Sage Journals: Discover world-class research

Abstract

Background:

Lung cancer remains the leading cause of cancer-related morbidity and mortality all over the world, with high rates of locoregional recurrence and distant metastasis even after curative-intent surgical resection. The mechanisms of the tumor microenvironment’s role in supporting metastasis through the formation of pre-metastatic niches are crucial areas of investigation.

Methods:

Lung metastasis models were established by injecting Lewis lung cancer cells (LLCs) into the tail vein of 20 specific pathogen free (SPF)—grade male C57BL/6 mice. The mice were divided into 4 groups: control (physiological saline), GuBenPeiYuan (GBPY) medium-dose (25 g/kg), GBPY high-dose (50 g/kg), all administered by gavage, and gemcitabine (50 mg/kg, administered by intraperitoneal injection on days 1, 4, 7, 10, and 13), the total treatment duration was 14 days. Qualitative and quantitative analyses of GBPY were performed using Ultra-Performance Liquid Chromatography (UPLC). Metastasis was observed using hematoxylin and eosin (H&E) staining, and the expression of immune cells was assessed by flow cytometry and immunofluorescence staining. Mechanistic insights were gained through Western blot.

Results:

The high-dose GBPY and gemcitabine groups showed significantly fewer lung metastatic tumors (P = .002; P < .001), while no significant difference was observed between the medium-dose group and control group (P = .438). Flow cytometry results indicated that high-dose GBPY significantly downregulated Myeloid-Derived Suppressor Cells (MDSCs) and G-MDSCs (P = .002 and P = .001, respectively), upregulated dendritic cells (DCs; P = .021), increased M1 macrophages (F4/80⁺/iNOS⁺; P = .001) and decreased M2 macrophages (CD206⁺ F4/80⁺) (P < .001). Furthermore, Western blot results showed that the high-dose GBPY group significantly inhibited the expression of p-JAK2, p-STAT3 (P = .013, P = .001 respectively).

Conclusions:

The GBPY Formula may reduce lung cancer metastasis and recurrence by inhibiting the JAK2/STAT3 pathway, downregulating the presence of MDSCs, upregulating the proportion of DCs, and promoting the polarization of M2 macrophages to M1 macrophages. These changes enhance the anti-tumor immune response, contributing to the reduction of lung cancer metastasis and recurrence.

Keywords

macrophages myeloid-derived suppressor cells dendritic cells tumor microenvironment traditional Chinese medicine

Introduction

The latest epidemiological data indicate that China recorded approximately 1 060 600 new cases of lung cancer and 733 300 deaths in 2022, making it the leading cause of morbidity and mortality among people with malignant tumors.¹ Metastasis is the leading cause of death in lung cancer, and only about 15% of those with advanced non-small cell lung cancer (NSCLC) survive for 5 years after diagnosis.² Even for early-stage NSCLC patients who have undergone curative-intent anatomic surgical resection, there is still a risk of locoregional recurrence or distant metastasis.³ Consequently, focusing on the underlying mechanisms of invasion, and migration is crucial for improving survival rates and developing more effective lung cancer treatment strategies.

The formation of a supportive microenvironment in distant tissues, known as the pre-metastatic niche, plays a key role in promoting cancer metastasis. The recruitment of immature Myeloid-derived suppressor cells (MDSCs) to this niche is important to lung cancer metastasis.⁴ MDSCs, originating from bone marrow myeloid progenitor cells, are a heterogeneous group with strong immunosuppressive functions. In both humans and mice, MDSCs are classified as monocyte (M)-MDSCs and Granulocytic (G)-MDSCs.⁵ These cells contribute to tumor metastasis by inhibiting effector T cells, stimulating regulatory T cells, and mediating immune escape.^6,7 It is noteworthy that in lung cancer patients, the G-MDSC subset is the predominant immunosuppressive cell type,⁸ playing a crucial role not only in immune suppression but also in promoting tumor cell proliferation, angiogenesis, and metastatic growth.^9,10 Studies have shown that high levels of MDSCs in solid tumors are linked to reduced patient survival rates.^11,12 Recently, targeted therapies against MDSCs have shown potential in enhancing immune response and inhibiting tumor growth.¹³

Tumor-associated macrophages (TAMs) are another kind of major suppressive immune cells in the tumor microenvironment (TME), including classically activated macrophages (M1-type) and alternatively activated macrophages (M2-type).¹⁴

TAMs promote tumor metastasis through various mechanisms.^15,16 Certain cytokines secreted by TAMs, such as interleukin-6 (IL-6), are implicated in inducing metastasis by activating the JAK2/STAT3 pathway.¹⁷ Research showed that interactions between MDSCs and macrophages further exacerbate suppression by these cells by altering cytokine production and expression of cell-surface molecules important for cellular function.¹⁸ Unlike MDSCs, dendritic cells (DCs) are a vital component of the normal immune system and play a key role in antigen presentation to naive T cells.¹⁹ However, DCs often exhibit an immature or tolerogenic phenotype due to MDSCs, which impair DC maturation by reducing antigen uptake, preventing the migration of both immature and mature DCs, and blocking the ability of DCs to induce IFNγ-producing T cells.²⁰ The cross-talk between MDSCs, TAMs, and DCs has gradually attracted attention.

