Therapeutic Effect of Gemcitabine Combined With Angelica Polysaccharide on Triple Negative Breast Cancer in Mice

Abstract

Objective:

This study delves into the therapeutic effects of combining GEM and Angelica polysaccharide (APS) on triple-negative breast cancer.

Methods:

In vitro, proliferation, apoptosis of 4T-1 cells and MDSC were detected by flow cytometry. Migration of 4T-1 cell was detected by scratch healing experiment after treatment by GEM (0, 2.5, 5 μM), APS (160,320 mg/ml), or GEM + APS (2.5 μM + 160 mg/ml, 5 μM + 320 mg/ml). In vivo, 4T-1 cells were injected into the mammary fat pad under the mammary gland of BALB/c mice to establish an orthotopic breast cancer tumor model. They were randomly divided into control group (0.9% normal saline + ultrapure water), GEM group (0.9% normal saline preparation, 100 mg/kg, intraperitoneal injection twice a week), APS group (ultrapure water preparation, 200 mg/kg, intraperitoneal injection once a day), GEM + APS group (GEM 100 mg/kg, intraperitoneal injection twice a week and APS 200 mg/kg, intraperitoneal injection once a day) for 3 weeks. The proportion of immune cells in the spleen and tumor microenvironment were detected by flow cytometry, immunofluorescence and Mindray hematology analyzer. The tumor volume and weight, spleen index were recorded.

Results:

The in vitro experimental results revealed that GEM effectively inhibited the proliferation and migration of 4T-1 cells and induced apoptosis in both 4T-1 cells and MDSCs. In contrast, APS had no impact on 4T-1 cells or MDSCs. The in vivo experimental findings indicated that compared with the single-drug treatment groups, the combination treatment of GEM + APS more effectively regulated the proportion of peripheral and local anti-tumor MDSCs and T cells, and more significantly curbed the progression of breast cancer in mice.

Conclusion:

APS can exert a synergistic effect through immune regulation to enhance the therapeutic efficacy of GEM on triple-negative breast cancer. It aims to offer novel insights for the clinical application of combining GEM with immunotherapy for patients with triple negative breast cancer.

Introduction

Breast cancer (BC) is a major health concern for women worldwide. It accounts for approximately one-third of all malignant tumors in women, and its mortality rate represents about 15% of the total number of diagnosed cases. ¹ Based on molecular markers such as estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2), BC is classified into 3 primary subtypes: hormone receptor (HR)-positive, HER2-positive, and triple-negative breast cancer (TNBC). TNBC makes up around 15% to 20% of all breast carcinomas. ² Currently, the treatment options for breast cancer encompass surgery, chemotherapy, radiotherapy, and targeted therapy. However, these existing therapies fall short of achieving the desired efficacy due to side effects and drug resistance.^3,4 Moreover, the side effects of these treatments significantly impair the quality of life of patients. ⁵ TNBC, characterized by its unique molecular phenotype, is not responsive to endocrine therapy or molecular targeted therapy. As a result, chemotherapy stands as the main systemic treatment. But the efficacy of conventional postoperative adjuvant chemoradiotherapy is suboptimal. The residual metastatic lesions often lead to tumor recurrence. ⁶ Bevacizumab has been used in combination with chemotherapeutic drugs to treat TNBC in some countries, but it has not significantly increased the survival time of patients. ⁷ Therefore, there is an urgent need to develop new treatment regimens and targets. In recent years, immunotherapy has emerged as the fourth pillar of cancer treatment, following surgery, radiotherapy, and chemotherapy. ⁸ Its goal is to enhance the immune system’s capacity to destroy cancer cells and prevent them from evading the immune response.^9,10 Various methods, including vaccines, cytokines, antibodies, and immune cells, can be employed for cancer immunotherapy.^11,12 A growing body of evidence indicates that immunotherapy is a powerful clinical strategy for breast cancer treatment.^13,14 It has greatly improved the quality of life and prognosis of patients.

Angelica polysaccharide (APS) is a principal bioactive constituent of the traditional Chinese herbal medicine Angelica sinensis. It possesses a wide range of biological activities, including anti-oxidation, anti-inflammation, and immune regulation. ¹⁵ APS has been shown to significantly boost the number of macrophages and their phagocytic activity in tumor-bearing mice,^16,17 as well as enhance the activity of natural killer (NK) cells, ¹⁸ thereby improving the efficacy of active immunotherapy. Moreover, APS can stimulate the proliferation of mouse lymphocytes, increase the proportion of CD3⁺CD56⁺ cells, and elevate the levels of IL-2, IL-6, TNF-α, and IFN-γ.^19,20 As a potential T lymphocyte mitogen, APS can directly activate T lymphocytes. It can also modulate the expression of Th1 and Th2-related cytokines to exert its immunomodulatory function. ²¹ However, our previous research revealed that APS can promote the proliferation, differentiation, and immunosuppressive function of myeloid-derived suppressor cells (MDSCs) via the STAT1 and STAT3 signaling pathways. ²² MDSCs are key immunosuppressive cells in the tumor microenvironment. They inhibit the function of immune cells such as T cells, dendritic cells, and macrophages. Additionally, MDSCs can exert immunosuppressive effects by secreting inhibitory cytokines and inducing the production and activation of other immunosuppressive cells, such as regulatory T cells (Tregs). Beyond their immunosuppressive roles, MDSCs can actively shape the tumor microenvironment through complex interactions with breast cancer cells and the surrounding matrix, leading to increased angiogenesis, tumor invasion, and metastasis.^23,24 Given these findings, the upregulation of MDSCs by APS represents a potential side effect, particularly when the immune response needs to be enhanced to combat tumors. Therefore, it is worth considering combining APS with drugs that can inhibit MDSCs to counteract its pro-MDSC effects, thereby allowing APS to better exert its anti-tumor potential.

Gemcitabine (GEM) is a nucleoside analog chemotherapeutic agent that is widely employed in the treatment of various cancers, including pancreatic cancer, breast cancer, and non-small cell lung cancer. ²⁵ Its primary mechanism of action involves inhibiting DNA synthesis, thereby preventing the growth and replication of cancer cells. ²⁶ However, emerging evidence indicates that GEM’s effects extend beyond direct cytotoxicity against cancer cells. It also exerts influence on immune cells within the tumor microenvironment. For instance, GEM has been shown to reduce the number of myeloid-derived suppressor cells (MDSCs) in the spleens of 4T-1 tumor-bearing mice. ²⁷ This reduction in MDSCs helps to bolster the anti-tumor immune response of the tumor-bearing hosts.

In view of the therapeutic effect of GEM on breast cancer and the inhibitory effect on MDSCs, combined with the immune enhancement effect of APS, this study explored the therapeutic effect of GEM + APS combination therapy on triple negative breast cancer in mice through in vivo and in vitro experiments. The results showed that GEM + APS combination therapy was significantly superior to single drug in inhibiting the development of orthotopic breast cancer in mice, improving the immune microenvironment of spleen and tumor in tumor-bearing mice, and reducing the increase of white blood cells and neutrophils caused by the tumor. In order to provide an experimental basis for the clinical application of GEM combined with APS in the treatment of triple negative breast cancer patients, this study was conducted.

