drug combination, which would not be possible in human xenograft models using immunodeficient mice. Therefore, our in vivo data should be interpreted primarily as a robust demonstration of the synergistic antitumor efficacy of metformin and palbociclib in a complex, immunologically intact microenvironment. Although the regulation of drugs on immune function has not been deeply studied in this study, it represents an important direction for our future research. The primary aim of this study was to provide a proof-of-concept and preclinical rationale for the synergy between metformin and palbociclib. Subsequent efforts focusing on advanced delivery strategies or more extensive dose-escalation and pharmacokinetic studies could further enhance its translational potential.45,46

Conclusion

In conclusion, our current study demonstrates that metformin synergizes with palbociclib against HR+/HER2- breast cancer, with its mechanism of action primarily related to the inhibition of the PI3K/AKT/mTOR pathway. These findings highlighted the novel uses of metformin and suggested its use as an adjunctive drug to enhance the efficacy of the CDK4/6 inhibitor. The addition of metformin in CDK4/6 inhibitor treatment of HR+/HER2- breast cancer may be an effective strategy to improve treatment efficacy, and it is promising to be further studied in clinical practice.

Funding Statement

This work was supported by the National Natural Science Foundation of China (81402266), the Medical Science and Technology Research Projects of Henan Province (SBGJ202002082), the Young and Middle aged Discipline Leader Project of Henan Health Commission (HNSWJW-2021029), the Outstanding Young Talent Project of Scientific and Technological Innovation in Henan Health (YXKC2020032), Special Scientific Research Project for Post-marketing Clinical Research of Innovative Drugs (WKZX2024CX501221) and Wu Jieping medical foundation for clinical scientific research special funds (320.6750.2025-6-66).

Data Sharing Statement

All data generated or analysed during this study are included in this published article and its Supplementary Information Files.

Ethics Statement

All animal studies were ethically approved by Zhengzhou University Academy of Medical Sciences (Approval NO.: 2023101701). All procedures were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and complied with the 3R principles (Replacement, Reduction, Refinement) to ensure animal welfare.

Author Contributions

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Disclosure

The authors have no relevant financial or non-financial interests to disclose.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

All data generated or analysed during this study are included in this published article and its Supplementary Information Files.

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