Drawing a long history of clinical practice, Traditional Chinese medicine (TCM) has evolved unique approaches to prevent tumor metastasis. TCM and its compounds can promote the differentiation of MDSCs into mature myeloid cells, reduce the proliferation of MDSCs, and inhibit their suppressive function, thereby preventing tumor metastasis. Icariin (ICA) could reduce the expression of IL-10, IL-6, and iNOS in MDSCs, which weakens their function by inhibiting STAT3 activation.²¹ Bao-Yuan-Jie-Du decoction has been shown to downregulate the protein levels of TGF-β, Smad2, Smad3, and Smad4 through the TGF-β/Smads pathway, which prevents the recruitment of MDSCs to the lung.²² For lung cancer patients with metastasis, clinical research has shown that they often present with Qi deficiency syndrome. Targeted use of tonic herbs that strengthen the body’s foundations and enhance regeneration can enhance the body’s immune function, and extend progression-free survival.²³ Our previous study also demonstrated that the Gu-Ben-Pei-Yuan (GBPY) Formula (originally named Spleen and Kidney Warming Formula), known for boosting immune function, can decrease the levels of miR-18a-5p and miR-182-5p, which are related to the development, invasion, and metastasis in a lung cancer metastasis model.²⁴ However, the specific mechanisms by which tonic herbs reduce MDSCs and regulate tumor-infiltrating myeloid precursors (TIMPs) in lung cancer patients remain unclear. Therefore, our study aims to examine the effects of the GBPY Formula on MDSCs, TAMs, and DCs.

Materials and Methods

Cell Culture

Mouse-derived LLCs (Cat# RRID: CVCL_S007) were purchased from the ATCC (American Type Culture Collection, Manassas, VA, USA). LLC cells were cultured at 37°C and 5% CO₂ in a humidified incubator and passaged every 2 to 3 days (ie, when about 90% confluent), using Dulbecco’s Modified Eagle Medium (DMEM) with 10% fetal bovine serum, penicillin (100 U/mL), streptomycin (100 mg/mL), L-glutamine (2 mM), and pyruvate (1 mM).

Preparation of GBPY Formula

GBPY is composed of 11 Chinese herbal medicines, all of which were purchased from Sichuan Xinye Pharmaceutical Technology Development Co., Ltd. Quality control was carried out. The composition and detailed batch number are shown in Table 1. The preparation of GBPY was completed by Shanghai Luming Biotechnology Co., Ltd. It has undergone strict quality control following the “Chinese Pharmacopeia” standard (SYZ-QB-0004-2019). The preparation process is to add 10 times the amount of water to soak and decoct all the herbs for 1 h and filter them; after drying with spray powder, it is made with 80% ethanol as a wetting agent, granulated through a 20-mesh sieve, and dried at 60ºC.

Table 1.

GuBenPeiYuan formula components.

Latin name	Chinese name	Medicinal part	Mass (g)	Batch number
Astragalus mongholicus Bunge [Fabaceae]	Huang Qi	Dry rhizoma	30	21 090 158
Codonopsis pilosula (Franch.) Nannf. [Campanulaceae]	Dang Shen	Dry rhizoma	15	21 100 334
Atractylodes macrocephala Koidz. [Asteraceae]	Bai Zhu	Dry rhizoma	9	21 100 332
Poria cocos (Schw.) Wolf. [Polyporaceae]	Fu Ling	Dry sclerotia	15	21 100 369
Morinda lucida Benth. [Rubiaceae]	Ba Jitian	Fleshy root	15	21 100 109
Epimedium brevicornu Maxim. [Berberidaceae]	Xian Lingpi	Herbal	15	21 100 266
Cuscuta chinensis Lam. [Convolvulaceae]	Tu Sizi	Mature seeds	15	21 080 067
Prunella vulgaris L. [Lamiaceae]	Xia Kucao	Dry spikes	9	21 090 057
Cremastra appendiculata (D.Don) Makino [Orchidaceae]	Shan Cigu	Dry pseudobulb	15	21 090 024
Selaginella doederleinii Hieron. [Selaginellaceae]	Shi Shangbai	Herbal	30	20 080 206
Ostreae Concha	Mu Li	Oyster shell	30	21 080 135

Ultra-Performance Liquid Chromatography (UPLC)

About 2 μL of GBPY extract was analyzed by UPLC using a Waters ACQUITY UPLC I-Class Plus system at a flow rate of 0.3 mL/min. An ACQUITY UPLC HSS T3 (2.1 × 100 mm, 1.8 μm) chromatography column was employed with a mobile phase A of acetonitrile and a mobile phase B of 0.1% formic acid aqueous solution.²⁵ Gradient elution was performed, and the monitoring wavelength was 254 nm.

Animal studies

A total of 20 male mice (C57BL/6; 6-week-old; Shanghai Laboratory Animal Center, CAS Service Shanghai) were divided into 4 groups (5 mice per group): A: Control group; B: GBPY medium-dose group (25 g/kg); C: GBPY high-dose group (50 g/kg); D: Gemcitabine group (as a positive control). Animals were acclimated to the animal facility for 1 week before surgery on a 12 hours light-dark cycle with access to food and water ad libitum in SPF barrier. For the lung metastasis model establishment, LLCs (1 × 10⁷/1 mL phosphate-buffered saline (PBS)/mouse) were injected into the tail vein of C57BL/6 mice.²⁶ Once the model was established, the mice in each group were treated according to the above specifications. Briefly, GBPYM and GBPYH-treated mice were administered 0.2 mL of GBPY daily (25 and 50 g/kg, respectively) by oral gavage for 14 days. Gemcitabine-treated mice were injected intraperitoneally 5 times at a dose of 50 mg/kg body weight/day, on days 1, 4, 7, 10, and 13 following tumor cell injection. Control animals received 0.2 mL of normal saline by oral gavage daily for 14 days. This study was approved by Ethics Committee of Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai University of Traditional Chinese Medicine (animal ethics approval No. YYLAC-2022-162) and was performed following principles for Institutional Animal Care and Use Committee of Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai University of Traditional Chinese Medicine.

Sample Collection and Processing

Fourteen days after the LLC cell injection, the mice were anesthetized with 2% isoflurane and euthanized by cervical dislocation. The lungs were excised and gross metastatic tumors visible on the lung surface were counted and documented through photographic records. For further quantitative and qualitative analyses, the metastatic lesions were subjected to H&E staining, Western blot, flow cytometric analysis and immunofluorescence staining. All animal procedures were performed in compliance with Shanghai University of Traditional Chinese Medicine Institutional Animal Care and Use Committee (IACUC) guidelines.