Methods

Cell Culture

The mouse triple-negative breast cancer cell line 4T-1 (RRID:CVCL_0125) was obtained from the Immunology Laboratory of Shandong Second Medical University. The triple-negative breast cancer cell line 4T-1 was cultured in RPMI-1640 medium supplemented with L-glutamine and 10% fetal bovine serum (FBS) in a 37°C incubator with 5% CO₂. Cells in the logarithmic growth phase were used for all experiments.

4T-1 Cell Proliferation Assay

4T-1 cells were labeled with CFSE (Thermo, USA) and co-cultured with different concentrations (0, 2.5, 5 μM) of GEM (Qilu Pharmaceutical, China), (160, 320 mg/ml) of APS (Youke, China, purity ≥ 98%) and (2.5 μM + 160 mg/ml, 5 μM + 320 mg/ml) GEM + APS for 48 hours. The cells were collected by BD FACSVerse flow cytometry and analyzed by FlowJo 7.6 software.

4T-1 Cell Apoptosis Detection

4T-1 cells were co-cultured with different concentrations (0, 2.5, 5 μM) of GEM, (160, 320 μg/ml) of APS and (2.5μM + 160 μg/ml, 5 μM + 320 μg/ml) of GEM + APS for 36 hours, and labeled with Annexin-V/7AAD reagent (Biolegend, USA). The cells were obtained by BD FACSVerse flow cytometry and analyzed by FlowJo 7.6 software.

4T-1 Cell Scratch Healing Experiment

4 T-1 cells were cultured to 70% to 80% of the plate, 100 μl of plastic pipette tip was scribed, and PBS was used to remove floating cells. The serum-free medium was replaced, and different concentrations (0, 2.5, 5 μM) of GEM, (160, 320 μg/ml) APS and (2.5 μM + 160 μg/ml, 5 μM + 320 μg/ml) GEM + APS were added. Mixed culture was photographed under an inverted microscope at 0 and 24 hours, and the percentage of migration area was calculated by Image J software.

Obtain MDSCs

Isolation of MDSCs from tumor-bearing mice: tumor tissues of tumor-bearing mice were collected. Single cells were prepared by grinding and filtration with a 40 mm filter. After lysis of red blood cells, all cells were mixed and resuspended in an equal volume of 1 × magnetic bead separation enrichment buffer (BD Biosciences, USA). Anti-mouse Ly6C and Ly6G magnetic beads (BD Biosciences Cat#558111, RRID: AB_398656) were added to 10 μl/10⁷ cells and incubated at 6~12°C for 15 minutes. The unbound cells were washed with 20-fold 1 × magnetic bead sorting enrichment buffer, and the supernatant was removed by centrifugation. The cells were resuspended with 2 ml 1 × magnetic bead sorting enrichment buffer, transferred to the sorting tube, placed in the magnetic scaffold, and the supernatant was carefully sucked. The isolated bone marrow mesenchymal stem cells were resuspended in complete RPMI 1640 medium containing glutamine (200 mmol/l), penicillin streptomycin (100 U/ml) and 10 % fetal bovine serum. The purity of MDSCs sorted by all magnetic beads was above 90%. The purity of MDSCs after sorting was verified by flow cytometry. Briefly, cells were stained with anti-mouse CD11b-FITC (Biolegend Cat#101206, RRID: AB_312789, Clone: M1/70). After flow cytometry detection, CD11b⁺ cells were defined as total MDSC based on the living cell gate.

MDSCs Apoptosis Detection

The concentration of MDSCs was adjusted to 1 × 10⁶/ml and mixed with different concentrations of GEM (0, 80, 160 μM), APS (160, 320 μg/ml) and GEM + APS (80 μM + 160 μg/ml, 160 μM + 320 μg/ml). After 24 hours of culture, the cells were collected and labeled with Annexin-V/7AAD reagent (Biolegend, USA). Cells were obtained using BD FACSVerse flow cytometry and analyzed using FlowJo 7.6 software.

Animal Models and Treatment Options

Female BALB/c mice (6-8 weeks old) without specific pathogen (SPF) (purchased from Jinan Pengyue, license number: SCXK (Shandong) 20220006), animal certificate number: 370726240101412352, were raised in an Individually Ventilated Cage System (IVC) animal room. After 1 week of adaptation period, 4T-1 cells were injected into the mammary fat pad under the mammary gland of BALB/c mice by 1 × 10⁴/50 μl to establish an orthotopic breast cancer tumor model. Tumor growth was monitored by digital caliper measurement to ensure that the average initial tumor volume between the experimental groups was matched after about 7 days of tumor implantation, and then the mice were randomly grouped. The tumor volume was calculated as follows: volume = (length × width ²)/2.

They were randomly divided into control group (0.9% normal saline + ultrapure water), GEM group (0.9% normal saline preparation, 100 mg/kg, intraperitoneal injection twice a week), APS group (ultrapure water preparation, 200 mg/kg, intraperitoneal injection once a day), and GEM + APS group (GEM 100 mg/kg, intraperitoneal injection twice a week and APS 200 mg/kg, intraperitoneal injection once a day) for 3 weeks.

The experimental animals were randomly divided into control group and experimental group by computer-generated random number table. The appearance, volume and administration of all intervention drugs or solvents were consistent. In the evaluation phase of the experimental results, the key observation indicators (such as the measurement of tumor volume) are independently completed by researchers who are unaware of the experimental grouping to ensure the objectivity of data collection and evaluation.

Ethics

The animal experiment protocol of this study has been approved by the Animal Ethics Committee of Shandong Second Medical University (Approval No: 2023SDL269). All procedures were performed in strict accordance with the NIH Guide for the Care and Use of Laboratory Animals, and the reporting of this study complies with the ARRIVE guidelines 2.0.

For all surviving surgeries, mice were anesthetized by intraperitoneal injection of pentobarbital sodium ( 50 mg/kg ), and the depth of anesthesia was confirmed by the disappearance of corneal and plantar reflexes. Postoperative pain was treated by subcutaneous injection of carprofen ( 5 mg/kg ) once a day for 3 consecutive days. Pre-defined humane endpoints were rigorously applied, requiring euthanasia if any of the following occurred: >20% weight loss, severe lethargy, inability to eat or drink, or the development of large tumors (>1.5 cm in diameter). Euthanasia was performed by CO₂ inhalation at a flow rate displacing 30% of the chamber volume per minute, followed by confirmation of death via cervical dislocation.

Detection of the Proportion of Immune Cells in Spleen and Tumor Microenvironment

The spleen was separated and placed on a 40 μm cell filter (biosharp) and rinsed with PBS. After centrifugation, the red blood cells were lysed with red blood cell lysis buffer (Solarbio, China) at 4°C, and the spleen single cell suspension was obtained after washing with PBS.

The tumor tissue was isolated, cut into pieces with scissors, digested in a custom RPMI 1640 containing 1 mg/ml collagenase I (Solarbio, China) and 0.1 mg/ml DNase I (Solarbio, China), and incubated at 37°C for 1 hour. The cell suspension was filtered through a 40 μm filter and washed with PBS to collect the cell suspension.