H&E Staining

Some metastatic tumors were fixed in formalin, embedded in paraffin, and sectioned at 3 μm thickness. Sections were placed in a 60°C oven for 1 hour, deparaffinized with xylene, and rehydrated in PBS. H&E staining involved Harris’s hematoxylin for 5 minutes, differentiation in 1% hydrochloric acid in ethanol, and dehydration through ethanol series. After clearing in xylene, sections were mounted with mounting medium and examined under an optical microscope.²⁷

Western Blot

Protein samples were extracted from metastatic tumor tissues using RIPA Lysis Buffer (Thermo, Cat# 89900) and quantified using a bicinchoninic acid assay kit (Shanghai Epizyme Biomedical, Cat# ZJ101). Proteins (30 µg) were separated by 10% SDS-PAGE and transferred to polyvinylidene fluoride membranes (EMD Millipore). Membranes were blocked with 5% skim milk for 2 hours at room temperature and incubated with primary antibodies (1:1000) for p-STAT3 (Cat# 9145, RRID: AB_2491009), p-JAK2 (Cat# 3776, RRID: AB_2617123), STAT3 (Cat# 9139, RRID: AB_331757), and JAK2 (Cat# 3230, RRID: AB_2128522) from Cell Signaling Technology overnight at 4°C. Afterward, membranes were treated with secondary antibodies (anti-rabbit Cat# 7074, RRID: AB_2099233 or anti-mouse Cat# 7076, RRID: AB_330924) from Cell Signaling Technology for 2 hours at room temperature. Band intensities were semi-quantified using an enhanced chemiluminescence kit (New Cell & Molecular Biotech, Cat# P10060). GAPDH (Proteintech Group, Cat# 60004-1-Ig, RRID: AB_2107436) was used as a loading control.²⁸

Flow Cytometric Analysis

Metastatic tumor tissues and peritumoral tissues were collected from murine metastasis models. After mechanical dissociation and enzymatic digestion, single-cell suspensions were prepared. Flow cytometry identified MDSCs, macrophages, and DCs. MDSCs were broadly classified using CD11b and Gr-1 markers and further differentiated into monocytic M-MDSCs (CD11b⁺Ly6C⁺Ly6G⁻) and granulocytic G-MDSCs (CD11b⁺Ly6C⁻Ly6G⁺) with Ly6C and Ly6G markers. Tumor-associated DCs (CD11c⁺MHCII⁺) and macrophages were categorized into M1 (F4/80⁺iNOS⁺) and M2 (CD206⁺F4/80⁺) phenotypes using specific markers. Samples were analyzed using BD flow cytometry and FlowJo software.²⁹ Antibodies used included anti-CD11b (Cat# 17800, RRID: AB_3665018), anti-Gr-1 (Cat# 31469, RRID: AB_2888647), anti-iNOS (Cat# 13120, RRID: AB_2687529) from Cell Signaling Technology, and anti-Ly6C (Cat# GB115601, RRID: AB_3665019), anti-Ly6G (Cat# GB11229, RRID: AB_2814689), anti-CD11c (Cat# GB11059, RRID: AB_2905514), anti-MHCII (Cat# GB115523, RRID: AB_3665020), anti-F4/80 (Cat# GB113373, RRID: AB_2938980), anti-CD206 (Cat# GB113497, RRID: AB_3665021) from Servicebio.

Immunofluorescence (IF) Staining

Mouse tumor tissues were washed with PBS, fixed in 4% paraformaldehyde, and permeabilized with 0.1% Triton X-100 (Sigma, USA) for 10 minutes. Non-specific binding was blocked using 3% Bovine Serum Albumin (BSA) for 30 minutes.³⁰ Tissues were then incubated overnight at 4°C with primary antibodies: anti-Gr-1 (Cat# 31469, RRID: AB_2888647), anti-CD11b (Cat# 17800, RRID: AB_3665018) from Cell signaling technology, and anti-CD206 (Cat# GB113497, RRID: AB_3665021), anti-F4/80 (Cat# GB113373, RRID: AB_2938980), anti-MHCII (Cat# GB115523, RRID: AB_3665020), anti-CD11c (Cat# GB11059, RRID: AB_2905514) from Servicebio at 1:100 dilution. This was followed by incubation with secondary antibodies (Alexa Fluor® 488, Cat# GB25303 and Cat# GB28301) at 1:100 dilution. After PBS washes, cell nuclei were stained with DAPI (Cat# GDP1024) for 10 minutes. Images were captured using a confocal microscope (Leica TCS SP5II, Germany).

Statistical Analysis

Statistical significance was assessed by using GraphPad Prism 9.0 software (GraphPad Software, Inc). Differences between the 2 groups were compared using a two-tailed unpaired Student’s t-test. Differences between multiple groups were compared using one-way ANOVA, followed by Tukey’s post hoc analysis. P-values < .05 were considered significant.

Results

Analysis of GBPY Formula Compounds

The chemical compositions of the GBPY Formula extracts were determined by UPLC-Q-TOF-MS in both positive and negative-ion modes, as shown in Figure 1. A total of 64 compounds were identified based on the peaks with high response values in the BPI (Base peak ion) diagram. These compounds were confirmed by comparing their retention times, molecular weights, and MS data with those of the reference standards, and the detailed list of the compounds, determined based on the peak areas, is shown in Table S1. Among these, Epimedin C and Icariin were the main components identified in the extracts, with retention times of 28.34 and 28.71 minutes.

Figure 1.

Mass spectrum chromatograms of GBPY formula ingredients. (A) Positive ion pattern. (B) Negative ion pattern.

GBPY Significantly Inhibited Lung Metastasis Tumor Growth

The inhibitory effect of medium and high doses of GBPY formula on tumor nodules in the lung metastasis model was observed. To confirm lung metastasis, photography and HE staining were employed, and the number of metastatic nodules and tumor size were counted (Figure 2A). As illustrated in Figure 2B, the mean number of pulmonary metastases was significantly reduced in the high-dose GBPY group (10.40 ± 2.06) and the Gemcitabine group (7.20 ± 1.72) compared to the control group (17.40 ± 2.33), with P = .002 and P < .001, respectively. Although the medium dose of GBPY did not significantly reduce the number of metastases compared to the control (P = .439), the overall results suggest that GBPY inhibits lung cancer metastasis in a dose-dependent manner.

Figure 2.

LLCs were intravenously injected into C57BL/6 mice via the lateral tail vein. (A) Representative images of lungs (top row) and lung sections stained with H&E (bottom row) were collected 2 weeks after injection. Arrows highlight the metastatic tumors. (B) Surface metastatic nodules in the lungs were quantified. Data represent mean ± SD (n = 5 per group).

To demonstrate the adequacy of the sample size, we performed a power analysis using the number of lung metastases in mice with the R packages effsize and pwr. The calculated power was 0.999 for the Control versus Gemcitabine groups and 0.975 for the Control versus High-dose GBPY group, both exceeding 0.8, indicating sufficient sample size and robust study design.

GBPY Reduces MDSC Expression in Lung Metastases and Peritumoral Areas

Initially, immunofluorescence (IF) was employed to identify CD11b and Gr-1 in mice, enabling visual confirmation of downregulation of MDSC expression in the tumor and peritumoral areas in both the gemcitabine group and the medium to high-dose GBPY groups (Figure 3A). Flow cytometry further quantified this observation, revealing that a medium dose of GBPY reduced MDSC levels to 9.33% ± 1.23%, while a high dose further decreased it to 6.32% ± 0.88%, both showing statistical significance compared with the control group (13.53% ± 0.85%, P = .035 and P = .002, respectively; Figure 3B). This indicates a dose-dependent decrease in the percentage of MDSCs after GBPY treatment.

Figure 3.

GBPY Reduces MDSC Presence. (A) The immunofluorescence images of Gr-1 and CD11b in tumor and peritumoral tissues from lung metastasis mice. (B) The percentage of MDSCs (defined as Gr-1⁺, CD11b⁺) in the metastatic tumor tissue of lung metastasis mice was measured by flow cytometry. (C) The percentage of Ly6C⁻Ly6G⁺ (G-MDSCs, lower right quadrant) and Ly6C⁺Ly6G⁻ (M-MDSCs, upper left quadrant) in tumor and peritumoral tissues from lung metastasis mice was measured by flow cytometry (N = 5).

Further analysis was conducted based on the binding specificity of antibodies to different epitopes of Gr-1 (Ly6G, Ly6C), assessing the impact on M-MDSCs (CD11b⁺Ly6C⁺Ly6G⁻) and G-MDSCs (CD11b⁺Ly6C⁻Ly6G⁺) in the tumor and peritumoral areas. It was found that high doses of GBPY significantly downregulated G-MDSCs, with Ly6G levels reaching 5.27% ± 0.47% (P = .001), and upregulated M-MDSC with Ly6C expression levels reaching 15.10% ± 1.00% (P = .005). Additionally, the high-dose GBPY group showed a significant reduction in G-MDSC expression compared to the medium-dose GBPY group (5.27% ± 0.47% vs 9.64% ± 0.53%, P = .026), as shown in Figure 3C. This indicates that GBPY reduces the immunosuppressive effects of MDSCs in TME.

GBPY Enhances Dendritic Cells Activation in Lung Metastatic Tumors

Immunofluorescence analysis revealed a marked increase in DCs maturation within the lung metastatic tumors following treatment with GBPY. Staining for CD11c and MHC II showed that both medium and high doses of GBPY significantly boosted the presence of mature DCs, with the high-dose GBPY group exhibiting a notable increase in the density of CD11c⁺MHCII⁺ cells, compared to the control group (Figure 4A). Subsequent flow cytometry analysis further confirmed these IF findings. The Gemcitabine group showed a substantial increase in mature DCs, with a mean of 23.03% ± 1.11% (P = .001), while the high-dose GBPY treatment group also demonstrated a significant enhancement, with a mean of 20.87% ± 0.51% (P = .021), as shown in Figure 4B. These results suggest that GBPY, especially at higher doses, exerts a strong influence on the maturation of DCs within the TME, a critical factor for the efficacy of antigen presentation and subsequent activation of the adaptive immune response.

Figure 4.

GBPY enhances dendritic cell activation. (A) The immunofluorescence images of MHCII and CD11c in tumor and peritumoral tissues from lung metastasis mice. (B) The percentage of DCs (defined as CD11c⁺, MHC II⁺) in the metastatic tumor tissue of lung metastasis mice was measured by flow cytometry (N = 5).

GBPY Induces a Dose-Dependent Shift in Macrophage Polarization Within Lung Metastases

Immunofluorescence and flow cytometry analyses were conducted to evaluate the effects of GBPY on macrophage polarization within lung metastatic tumors in mouse models. IF analysis initially demonstrated an increase in F4/80⁺ macrophages, particularly in Gemcitabine and high-dose GBPY treatment groups (Figure 5A). Further flow cytometry analysis revealed a significant shift in macrophage polarization. A significant increase in the M1 phenotype, marked by F4/80⁺iNOS⁺ cells, was observed in the medium-dose group (24.73% ± 1.22%, P = .020) and the high-dose group (28.47% ± 3.10%, P = .001), with the Gemcitabine group serving as a positive control (36.63% ± 1.69%, P < .001), as illustrated in Figure 5B. Conversely, the medium-dose (12.6% ± 0.95%, P = .002) and high-dose (9.06% ± 0.21%, P < .001) GBPY groups showed a reduction in M2 macrophage phenotype, as indicated by the decreased CD206⁺ F4/80⁺ population compared to the control group (17.77% ± 1.55%; Figure 5C).