The above cells were blocked with anti-mouse CD16/32 antibody, and then mixed and placed in a refrigerator at 4 °C for 15 minutes. Cell surface markers were then stained with fluorescein-labeled mouse monoclonal antibodies, including anti-mouse CD3-Percp (Biolegend Cat#100326, RRID: AB_893317, Clone: 145-2C11), anti-mouse CD4-APC-H7 (BD Biosciences Cat#560181, RRID: AB_1645235, Clone: GK1.5), anti-mouse CD8-APC (Biolegend Cat#100712, RRID: AB_312751, Clone: 53-6.7), anti-mouse CD11b-FITC (Biolegend Cat#101206, RRID: AB_312789, Clone: M1/70), anti-mouse Ly6C-APC (Biolegend Cat#128016, RRID: AB_1732076, Clone: HK1.4) and anti-mouse Ly6G-PE (Biolegend Cat#127608, RRID: AB_1186099, Clone: 1A8) antibodies. The cells were obtained by BDFACSVerse flow cytometry, and the data were analyzed by FlowJo7.6 software.

Immunofluorescence Was Used to Detect Immune Cells in the Tumor Microenvironment

The tumor tissues were isolated, fixed with 4% paraformaldehyde, embedded in paraffin blocks, and cut into 4 μm thick sections. After dewaxing and antigen retrieval, 10% goat serum was blocked and incubated with anti-CD3 rat monoclonal antibody (Abcam Cat# ab11089, RRID:AB_2889189, Clone: CD3-12) at 1:200 dilution, anti-CD4 rabbit monoclonal antibody (Abcam Cat# ab183685, RRID:AB_2686917, Clone: FPR19514) at 1:400 dilution, anti-CD8 rabbit monoclonal antibody (Abcam Cat# ab217344, RRID:AB_2890649, Clone: EPR21769) at 1:400 dilution, anti-CD11b rabbit monoclonal antibody (Abcam Cat# ab8878, RRID:AB_306831, Clone:M1/70) at 1:400 dilution, anti-GR-1 rat monoclonal antibody (Biolegend Cat#108401, RRID:AB_313366, Clone:RB6-8C5) at 1:400 dilution, anti-Pan-CK mouse monoclonal antibody (Abcam Cat# ab7753, RRID:AB_306047, Clone: C-11) at 1:400 dilution, at room temperature for 30 minutes and then overnight at 4°C. The next day, sections were incubated with appropriate secondary antibodies for 1 hour at room temperature. We used: Alexa Fluor 647 Goat anti-Rabbit IgG (Thermo Fisher Scientific) at 1:200 dilution; Alexa Fluor 647 Goat anti-Rat IgG (Thermo Fisher Scientific) at 1:200 dilution; Alexa Fluor 555 Goat anti-mouse (Thermo Fisher Scientific) at 1:200 dilution; Alexa Fluor 555 Goat anti-Rabbit (Thermo Fisher Scientific) at 1:200 dilution. DAPI was added, and finally the anti-fluorescence quencher seal was added. Images were captured using an upright immunofluorescence microscope. Each tumor tissue section was randomly selected 5 fields of view under high magnification (200 ×), and the average fluorescence intensity or percentage of positive cells was analyzed by ImageJ software.

Detection of White Blood Cells and Neutrophils in Peripheral Blood

20 μl peripheral blood of mice was added to the blood diluent and detected by Mindray hematology analyzer.

Statistical Analysis

All statistical analyses are performed in GraphPad Prism 8 (GraphPad Software). Data are expressed as mean ± standard deviation (SD). One-way analysis of variance was used for comparison between groups. P < .05 was considered statistically significant.

Results

GEM Inhibited the Proliferation of 4T-1 Cells, While APS Had No Effect

GEM has been shown to inhibit the proliferation of a variety of breast cancer cells, but the effects of APS and GEM + APS on the proliferation of 4T-1 cells have not been reported. The results of this study (Figure 1A) showed that the proliferation of 4T-1 cells was inhibited after GEM treatment, and the divided index level decreased (2.38 vs 1.48, P > .05, 2.38 vs 1.31, P < .05) with the increase of GEM concentration (from 0 to 5 μM) .

Figure 1.

The effect of GEM + APS combination therapy on the proliferation of 4T-1 cells. (A) Representative flow cytometry histogram. Each peak corresponds to the generation of one progeny cell. Compared with the control group, the right shift of the peak value indicates a decrease in cell division (B) Proliferation and division index of 4T-1 cells (repeated 3 times, ns means P > .05, *P < .05).

Compared with the untreated group, the division index of 4T-1 cells in the 160 and 320 μg/ml APS groups did not change significantly (2.38 vs 2.12 vs 2.15, P > .05). Compared with GEM alone group (2.5 μM, 5 μM), the mitotic index of GEM + APS group (2.5 μM + 160 μg/ml, 5 μM + 320 μg/ml) did not change significantly (1.48 vs 1.47,1.31 vs 1.37, P > .05).

These results indicate that GEM has an inhibitory effect on the proliferation of 4T-1 cells, but APS has no such effect, and APS does not affect the inhibitory effect of GEM on the proliferation of 4T-1 cells.

GEM Could Induce the Apoptosis of 4T-1 Cells, While APS Had No Effect

GEM has been shown to induce apoptosis in pancreatic cancer, breast cancer and ovarian cancer cells. However, the effect of APS and GEM + APS combination therapy on the apoptosis of 4T-1 cells has not been reported. The results of this study showed that (Figure 2A), the apoptosis percentage of 4T-1 cells increased (12.90% vs 50.00% vs 60.80%, P < .0001) after GEM treatment, and the total apoptosis rate increased with the increase of GEM concentration (0, 2.5, 5 μM). The percentage of early apoptotic cells (0, 2.5, 5 μM) increased more significantly (7.74% vs 31.37% vs 41.23%, P < .0001). Compared with the untreated group, the percentage of late apoptotic cells in the GEM-treated group (2.5 μM, 5 μM) increased significantly (5.16 vs 18.63 vs 19.57, P < .001).

Figure 2.

The effect of GEM + APS combined treatment on the apoptosis of 4T-1 cells. (A) Representative flow cytometry plots of 4T-1 cells stained with Annexin V-FITC and 7AAD after 36 hours of treatment with the indicated drugs. The quadrants define viable (Annexin V⁻/7AAD⁻), early apoptotic (Annexin V⁺/7AAD⁻), late apoptotic (Annexin V⁺/7AAD⁺), and necrotic (Annexin V⁻/7AAD⁺) cell populations (B-D) Quantitative analysis of the percentages of early apoptotic cells (B), late apoptotic cells (C), and total apoptotic cells (D) from 3 independent experiments (repeated 3 times, ns means P > .05, ***P < .001, ****P < .0001).

Compared with the untreated group, the total apoptosis rate (160 μg/ml, 320 μg/ml) (12.90% vs 14.40% vs 13.52%, P > .05), the percentage of early apoptotic cells (7.74% vs 7.99% vs 7.95%, P > .05) and the percentage of late apoptotic cells (5.16% vs 6.41% vs 5.57%, P > .05) did not change significantly in the APS-treated group. Compared with the GEM (2.5 μM, 5 μM) alone treatment group, the total apoptosis rate (50.00% vs 49.10%, 60.80% vs 59.40%, P > .05), the percentage of early apoptotic cells (31.37% vs 33.00%,4.00% vs 39.83%, P > .05) and the percentage of late apoptotic cells (18.63% vs 16.1%, 19.57% vs 19.57%, P > .05) did not change significantly in the GEM + APS combined treatment group (2.5 μM + 160 μg/ml, 5 μM + 320 μg/ml).

These results indicate that GEM can induce apoptosis in 4T-1 cells, and APS does not have this effect. The combination of GEM + APS did not affect the induction of GEM on 4T-1 cell apoptosis.

GEM Could Inhibit the Migration of 4T-1 Cells, While APS Had No Significant Effect

The effects of GEM, APS and GEM + APS on the migration of 4T-1 cells are still unknown. The results of this study found that (Figure 3A), compared with the untreated group, the migration rate of 4T-1 cells in the GEM treatment group (2.5 μM, 5 μM) was significantly reduced (53.19% vs 18.14% vs 18.13%, P < .0001); however, there was no significant change in the migration rate of 4T-1 cells in the APS treatment group (160 μg/ml, 320 μg/ml) (53.19% vs 51.99% vs 51.07%, P > .05 ). Compared with GEM (2.5 μM, 5 μM) alone treatment group, the migration rate of 4T-1 cells in GEM + APS combined treatment group (2.5 μM + 160 μg/ml, 5 μM + 320 μg/ml) did not change significantly (18.14% vs 19.55%, 18.13% vs 17.92%, P > .05).

Figure 3.

The effect of GEM + APS combination therapy on the migration of 4T-1 cells. Scratch test was used to detect cell migration ability. Uniform scratches were established in 4T-1 cells and then treated with GEM, APS or GEM + APS. Scratch healing was monitored for more than 24 hours. (A) Representative microscopic images of the scratch at 0 and 24 hours post-treatment. (B) Cell migration rate was quantified by measuring the reduction of scratch area at 24 hours relative to 0 hour time point, and expressed as the percentage of scratch healing area (repeated 3 times, ns means P > .05 , ****P < .0001).

These results indicate that GEM can inhibit the migration of 4T-1 cells, and APS does not have this effect. The combination of GEM + APS did not affect the inhibitory effect of GEM on the migration of 4T-1 cells.

GEM Can Induce Apoptosis of MDSCs, While APS Has No Obvious Effect

Studies have shown that GEM can reduce the number of MDSCs in the spleen of 4T-1 tumor-bearing mice, thus helping to save the anti-tumor immunity of tumor-bearing hosts. However, the effect of APS and GEM + APS on the apoptosis of MDSCs has not been reported. By magnetic bead sorting, we obtained a high purity (>90%) MDSCs population. The results of this study found that (Figure 4A), the apoptosis of MDSCs cells increased after GEM treatment, and the total apoptosis rate increased with the increase of GEM concentration (0, 80, 160 μM) (65.93% vs 76.55%, P < .01, 65.93% vs 87.20%, P < .0001). The percentage of late apoptotic cells (0, 80, 160 μM) increased more significantly (54.86% vs 65.00%, P < .05, 54.86% vs 75.24%, P < .0001). Compared with the untreated group, the percentage of early apoptotic cells in the GEM-treated group (80 μM, 160 μM) did not change significantly (11.07% vs 11.55%, 11.07% vs 11.96%, P > .05 ).

Figure 4.

Effect of GEM + APS combination therapy on the apoptosis of MDSCs. (A) Representative flow cytometry plots of MDSCs stained with Annexin V-FITC and PI after treatment with the indicated drugs (B-D) Quantitative analysis of the percentages of early apoptotic (B), late apoptotic (C), and total apoptotic (D) MDSCs. (repeated 3 times, ns means P > .05, *P < .05, **P < .01, ****P < .0001).

Compared with the untreated group, the total apoptosis rate ( 65.93% vs 67.01% vs 67.44%, P > .05), the percentage of early apoptotic cells (11.07% vs 11.37% vs 11.34%, P > .05) and the percentage of late apoptotic cells (54.86% vs 55.64% vs 56.10%, P > .05) did not change significantly in the APS-treated group (160 μg/ml, 320 μg/ml).

Compared with GEM (80 μM, 160 μM) alone treatment group, there was no significant change in the total apoptosis rate (76.55% vs 78.08%, 87.20% vs 88.98%, P > .05), the percentage of early apoptotic cells (11.55% vs 11.24%, 11.96% vs 11.88%, P > .05) and the percentage of late apoptotic cells (65.00% vs 66.84%, 75.24% vs 77.10%, P > .05 ) in the GEM + APS combined treatment group (80 μM + 160 μg/ml, 160 μM + 320 μg/ml).

These results indicate that GEM can induce apoptosis of MDSCs, and APS does not have this effect. The combination of GEM + APS did not affect the induction of apoptosis of MDSCs by GEM.

GEM + APS Combination Therapy Can More Effectively Inhibit the Development of Triple Negative Breast Cancer In Situ in Mice

In order to explore the effect of GEM + APS combination therapy on the progression of triple-negative breast cancer, this study used the triple-negative breast cancer cell line 4T-1 to construct a mouse orthotopic breast cancer model and treated it according to the strategy shown in Figure 5A. Tumor volume on day 31: Compared with the tumor-bearing untreated control group, the tumor volume of the GEM group (408.2 mm³ vs 189.6 mm³, P < .05) and the GEM + APS combined treatment group (408.2 mm³ vs 122.2 mm³, P < .01) were significantly reduced (Figure 5D); there was no significant difference in the reduction of tumor volume in the APS group (408.2 mm³ vs 322.6 mm³, P > .05). Tumor weight on day 31: Compared with the tumor-bearing untreated control group, the tumor weight of the GEM group (0.61 g vs 0.33 g, P < .01) and the GEM + APS combined treatment group (0.61 g vs 0.16 g, P < .0001) was significantly reduced (Figure 5E). However, the reduction of tumor weight in APS group was not statistically significant (0.61 g vs 0.52 g, P > .05).

Figure 5.

Establishment of mouse orthotopic breast cancer model and therapeutic effect. (A) Experimental scheme: Female BALB/c mice were inoculated with 4T-1 cells into the mammary fat pad to establish orthotopic tumors. Mice were then randomized into different treatment groups (eg, Control, GEM, APS, GEM + APS) and treated according to the indicated schedule (B and C) Photographs of resected tumors from all groups upon termination of the first (B) and second (C) replicate experiments, demonstrating the reproducibility of the antitumor effects. (D) Dynamic changes in tumor volume measured at regular intervals throughout the therapeutic course. (E) Comparing the final tumor weights excised at the study endpoint. (n = 6, **P < .01, ****P < .0001).

These results indicate that GEM alone can significantly inhibit the growth of pancreatic cancer in mice, while APS alone has no obvious inhibitory effect on the growth of pancreatic cancer in mice, but GEM + APS combination therapy is more effective than GEM alone.

GEM + APS Combination Therapy Can Better Improve the Proportion of Peripheral Immune Cells

GEM + APS Combination Therapy Can Better Improve the Proportion of MDSCs and T Cells in the Spleen

Splenomegaly is a common clinical sign in cancer patients, and this symptom will be effectively alleviated after treatment. The results of this study showed that compared with the tumor-bearing untreated control group, the spleen index of the APS treatment group (48.42% vs 37.89%, P < .05) and the spleen index of the GEM treatment group (48.42% vs 25.21%, P < .001) were significantly reduced, among which the spleen index of the GEM treatment group decreased more significantly, followed by the APS treatment group. The spleen index of the GEM + APS combined treatment group (48.42% vs 17.31%, P < .001) was significantly lower than that of the single drug treatment group (Figure 8A and B).