Figure 5.

GBPY promotes polarization from M2 to M1 macrophages in tumor tissue. (A) The immunofluorescence images of CD206 and F4/80 in tumor and peritumoral tissues from lung metastasis mice. (B) The percentage of M1 macrophages (defined as F4/80⁺, iNOS⁺) in the metastatic tumor tissue of lung metastasis mice was measured by flow cytometry. (C) The percentage of M2 macrophages (defined as F4/80⁺, CD206⁺) in the metastatic tumor tissue of lung metastasis mice was measured by flow cytometry (N = 5).

GBPY May Influence Immune-Related Cells by Inhibiting the JAK2/STAT3 Pathway

Aberrant STAT3 expression, activated by JAK2, plays a key role in immune suppression and is involved in various inflammatory, immune-related diseases, and tumors.^31,32 To further investigate the mechanism by which GBPY modulates immune-suppressive cells, we performed western blot analysis to examine the JAK2/STAT3 signaling pathway. There was a significant downregulation in the p-STAT3 expression levels and p-STAT3/STAT3 ratios in both the medium and high-dose GBPY groups compared with the control (Figure 6A), with the p-STAT3 signal intensity normalized to GAPDH expression. The lowest expression levels were detected in the high-dose GBPY group (P = .001, P < .001, respectively; Figure 6B). Additionally, the p-JAK2 expression level and the p-JAK2/JAK2 ratio showed a decrease in the high-dose GBPY group (P = .013, P = .010, respectively), with a notable difference in the p-JAK2/JAK2 ratio between the medium and high-dose groups (P = .030; Figure 6B). It is suggested that GBPY formula may inhibit the JAK2/STAT3 pathway, modulating the polarization of macrophages in mouse lung metastatic tumors, with a clear dose-dependent effect.

Figure 6.

Whole protein cell lysates were prepared randomly from mouse lung metastatic tumors in each group, followed by Western blot analysis of p-STAT3, STAT3, p-JAK2, and JAK2. The reference proteins are GAPDH. (A) Western blot detection of p-STAT3, STAT3, p-JAK2, and JAK2 protein expression in mouse lung metastatic tumors. (B) Statistical analysis of protein expression levels in mouse lung metastatic tumors in each group.

Discussion

Lung cancer remains one of the most lethal malignancies worldwide due to its high metastasis propensity and limited treatment efficacy.³³ Despite advancements in targeted therapies and immunotherapies, managing metastatic lung cancer remains challenging.³⁴ Bone marrow-derived myeloid cells (BMDC) are crucial in forming the premetastatic microenvironment necessary for disseminated tumor cells to colonize distant sites, as demonstrated in murine pulmonary metastasis models.^4,35,36 MDSCs, a diverse group of immature myeloid cells, proliferate and significantly contribute to tumor progression.³⁷ Despite their known involvement, the specific role of MDSCs in premetastatic niche formation is not fully understood.

Research suggests that cytokines, chemokines, and enzymes released by activated MDSCs enhance tumor cell invasion, proliferation, adhesion, and chemotaxis, thereby facilitating tumor invasion and metastasis.³⁸ This understanding resonates with insights from the Inner Canon of the Yellow Emperor,³⁹ which describes metastasis as the transmission of disease to different locations due to cancer toxins and deficiency of vital energy (Zheng Qi). According to this theory, tumor metastasis occurs due to the malignancy of cancer and weakened vital energy in target organs. Therefore, for patients with lung cancer, the treatment emphasizes strengthening the foundation and cultivating primordial energy to nurture and protect vital energy, enhancing resistance to external pathogens. The GBPY formula in our study is a distinctive TCM compound that integrates the effects of cultivating primordial energy, strengthening postnatal essence, and detoxifying to fight cancer.

To test the role of GBPY formula in regulating the premetastatic niche, we used a mouse tail vein lung metastasis model, whose time and process of metastasis were relatively clear.⁴⁰ Our results showed that GBPY formula significantly reduces the number of spontaneous metastatic tumors in a dose-dependent manner. Following GBPY formula treatment, there was a downregulation in the proportion of MDSCs, particularly the G-MDSC subset.

We also observed significant changes in other immune cell proportions. MDSCs are known to suppress DCs function by inducing the expansion of regulatory T cells (Tregs) and promoting the negative regulation of immune responses by T cells.⁴¹ DCs are crucial for initiating and regulating immune responses, including promote anti-tumor immunity by activating cytotoxic T lymphocytes that target and kill cancer cells.⁴² Sanguinarine, found in traditional herbal plants, has been observed to induce the differentiation of MDSCs into DCs through the NF-κB pathway in vitro,⁴³ thereby inhibiting the growth of lung cancer. Consistent with these findings, our results demonstrated an increase in DCs in the GBPY-treated group. Moreover, recent studies have shown that MDSCs can further differentiate into TAMs within TME.¹⁸ TAMs are classified into 2 categories: the M1 subtype, which inhibits tumor growth, and the M2 subtype, which promotes tumor growth. MDSCs tend to differentiate into the M2 phenotype due to growth factors and cytokines in tumor tissues.⁴⁴ Inducing MDSCs to differentiate into the M1 phenotype can slow tumor growth, increase tumor-specific immune cells, reduce neovascularization, and inhibit metastasis.⁴⁰ Research indicates that many anti-tumor Chinese herbal medicines modulate M2 macrophage polarization. Pulsatilla saponins extracted from Pulsatilla chinensis (Bge.) Regel has been shown to suppress lung metastasis of melanoma by inhibiting the polarization of M2 macrophages via the STAT6 signaling pathway.⁴⁵ Our study found that the GBPY formula decreases the proportion of M2-type TAMs and increases the proportion of M1-type TAMs following intervention.