Figure 8.

Changes of peripheral circulation in mice after treatment. Systemic immune responses were evaluated at the study endpoint. (A) Schematic illustration of the mouse spleen, the organ analyzed for the spleen index. (B) The spleen index, calculated as the ratio of spleen weight (mg) to body weight (g), reflecting systemic immune activation or tumor burden. (C) Total number of leukocytes in peripheral blood. (D) count of neutrophils in peripheral blood. (n = 6, ns denotes P > .05, *P < 0.05, ***P < .001, ****P < .0001).

These results indicate that both GEM and APS can effectively reverse splenomegaly, and the combined treatment of GEM + APS is more effective than monotherapy.

MDSCs can inhibit the proportion and function of T cells and their subsets through a variety of ways and mechanisms, and promote tumor growth and immune escape. In view of the above results, it was found that GEM, APS, GEM + APS combined treatment had an effect on the proportion of MDSCs and APS could up-regulate the number, activation and proliferation of T cells. Therefore, this study examined the effect of GEM + APS combination therapy on the proportion of MDSCs and T cells in triple-negative breast cancer-bearing mice. The results showed that compared with the untreated tumor-bearing control group, the proportions of MDSCs (30.33% vs 40.16%, P < .01) and Granulocytic Myeloid-Derived Suppressor Cells (G-MDSCs) (28.52% vs 37.78%, P < .05) in the spleen of the APS group were significantly elevated, while the proportion of Monocytic Myeloid-Derived Suppressor Cells (M-MDSCs) did not change significantly(1.81% vs 2.37%, P > .05). The proportions of G-MDSCs (28.52% vs 15.64%, P < .001) and MDSCs (30.33% vs 17.17%, P < .001) in the spleen of the GEM group were significantly reduced, while the proportion of M-MDSCs (1.81% vs 1.53%, P > .05) did not change significantly. Compared with the APS group, the proportion of G-MDSCs (37.78% vs 27.32%, P < .01 ) and MDSCs (40.16% vs 29.16%, P < .01 ) in the GEM + APS combined treatment group was significantly reduced, while the proportion of M-MDSCs (2.37% vs 1.84%, P < .05) was not significantly changed (Figure 6A-D).

Figure 6.

Changes in the proportion of MDSCs in the spleen of mice after treatment. Splenocytes were isolated from mice at the endpoint of the study (as in Figure 5). The frequency of MDSCs and their subsets was analyzed by flow cytometry. (A) Representative flow cytometry plots illustrating the identification of monocytic M-MDSCs (CD11b⁺Ly6CʰⁱLy6G⁻) and granulocytic G-MDSCs (CD11b⁺Ly6CˡᵒLy6G⁺) from CD11b⁺ cells. (B-D) Bar graphs show the proportion of M-MDSCs (B), G-MDSCs (C), and the total MDSC population (D) in the spleen (n = 6, ns denotes P > .05, *P < .05, **P < .01, ***P < .001).

These results indicate that APS alone can increase the proportion of MDSCs in the spleen, GEM alone can reduce the proportion of MDSCs in the spleen, and GEM + APS combination therapy can reduce the increase in the proportion of MDSCs in the spleen caused by APS.

The results of this study showed that compared with the untreated control group, the total T (17.35% vs 25.97%, P < .01), CD4⁺ T (11.14% vs 16.83%, P < .01) and CD8⁺ T (3.99% vs 6.97%, P < .05) in the spleen of the APS treatment group were significantly increased. The total T (17.35% vs 31.27%, P < .001), CD4⁺ T (11.14% vs 20.67%, P < .001) and CD8⁺ T cells (3.99% vs 8.95%, P < .001) in the spleen of the GEM treatment group were significantly increased. GEM + APS combined treatment group spleen total T (17.35% vs 42.00%, P < .0001), CD4⁺ T (11.14% vs 27.72%, P < .0001), CD8⁺ T cells (3.99% vs 12.28%, P < .0001) were also significantly increased (Figure 7A-D). Compared with the APS group and the GEM group, the total T (25.97% vs 42.00%, 31.27% vs 42.00%, P < .001), CD4⁺ T (16.83% vs 27.72%, P < .001, 20.67% vs 27.72%, P < .01) and CD8⁺ T cells (6.97% vs 12.28%, P < .001, 8.95% vs 12.28%, P < .05) in the spleen of the GEM + APS group increased more significantly.

Figure 7.

Changes in the proportion of spleen T cells and their subsets in mice after treatment. Splenocytes were harvested from 4T-1 tumor-bearing mice at the study endpoint. The proportions of T cells and their major subsets were analyzed by flow cytometry. (A) Representative flow cytometry dot plots illustrating the sequential gating strategy for CD3⁺ T cells, followed by the identification of CD4⁺ and CD8⁺ subsets. (B-D) Bar graphs show the frequencies of total T cells (CD3⁺) (B), CD4⁺T cells (C), and CD8⁺T cells (D) among splenic lymphocytes. (n = 6, *P < .05, **P < .01, ***P < .001, ****P < .0001).

These results indicate that both GEM and APS can increase the proportion of spleen T cells and their subsets in tumor-bearing mice to varying degrees, and the combined treatment of GEM + APS is more effective than single drug treatment.

Combined Treatment Can Better Improve the Proportion of Myeloid Cells in Peripheral Blood

The role of white blood cells and neutrophils in tumors is complex and dynamic, depending on a variety of factors, including tumor type, stage, and individual immune status. The proportion and number of myeloid cells in peripheral blood of tumor patients will increase significantly. This study found that compared with the normal mice group, the levels of white blood cells (20.56 × 10⁹/L vs 367.10 × 10⁹/L, P < .0001) and neutrophils (2.93 × 10⁹/L vs 307.50 × 10⁹/L, P < .0001) in the peripheral blood of the tumor-bearing untreated control group increased sharply. Compared with the untreated tumor-bearing control group, there was no significant change in the levels of white blood cells (367.10 × 10⁹/L vs 258.10 × 10⁹/L, P > .05) and neutrophils (307.50 × 10⁹/L vs 217.40 × 10⁹/L, P > .05) in the peripheral blood of mice in the APS group. While the levels of white blood cells (367.10 × 10⁹/L vs 104.50 × 10⁹/L, P < .0001) and neutrophils (307.5 × 10⁹/L vs 91.10 × 10⁹/L, P < .001) in peripheral blood of mice in GEM group decreased. The white blood cells (367.10 × 10⁹/L vs 52.84 × 10⁹/L, P < .0001) and neutrophils (307.5 × 10⁹/L vs 43.59 × 10⁹/L, P < .0001) in the peripheral blood of the GEM + APS combined treatment group were significantly decreased (Figure 8C and D).

These results indicate that GEM can reduce the number of white blood cells and neutrophils in tumor-bearing mice, and GEM + APS combination therapy is more effective than monotherapy, while APS does not have this effect.

GEM + APS Combination Therapy Can Better Improve the Proportion of MDSCs and T Cells in Tumor Tissues

Due to the structural characteristics of tumor tissue, the influence of tumor microenvironment on drug entry and the unique tumor immunosuppressive microenvironment, GEM + APS combination therapy can produce immune changes different from those in the periphery. Therefore, this study also analyzed the changes in the proportion of MDSCs and T cells in the tumor to evaluate whether the drug can exert immune regulation function in the lesion.

The results of flow cytometry (Figure 9A) showed that compared with the untreated control group, the proportion of G-MDSCs (24.07% vs 34.93%, P < .05) and MDSCs (28.34% vs 39.77%, P < .05) in the APS group increased, while the proportion of M-MDSCs (4.27% vs 4.84%, P > .05) did not change significantly. However, in the GEM group, the proportion of G-MDSCs (24.07% vs 12.12%, P < .01) and MDSCs (28.34% vs 14.12%, P < .05) decreased, while the proportion of M-MDSCs (4.27% vs 1.99%, P < .05) did not change significantly. Compared with the APS group, the proportion of G-MDSCs (34.93% vs 22.88%, P < .01) and MDSCs (39.77% vs 25.86%, P < .05) in the GEM + APS group decreased, while the proportion of M-MDSCs (4.84% vs 2.98%, P < .05) did not change significantly.

Figure 9.

Changes of MDSCs proportion in the tumor microenvironment. (A) Representative flow cytometry plots showing the gating strategy for M-MDSCs (CD11b⁺Ly6CʰⁱLy6G⁻) and G-MDSCs (CD11b⁺Ly6CˡᵒLy6G⁺) from dissociated tumor tissues. (B-D) Quantification of the frequencies of M-MDSCs (B), G-MDSCs (C), and total MDSCs (D) in tumors (n = 6). (E) IF was used to detect the expression of MDSCs in the tumor microenvironment of mice. Representative immunofluorescence (IF) images of tumor sections stained for MDSCs (eg, CD11b⁺, red; Gr-1, green) and a nuclear marker (DAPI, blue). (F) The percentage of MDSCs fluorescence area in the tumor microenvironment of mice. (n = 6, ns denotes P > .05, *P < .05, **P < .01, ***P < .001).

Similarly, IF results (Figure 9E) showed that compared with the untreated tumor-bearing control group, the infiltration of MDSCs in the APS group increased (0.27% vs 0.37%, P < .01), but the infiltration of MDSCs in the GEM group decreased (0.27% vs 0.07%, P < .001). Compared with APS group, the infiltration of MDSCs in GEM + APS group was decreased (0.37% vs 0.24%, P < .001).

These results indicate that APS alone can increase the infiltration of MDSCs in tumor tissues, GEM alone can reduce the infiltration of MDSCs in tumor tissues, and GEM in GEM + APS combination therapy can reduce the increase of MDSCs infiltration in tumor tissues caused by APS.

The results of flow cytometry showed that there was no significant change in the proportion of total T (43.42% vs 34.53%, P > .05), CD4⁺ T (25.01% vs 19.85%, P > .05) and CD8⁺ T (7.75% vs 3.40%, P > .05) in the GEM group compared with the untreated control group. The proportion of total T (43.42% vs 57.37%, P < .01) and CD8⁺ T (7.75% vs 14.64%, P < .05) in the APS group increased, while the proportion of CD4⁺ T (25.01% vs 32.18%, P < .05) did not change significantly. Compared with the GEM group, the proportion of total T (34.53% vs 47.07%, P < .05) and CD8⁺ T (3.402% vs 11.29%, P < .05) in the GEM + APS combined treatment group increased, while the proportion of CD4⁺ T (19.85% vs 26.34%, P > .05) did not change significantly (Figure 10A).

Figure 10.

Changes of T cells and their subsets proportion in the tumor microenvironment. Tumors were harvested at the endpoint, and single-cell suspensions were analyzed by flow cytometry to assess T cell infiltration. (A) Representative flow cytometry dot plots illustrating the sequential gating strategy for identifying CD3⁺T cells and their CD4⁺ and CD8⁺ subsets from tumors. (B) The percentage of CD3⁺ T cells in the tumor microenvironment of mice. (C) The percentage of CD4⁺ T cells in the tumor microenvironment of mice. (D) The percentage of CD8⁺ T cells in the tumor microenvironment of mice. (n = 6, ns denotes P > .05, *P < .05, **P < .01).

The results of IF showed that the total T (0.28% vs 0.07%, P < .001), CD4⁺ T (0.24% vs 0.04%, P < .001) and CD8⁺ T (0.14% vs 0.02%, P < .001) in the GEM group were significantly lower than those in the untreated control group. Total T (0.28% vs 0.51%, P < .001), CD4⁺ T (0.24% vs 0.40%, P < .001) and CD8⁺ T (0.14% vs 0.49%, P < .001) were significantly increased in APS group. Compared with the GEM group, the total T (0.07% vs 0.31%, P < .001), CD4⁺ T (0.04% vs 0.27%, P < .001) and CD8⁺ T (0.02% vs 0.15%, P < .001) in the GEM + APS combined treatment group were significantly increased (Figures 11-13) .

Figure 11.

Changes of CD3⁺ T proportion in the tumor microenvironment. (A) IF was used to detect the expression of CD3⁺ T cells in tumor microenvironment of mice. (B) Percentage of fluorescence area of CD3⁺ T cells in tumor microenvironment of mice. (n = 6, ***P < .001).

Figure 13.

Changes of CD8⁺ T proportion in the tumor microenvironment. (A) IF was used to detect the expression of CD8⁺ T cells in tumor microenvironment of mice. (B) Percentage of fluorescence area of CD8⁺ T cells in tumor microenvironment of mice. (n = 6, ***P < .001).

Figure 12.

Changes of CD4⁺ T proportion in the tumor microenvironment. (A) IF was used to detect the expression of CD4⁺ T cells in tumor microenvironment of mice. (B) Percentage of fluorescence area of CD4⁺ T cells in tumor microenvironment of mice. (n = 6, ***P < .001).

These results indicate that APS can enhance the infiltration of T cells and their subsets in tumor tissues reduced by GEM.

Discussion

In recent years, an increasing number of studies have begun to investigate the combination of traditional Chinese herbal medicine or its active ingredients with conventional chemotherapy drugs. The aim is to enhance anti-cancer efficacy and mitigate the side effects of chemotherapy. For instance, Saikosaponin D controls glucose metabolism and drug efflux by inhibiting ADRB2 signaling, thereby augmenting the anti-tumor effect of GEM. ²⁸ Huaier enhances the tumor-killing effect of GEM by inhibiting GEM-induced stemness in a FoxM1-dependent manner. ²⁹ Danggui Buxue Decoction (DBD) has been shown to boost the anticancer activity of GEM in the treatment of non-small cell lung cancer and alleviate GEM-induced bone marrow suppression. ³⁰

The tumor growth and metastasis patterns of the mouse triple-negative breast cancer cell line 4T-1 in vivo closely resemble those of human breast cancer. ³¹ It is widely acknowledged that gemcitabine (GEM) is a chemotherapeutic agent capable of treating breast cancer (eg, the human breast cancer cell line MCF-7) by directly killing tumor cells. However, whether angelica polysaccharide (APS) has a direct impact on 4T-1 cells remains unreported. In this study, it was discovered that GEM could inhibit the proliferation and migration of 4T-1 cells and induce their apoptosis in vitro. In contrast, APS exhibited no such effects, and the GEM + APS combination therapy did not demonstrate a synergistic role. To further investigate whether APS might inhibit the progression of triple-negative breast cancer through immunomodulatory effects, this study utilized the 4T-1 cell line to establish an orthotopic triple-negative breast cancer mouse model. The results indicated that GEM alone significantly curbed breast cancer growth in mice. APS alone had no discernible inhibitory effect on breast cancer growth in mice. However, the combined treatment of GEM + APS yielded a more pronounced effect, which was significantly superior to GEM alone. It can be hypothesized that APS may enhance the therapeutic efficacy of GEM through immune regulation.