Other studies have found that TME induces immunosuppressive characteristics in MDSCs through transcription factors such as STAT1, STAT3, and STAT6.^46,47 Among these, STAT3 is crucial for regulating MDSC functions and expansion,⁴⁸ which is driven by tumor-derived factors.⁴⁹ Depleting STAT3 can eliminate immunosuppressive myeloid cells in cancer, significantly enhancing the differentiation and maturation of dendritic cells.^49
-51 Indeed, elevated STAT3 activation is observed in various cancers, including lung adenocarcinoma, and is associated with increased metastasis and poor patient prognosis.^52,53 Inhibiting the JAK2/STAT3 pathway in tumor tissues can reduce MDSCs-associated immunosuppressive functions. Recent research showed that Yanghe Decoction could modulate MDSCs functions via the JAK/STAT pathways, inhibiting 4T1 breast tumor growth.⁵⁴ Building on these findings, we analyzed key proteins in the JAK2/STAT3 pathway and found that the high-dose GBPY formula significantly downregulated the expression of p-STAT3 and p-JAK2 (P = .001, P = .013, respectively). The medium-dose GBPY formula also significantly downregulated p-STAT3 expression (P = .003). Our results demonstrate that the GBPY formula effectively regulates immune cells and downregulates the JAK2/STAT3 pathway, highlighting its potential in modulating tumor-induced immunosuppression.

However, our study has several limitations. Firstly, although we observed downregulation of the JAK2/STAT3 pathway and changes in immune cell proportions, the exact molecular mechanisms of GBPY remain unclear, requiring further detailed studies. Secondly, the study primarily focused on MDSCs, TAMs, and DCs. A broader assessment of other immune cell types and their interactions within the TME would better elucidate GBPY’s immunomodulatory effects.

Conclusion

In this study, we evaluated the immunoregulatory effects of the GBPY formula in LLCs mouse metastasis model. The GBPY formula inhibited lung cancer metastasis by suppressing MDSCs and modulating associated immune cells, whose mechanism is likely related to the JAK2/STAT3 pathway. Further research is recommended to better understand the role of the GBPY formula in the TME and its impact on tumor control in vivo and in vitro.

Supplemental Material

sj-xlsx-1-ict-10.1177_15347354251324650 – Supplemental material for GuBenPeiYuan Formula Inhibits Lung Cancer Metastasis by Suppressing Myeloid-Derived Suppressor Cells and Related Immune Cells

Supplemental material, sj-xlsx-1-ict-10.1177_15347354251324650 for GuBenPeiYuan Formula Inhibits Lung Cancer Metastasis by Suppressing Myeloid-Derived Suppressor Cells and Related Immune Cells by Yizhao Du, Yongming Zhou, Lijing Jiao, Wenxiao Yang, Ling Xu, Hailun Zhou, Jingwen Zhao, Quanyao Li, Yang Han, Yabin Gong and Qin Wang in Integrative Cancer Therapies

Footnotes

Acknowledgements

The authors would like to thank to Zhao Bei for the guidance of the experiment.

Authors’ Contributions

QW and YBG conceived and designed the experiments. The experiments were carried out by YZD, YMZ, LJJ, WXY, HLZ, and YH. The manuscript was drafted by YZD. Data collection and analysis were performed by YZD, XL, and QYL. Technical expertise was provided by QW. QW and YBG assisted in revising the manuscript. All authors reviewed and approved the final version of the manuscript.

Data Availability Statement

The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was supported by the National Natural Science Foundation of China (nos. 82104948, 82474588), the Project of Shanghai Municipal Public Health Bureau (no. 20214Y0177).

Ethical Approval

Ethical approval for the animal experiments was obtained from the Ethics Committee of Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai University of Traditional Chinese Medicine (Ethics Approval Number: YYLAC-2022-162).

ORCID iDs

Yizhao Du

Quanyao Li

Qin Wang

Supplemental Material

Supplemental material for this article is available online.

References

Han

Zheng

Zeng

, et al. Cancer incidence and mortality in China, 2022. J Natl Cancer Cent. 2024;4:47-53. doi:10.1016/j.jncc.2024.01.006

Zhang

Wang

Lin

Ding

Role of miRNA in lung cancer-potential biomarkers and therapies. Curr Pharm Des. 2018;23:5997-6010. doi:10.2174/1381612823666170714150118

Martini

Bains

Burt

, et al. Incidence of local recurrence and second primary tumors in resected stage I lung cancer. J Thorac Cardiovasc Surg. 1995;109:120-129. doi:10.1016/S0022-5223(95)70427-2

Kaplan

Riba

Zacharoulis

, et al. VEGFR1-positive haematopoietic bone marrow progenitors initiate the pre-metastatic niche. Nature. 2005;438:820-827. doi:10.1038/nature04186

Schroeter

Roesel

Matsunaga

, et al. Aging affects the role of myeloid-derived suppressor cells in alloimmunity. Front Immunol. 2022;13:917972. doi:10.3389/fimmu.2022.917972

Tong

Jiménez-Cortegana

Tay

AHM

, et al. NK cells and solid tumors: therapeutic potential and persisting obstacles. Mol Cancer. 2022;21:206. doi:10.1186/s12943-022-01672-z

Takeyama

Kato

Tamada

, et al. Myeloid-derived suppressor cells are essential partners for immune checkpoint inhibitors in the treatment of cisplatin-resistant bladder cancer. Cancer Lett. 2020;479:89-99. doi:10.1016/j.canlet.2020.03.013

Salehi-Rad

Crosson

, et al. Inhibition of granulocytic myeloid-derived suppressor cells overcomes resistance to immune checkpoint inhibition in LKB1-deficient non-small cell lung cancer. Cancer Res. 2021;81:3295-3308. doi:10.1158/0008-5472.CAN-20-3564

Mehmeti-Ajradini

Bergenfelz

Larsson

, et al. Human G-MDSCs are neutrophils at distinct maturation stages promoting tumor growth in breast cancer. Life Sci Alliance. 2020 Nov;3(11)pii:3/11/ e202000893. doi:10.26508/lsa.202000893

10.