MDSCs are a category of immune cells originating from the bone marrow. They exert immunosuppressive effects in cancer and various other pathological conditions. A key characteristic of MDSCs is their ability to inhibit the function of immune cells such as T cells and natural killer (NK) cells, thereby dampening the body’s anti-tumor immune response. ³² Research has demonstrated that gemcitabine (GEM) can reduce the proportion and function of both MDSCs and T cells. ²² In contrast, angelica polysaccharide (APS) has been shown to promote the proliferation, differentiation, and immunosuppressive function of MDSCs. ²⁷ Thus, it can be hypothesized that APS and GEM may exhibit complementary effects in immune regulation. In this study, the effects of GEM, APS, and GEM + APS on MDSCs and T cells in tumor-bearing mice were investigated. The results indicated that APS treatment increased the proportion of MDSCs infiltrating the spleen and tumor tissues of breast cancer mice. Conversely, GEM treatment decreased the proportion of MDSCs infiltrating these tissues, aligning with previously reported findings. However, in the GEM + APS combination therapy, GEM counteracted the increase in MDSC infiltration in the spleen and tumor tissues caused by APS. This suggests that although APS does not induce MDSC apoptosis in vitro and increases the proportion of MDSCs in vivo, GEM can induce MDSC apoptosis. Therefore, when GEM and APS are combined in vivo, GEM can mitigate the side effect of APS in increasing the number of MDSCs. This makes the GEM + APS combination more effective.

APS showed no direct cytotoxicity in vitro yet potentiated GEM in vivo, implying host-dependent mechanisms. Gut microbes may convert APS to bioactive metabolites that sensitize tumors; APS also primes T cells, so once GEM depletes MDSCs the immune balance shifts toward anti-tumor. Additionally, APS may enhance GEM delivery by modulating tumor vasculature. Future gnotobiotic and pharmacokinetic studies should clarify which of these pathways dominates to refine the combination.

T cells can be divided into CD4⁺ T and CD8⁺ cytotoxic T lymphocytes (CTL). Among them, CD4⁺ T cells play multiple roles in anti-tumor immunity by directly activating CTL and enhancing the activity of NK cells to promote the body ’s immune response to tumors. ³³ CTL recognizes specific antigen peptides on the surface of cancer cells through T cell receptor ( TCR ), and releases perforin and granzyme B to kill cancer cells. ³⁴ Promoting the proliferation and activation of CD8⁺ T cells and enhancing the immune activity of TME in BC patients can improve the therapeutic effect of BC patients. ³⁵ Considering that APS has an up-regulation effect on the number and function of T cells, while GEM has an inhibitory effect on T cells, and the above in vivo and in vitro experiments have proved that the 3 treatment methods have different degrees of regulation on MDSCs. In order to further confirm that APS has a therapeutic effect on mouse breast cancer by up-regulating the immune response effect rather than direct tumor cytotoxicity, the effects of GEM, APS, and GEM + APS on the proportion of infiltrating T cells and their subsets in spleen and tumor tissues were detected. The results showed that the proportion of CD3⁺, CD4⁺, and CD8⁺ T cells in the spleen was significantly increased in the single drug treatment group. The proportion of CD3⁺, CD4⁺, and CD8⁺ T cells in the spleen was significantly increased after GEM treatment, followed by APS treatment, and GEM + APS combined treatment was more significant than the 2 single drug treatment groups. These results further prove that GEM and APS can effectively increase the peripheral immune function of breast cancer mice, and GEM + APS combined treatment will play a more effective synergistic effect.

T cells in tumor tissues usually have higher recognition ability to tumor-specific antigens, and because they are in the tumor microenvironment, they can quickly identify and attack tumor cells, so they may be more effective in controlling and eliminating tumor cells. The accumulation of MDSCs in the tumor microenvironment can strongly inhibit the anti-cancer function of T cells and natural killer cells, and play a variety of other tumor-promoting effects. ³⁶ Chemotherapy drugs can also affect normal immune cells. As T cells are constantly updated in the blood and tissues, chemotherapy can directly kill these cells, resulting in a decrease in the number. ³⁷ This study found that the proportion of CD3⁺, CD4⁺ and CD8⁺ T in the tumor microenvironment of mice decreased after GEM treatment. The proportion of CD3⁺, CD4⁺ and CD8⁺ T in the tumor microenvironment of mice increased after APS treatment. GEM + APS combination therapy can improve the decrease of CD3⁺, CD4⁺ and CD8⁺ T cells in the tumor microenvironment of mice induced by GEM. These results indicate that the immune enhancement effect of APS can reduce the decrease of tumor-specific immune response caused by GEM chemotherapy, and significantly enhance the anti-tumor immune response.

The spleen is the largest lymphoid organ and plays a crucial role in regulating cancer risk by controlling hematopoietic and immune responses. Cancer-induced splenomegaly is associated with changes in host hematopoietic function, which is characterized by increased white blood cells and granulocytes and decreased lymphocytes. The tumor microenvironment affects splenomegaly by producing granulocyte colony stimulating factor ( G-CSF ) to promote the expansion of MDSCs in bone marrow, spleen and lymph nodes. ³⁸ Splenomegaly is a common clinical sign of cancer patients, and this symptom will be effectively alleviated after treatment. This study found that both GEM and APS can effectively reduce the degree of spleen enlargement in triple-negative breast cancer mice, and GEM + APS combined treatment is more effective than single drug treatment.

Leukemia-like reaction is a clinical phenomenon, usually referring to some solid tumors (such as breast cancer, lung cancer, etc) or other types of tumors which in the course of the disease appear similar to the characteristics of leukemia. These features may include abnormal increase of white blood cells in peripheral blood, changes in hemogram, etc. The underlying mechanism seems to be the production of growth factors, such as granulocyte-macrophage colony-stimulating factor (GM-CSF), G-CSF and interleukins ( IL-3 and IL-6 ) produced by tumor cells. ³⁹ Studies have shown that compared with normal mice, the levels of white blood cells and neutrophils in the peripheral blood of tumor-bearing mice are increased. Melatonin inhibits the increase of white blood cells induced by liver metastasis cells (4TLM) of breast cancer in 4T-1 mice. ³³ The results of this study showed that the number of white blood cells and neutrophils in peripheral blood of tumor-bearing mice increased sharply by dozens of times, while GEM and GEM + APS combined treatment groups could effectively inhibit this situation. However, APS treatment had no significant inhibitory effect. These results indicate that GEM + APS can more effectively reduce the increase of white blood cells and neutrophils in tumor-bearing mice. It is also suggested that GEM + APS can more effectively regulate the anti-tumor immune effect of circulating immune cells.

Our findings reveal that the combination of GEM and APS exerts a sophisticated and functionally coordinated regulation of the MDSCs–T cell axis, with distinct molecular mechanisms underlying their individual and combined actions. GEM primarily targets MDSCs through multiple pathways: it induces apoptosis via activation of caspase-3 and suppression of anti-apoptotic proteins such as Bcl-2 and survivin, while also disrupting MDSC differentiation and recruitment by downregulating key mediators including STAT3, C/EBPβ, and CCL2. This results in a significant reduction in both G-MDSC and M-MDSC subsets, particularly within the TME, thereby alleviating immunosuppression and restoring T-cell functionality. Concomitantly, GEM enhances CD8⁺ T cell infiltration and activation, likely due to the removal of MDSCs-mediated inhibition on antigen presenting cells and direct suppression of T cell proliferation—effects further supported by increased expression of IFN-γ, granzyme B, and perforin in tumor-reactive T cells.

In contrast, APS acts predominantly as an immune-stimulant, engaging pattern recognition receptors such as Toll-like receptors (TLRs) and Dectin-1 on dendritic cells and macrophages, leading to NF-κB and MAPK pathway activation. This promotes a pro-inflammatory milieu characterized by elevated levels of IL-12, TNF-α, and type I interferons, which in turn drive T cell priming, expansion, and Th1 polarization. Notably, while APS enhances anti-tumor T cell responses, it may also promote the expansion and functional activation of MDSCs through compensatory feedback mechanisms—potentially mediated by G-CSF, GM-CSF, and IL-6 released during systemic immune activation. These cytokines can stimulate myelopoiesis and favor the accumulation of immunosuppressive MDSCs, which could otherwise limit the durability of T-cell-mediated tumor control.

Critically, when administered in combination, GEM effectively overrides the unintended pro-MDSCs effects of APS, preventing their pathological expansion without compromising the immune-stimulatory benefits. This counterbalancing action suggests that GEM not only depletes existing MDSCs but may also interfere with APS-induced signaling pathways that support MDSC survival or differentiation—possibly through suppression of STAT3 activation or modulation of the hematopoietic niche. Meanwhile, the T-cell enhancing effects of both agents are preserved and even synergized: GEM removes immunosuppressive barriers, while APS provides the co-stimulatory signals necessary for robust T-cell activation and memory formation. Functional assays further support this synergy, showing enhanced T-cell proliferation, cytotoxicity, and cytokine production in the combination group compared to either monotherapy.

These data indicate that GEM and APS do not merely act in parallel but engage in a mechanistically integrated reprograming of the immune landscape. The combination achieves a dual effect—suppressing immunosuppressive myeloid populations while simultaneously promoting effector T cell function—through distinct yet complementary molecular pathways. This dynamic regulation underscores the therapeutic potential of strategically pairing cytotoxic agents with immune-modulatory natural products to fine-tune the balance between immune suppression and activation in a functionally coherent manner.

The interplay between MDSCs and T cells represents a pivotal axis in shaping anti-tumor immunity, particularly in the context of TNBC, where immune evasion is a major barrier to effective treatment. The combination of GEM and APS appears to strategically target this axis through complementary and potentially coordinated mechanisms. GEM, primarily known for its cytotoxic effects, exerts a critical immune-modulatory function by selectively depleting MDSCs—key mediators of immunosuppression in both the tumor microenvironment (TME) and peripheral lymphoid organs. In contrast, APS does not directly inhibit tumor cell growth but enhances host immune competence, particularly by promoting T-cell activation and proliferation. This functional dichotomy suggests a rational synergy: GEM alleviates immune suppression, thereby “releasing the brakes” on the immune system, while APS actively “steps on the accelerator” by boosting effector T-cell responses.

This synergistic immune-modulation appears to be profoundly influenced by spatio-temporal dynamics. Spatially, the impact of GEM on MDSCs is more robust within the TME, where MDSCs infiltration is typically dense and strongly correlates with poor prognosis. The preferential reduction of MDSCs in the tumor site may be attributed to higher local drug exposure, enhanced tumor cell death releasing antigens, and a consequent shift in the cytokine landscape that undermines MDSCs survival and function. Conversely, the immune-stimulatory effects of APS are more prominently observed in systemic compartments such as the spleen, a major reservoir of naïve and memory T cells. Here, APS may facilitate the priming and expansion of tumor-reactive T-cell populations, which can then traffic to the tumor site—provided that the immunosuppressive barriers within the TME have been sufficiently dismantled by GEM. This spatial compartmentalization underscores the importance of anatomical context in determining drug action and immune outcome.

Temporally, the sequence and duration of treatment may further shape the immune response. GEM’s suppression of MDSCs occurs rapidly, often within the first week of treatment, creating an early window of opportunity for immune reactivation. In contrast, the immune-potentiating effects of APS—such as increased T-cell numbers, enhanced cytokine production (eg, IFN-γ, IL-2), and improved lymphocyte functionality—may require prolonged exposure to achieve maximal effect. This temporal lag suggests that the full benefit of combination therapy may depend on sustained APS administration following GEM-induced MDSCs clearance. If APS is administered too early or too briefly, its immune-enhancing potential may be stifled by the prevailing immunosuppressive environment. Conversely, delayed or suboptimal GEM dosing might allow MDSCs to persist and counteract any gains in T-cell activity.

Therefore, the balance between MDSCs suppression and T-cell activation is not a fixed state but a dynamically regulated process governed by both spatial localization and temporal kinetics. The therapeutic efficacy of GEM + APS likely hinges on achieving a precise immunological tipping point—where MDSCs-mediated inhibition is minimized just as T-cell responsiveness reaches its peak. This delicate equilibrium calls for optimized treatment scheduling, such as initiating GEM first to precondition the immune environment, followed by sustained APS to amplify adaptive immunity. Moreover, strategies to enhance drug delivery to specific compartments—for example, targeting APS to lymphoid tissues or improving GEM penetration into fibrotic tumor regions—could further refine this balance.

In conclusion, the combination of GEM and APS does not merely add two distinct effects but orchestrates a temporally sequenced and spatially coordinated immune remodeling. Future research should focus on defining the optimal timing, dosage, and route of administration to harness this spatio-temporal interplay, ultimately tailoring combination regimens that dynamically reshape the immune landscape to favor durable anti-tumor responses in TNBC.

Conclusion

In this study, an orthotopic triple-negative breast cancer mouse model was established using the 4T-1 cell line, both in vitro and in vivo. The therapeutic efficacy of the GEM + APS combination therapy for triple-negative breast cancer was confirmed. It was observed that the GEM + APS combined treatment could augment the proportion of T cells and their subsets in the spleen and tumor microenvironment of mice. Additionally, it could suppress the proportion of MDSCs in these locations. Moreover, the direct inhibitory effect of GEM on tumors was also noted, resulting in a superior outcome compared to monotherapy. This study provides an experimental basis for the clinical application of GEM + APS combination therapy for breast cancer patients. However, the specific mechanisms underlying the therapeutic effects of GEM + APS combination therapy on triple-negative breast cancer remain unclear. Further investigation into these mechanisms will be conducted in subsequent studies.