Zhou

Nefedova

Lei

Gabrilovich

Neutrophils and PMN-MDSC: their biological role and interaction with stromal cells. Semin Immunol. 2018;35:19-28. doi:10.1016/j.smim.2017.12.004

11.

Zhang

Wang

, et al. Circulating and tumor-infiltrating myeloid-derived suppressor cells in patients with colorectal carcinoma. PLoS One. 2013;8:e57114. doi:10.1371/journal.pone.0057114

12.

Cui

Kryczek

Zhao

, et al. Myeloid-derived suppressor cells enhance stemness of cancer cells by inducing microRNA101 and suppressing the corepressor CtBP2. Immunity. 2013;39:611-621. doi:10.1016/j.immuni.2013.08.025

13.

Iclozan

Antonia

Chiappori

Chen

Gabrilovich

Therapeutic regulation of myeloid-derived suppressor cells and immune response to cancer vaccine in patients with extensive stage small cell lung cancer. Cancer Immunol Immunother. 2013;62:909-918. doi:10.1007/s00262-013-1396-8

14.

Locati

Curtale

Mantovani

Diversity, mechanisms, and significance of macrophage plasticity. Annu Rev Pathol. 2020;15:123-147. doi:10.1146/annurev-pathmechdis-012418-012718

15.

Zheng

Crosstalk between mesenchymal stromal cells and tumor-associated macrophages in gastric cancer. Front Oncol. 2020;10:571516. doi:10.3389/fonc.2020.571516

16.

Lin

Lan

Tumor-associated macrophages in tumor metastasis: biological roles and clinical therapeutic applications. J Hematol Oncol. 2019;12:76. doi:10.1186/s13045-019-0760-3

17.

Sui

Jin

, et al. IL6-STAT3-C/EBPβ-IL6 positive feedback loop in tumor-associated macrophages promotes the EMT and metastasis of lung adenocarcinoma. J Exp Clin Cancer Res. 2024;43:63. doi:10.1186/s13046-024-02989-x

18.

Ostrand-Rosenberg

Sinha

Beury

Clements

VK.

Cross-talk between myeloid-derived suppressor cells (MDSC), macrophages, and dendritic cells enhances tumor-induced immune suppression. Semin Cancer Biol. 2012;22:275-281. doi:10.1016/j.semcancer.2012.01.011

19.

Cabeza-Cabrerizo

Cardoso

Minutti

Pereira

Costa

Reis

Sousa

Dendritic cells revisited. Annu Rev Immunol. 2021;39:131-166. doi:10.1146/annurev-immunol-061020-053707

20.

Poschke

Mao

Adamson

, et al. Myeloid-derived suppressor cells impair the quality of dendritic cell vaccines. Cancer Immunol Immunother. 2012;61:827-838. doi:10.1007/s00262-011-1143-y

21.

Zhou

Chen

, et al. Icariin and its derivative, ICT, exert anti-inflammatory, anti-tumor effects, and modulate myeloid derived suppressive cells (MDSCs) functions. Int Immunopharmacol. 2011;11:890-898. doi:10.1016/j.intimp.2011.01.007

22.

Tian

Song

Wang

, et al. Chinese herbal medicine Baoyuan Jiedu decoction inhibits the accumulation of myeloid derived suppressor cells in pre-metastatic niche of lung via TGF-β/CCL9 pathway. Biomed Pharmacother. 2020;129:110380. doi:10.1016/j.biopha.2020.110380

23.

Liang

Wang

Zheng

Mei

A clinical study on the use of Yiqi Yangxue decoction combined with chemotherapy to promote rapid postoperative recovery in patients with non-small cell lung cancer. Emerg Med Int. 2022;2022:7073893. doi:10.1155/2022/7073893

24.

Wang

Lijing

jiaqi

Tonghai

Ling

Effects of Jianpi Wenshen Fang on expressions of miRNAs and related target genes mRNA in lung metastases of nude mice after subcutaneous transplantation. J Tradit Chin Med. 2018;59:1401-1404. doi:10.13288/j.11-2166/r.2018.16.014

25.

Wang

Geng

, et al. Pharmacokinetics of herb-drug interactions of plumbagin and tazemetostat in rats by UPLC-MS/MS. Drug Des Dev Ther. 2022;16:3385-3394. doi:10.2147/DDDT.S384156

26.

Singharajkomron

Yodsurang

Limprasutr

, et al. CAMSAP2 enhances lung cancer cell metastasis by mediating RASAL2 degradation. Life Sci. 2024;338:122391. doi:10.1016/j.lfs.2023.122391

27.

Travis

Brambilla

Noguchi

, et al. International association for the study of lung cancer/american thoracic society/european respiratory society international multidisciplinary classification of lung adenocarcinoma. J Thorac Oncol. 2011;6:244-285. doi:10.1097/JTO.0b013e318206a221

28.

Shen

, et al. Inhibition of ATM reverses EMT and decreases metastatic potential of cisplatin-resistant lung cancer cells through JAK/STAT3/PD-L1 pathway. J Exp Clin Cancer Res. 2019;38:149. doi:10.1186/s13046-019-1161-8

29.

Yang

Chuang

Tseng

, et al. Tumor promoting effect of PDLIM2 downregulation involves mitochondrial ROS, oncometabolite accumulations and HIF-1α activation. J Exp Clin Cancer Res. 2024;43:169. doi:10.1186/s13046-024-03094-9

30.

Han

Kim

, et al. Snail acetylation by autophagy-derived acetyl-coenzyme A promotes invasion and metastasis of KRAS-LKB1 co-mutated lung cancer cells. Cancer Commun. 2022;42:716-749. doi:10.1002/cac2.12332

31.

Sodji

Oyelere

AK.

Inflammation, fibrosis and cancer: mechanisms, therapeutic options and challenges. Cancers. 2022;14:552. doi:10.3390/cancers14030552

32.

O’Shea

Plenge

JAK and STAT signaling molecules in immunoregulation and immune-mediated disease. Immunity. 2012;36:542-550. doi:10.1016/j.immuni.2012.03.014

33.

Herbst

Morgensztern

Boshoff

The biology and management of non-small cell lung cancer. Nature. 2018;553:446-454. doi:10.1038/nature25183

34.

Duma

Santana-Davila

Molina

JR.

Non-small cell lung cancer: epidemiology, screening, diagnosis, and treatment. Mayo Clin Proc. 2019;94:1623-1640. doi:10.1016/j.mayocp.2019.01.013

35.

Bonapace

Coissieux

Wyckoff

, et al. Cessation of CCL2 inhibition accelerates breast cancer metastasis by promoting angiogenesis. Nature. 2014;515:130-133. doi:10.1038/nature13862

36.

Peinado

Zhang

Matei

, et al. Pre-metastatic niches: organ-specific homes for metastases. Nat Rev Cancer. 2017;17:302-317. doi:10.1038/nrc.2017.6

37.

Sato

Shimizu

Shinga

, et al. Characterization of the myeloid-derived suppressor cell subset regulated by NK cells in malignant lymphoma. OncoImmunology. 2015;4:e995541. doi:10.1080/2162402X.2014.995541

38.

Umansky

Blattner

Gebhardt

Utikal

The role of myeloid-derived suppressor cells (MDSC) in cancer progression. Vaccines. 2016;4:36. doi:10.3390/vaccines4040036

39.

Chinese Medicine Publishing Center. Inner Canon of the Yellow Emperor: Suwen. People’s Medical Publishing House; 2012.

40.

Zou

, et al. Epigenetic therapy inhibits metastases by disrupting premetastatic niches. Nature. 2020;579:284-290. doi:10.1038/s41586-020-2054-x

41.

Bhardwaj

Ansell

SM.

Modulation of T-cell function by myeloid-derived suppressor cells in hematological malignancies. Front Cell Dev Biol. 2023;11:1129343. doi:10.3389/fcell.2023.1129343

42.

Wang

Xiang

Xin

, et al. Dendritic cell biology and its role in tumor immunotherapy. J Hematol Oncol. 2020;13:107. doi:10.1186/s13045-020-00939-6

43.

Luo

Zhou

, et al. Role of sanguinarine in regulating immunosuppression in a Lewis lung cancer mouse model. Int Immunopharmacol. 2022;110:108964. doi:10.1016/j.intimp.2022.108964

44.

Brandau

Trellakis

Bruderek

, et al. Myeloid-derived suppressor cells in the peripheral blood of cancer patients contain a subset of immature neutrophils with impaired migratory properties. J Leukoc Biol. 2011;89:311-317. doi:10.1189/jlb.0310162

45.

Yang

Yan

, et al. Pulsatilla saponins inhibit experimental lung metastasis of melanoma via targeting STAT6-mediated M2 macrophages polarization. Molecules. 2023;28:3682. doi:10.3390/molecules28093682

46.

Gabrilovich

Ostrand-Rosenberg

Bronte

Coordinated regulation of myeloid cells by tumours. Nat Rev Immunol. 2012;12:253-268. doi:10.1038/nri3175

47.

Almand

Clark

Nikitina

, et al. Increased production of immature myeloid cells in cancer patients: a mechanism of immunosuppression in cancer. J Immunol. 2001;166:678-689. doi:10.4049/jimmunol.166.1.678

48.

Rébé

Végran

Berger

Ghiringhelli

STAT3 activation: a key factor in tumor immunoescape. JAKSTAT. 2013;2:e23010. doi:10.4161/jkst.23010

49.

Nefedova

Huang

Kusmartsev

, et al. Hyperactivation of STAT3 is involved in abnormal differentiation of dendritic cells in cancer. J Immunol. 2004;172:464-474. doi:10.4049/jimmunol.172.1.464

50.

Nefedova

Nagaraj

Rosenbauer

, et al. Regulation of dendritic cell differentiation and antitumor immune response in cancer by pharmacologic-selective inhibition of the janus-activated kinase 2/signal transducers and activators of transcription 3 pathway. Cancer Res. 2005;65:9525-9535. doi:10.1158/0008-5472.CAN-05-0529

51.

Kortylewski

Kujawski

Wang

, et al. Inhibiting stat3 signaling in the hematopoietic system elicits multicomponent antitumor immunity. Nat Med. 2005;11:1314-1321. doi:10.1038/nm1325

52.

Chai

Nan

Zhao

SHP2 mediates STAT3/STAT6 signaling pathway in TAM to inhibit proliferation and metastasis of lung adenocarcinoma. Aging. 2024;16:12498-12509. doi:10.18632/aging.205799

53.

Bickett

Knitz

Piper

, et al. Dichotomous effects of cellular expression of STAT3 on tumor growth of HNSCC. Mol Ther. 2022;30:1149-1162. doi:10.1016/j.ymthe.2021.11.011

54.

Mao

Feng

Gong

The antitumor and immunomodulatory effect of Yanghe decoction in breast cancer is related to the modulation of the JAK/STAT signaling pathway. Evid Based Complement Alternat Med. 2018;2018. doi:10.1155/2018/8460526

Supplementary Material

Please find the following supplemental material available below.

For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.

For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.

0.00 MB

0.02 MB