Abstract
The immunosuppressive tumor microenvironment and vascular abnormalities are important factors affecting the anticancer effects of chemotherapeutic drugs. Previous studies have shown that moxibustion combined with cisplatin can improve tumor immunity and vascular normalization. The aim is to further explore the pathways and molecular mechanisms by which moxibustion combined with cisplatin exerts anti-tumor effects through regulating the tumor immune-vascular microenvironment. Through RNA sequencing and bioinformatics analysis, we identified related signaling pathways and key molecules, which were subsequently validated at both cellular and molecular levels. The combination of moxibustion and cisplatin enhanced the infiltration of CD4+ T cells and myeloid dendritic cells in tumor tissues. Moreover, it elevated the M1/M2 and Th1/Th2 ratios along with enhanced Th1 cell polarization. This therapeutic approach modulated immune-related molecules through upregulation of Il2 and downregulation of Il1β at the mRNA level, accompanied by decreased protein expression of CXCL1, IL-13, CCL3, and CCL4. Furthermore, moxibustion upregulated Ifnγ and Pf4 mRNA levels and downregulated Vegfa and Flt1. Thus, the imbalance between pro-angiogenic and anti-angiogenic factors in the tumor microenvironment can be corrected. Notably, the anti-tumor effects of moxibustion combined with cisplatin were abrogated upon intratumoral injection of the IFN-γ neutralizing antibody, suggesting the critical role of IFN-γ. The findings demonstrate that the combination of moxibustion and cisplatin can enhance tumor immunity, inhibit tumor angiogenesis. This therapeutic effect is potentially mediated through the upregulation of IFN-γ.
Introduction
Cancer remains a major societal, public health, and economic challenge in the 21st century. Lung cancer was the most frequently diagnosed cancer in 2022 and remains the leading cause of cancer-related deaths. Non-small cell lung cancer (NSCLC) accounts for approximately 85% of all lung cancer cases and has a reported 5-year survival rate of less than 18%. 1 In the treatment of NSCLC, chemotherapy remains the cornerstone of systemic therapy. Whether combined with immunotherapy or serving as the core option when immunotherapy is contraindicated, it holds an indispensable position. 2 However, side effects of chemotherapy, such as myelosuppression, neurotoxicity, gastrointestinal toxicity, and nephrotoxicity, cannot be ignored. 3 Therefore, developing safer and more effective complementary and alternative therapies is of vital importance. 4
Moxibustion involves the stimulation of specific acupuncture points through the burning of mugwort. While sharing the same theoretical foundation as acupuncture in traditional Chinese medicine, moxibustion offers additional benefits through thermal stimulation, aromatic effects, herbal action, and biophysical properties.5,6 Zusanli (ST36) is a key point in the meridian system and has been extensively studied and applied in the treatment of lung cancer. Moxibustion at ST36 can be integrated with modern treatments such as chemotherapy and radiotherapy to reduce side effects and enhance therapeutic efficacy7,8
Evasion of immune surveillance and tumor angiogenesis are 2 critical hallmarks of cancer progression. Strategies aimed at enhancing the tumor microenvironment—including improving immune responses and vascular normalization—are essential for increasing the effectiveness of chemotherapeutic drugs or mitigating their adverse effects. Strengthening the body’s immune function and inhibiting tumor progression are key objectives in cancer therapy.
Studies have shown that moxibustion can improve the immunosuppressive tumor microenvironment by acting on various types of immune cells, such as T lymphocytes, natural killer (NK) cells, and M1 macrophages, thereby enhancing immune function. 8 There is also evidence suggesting that moxibustion can enhance immune function and improve quality of life in cancer patients.9,10 Cisplatin is a core drug for first-line chemotherapy of NSCLC, but it can cause immunosuppression, thereby weakening its own anti-tumor effect. 11 Moxibustion is expected to improve therapeutic efficacy and reduce side effects used in combination therapy.
Our previous study showed that moxibustion at ST36 in Lewis lung carcinoma (LLC) mice enhanced the infiltration of CD8+ cytotoxic T lymphocytes (CTLs), CD4+T cells, helper T (Th)1, Th9 cells, and M1 macrophages, and Th1+IFN-γ in tumor tissues. It also reduced vascular endothelial growth factor (VEGF) expression, increased pericyte coverage, and normalized the tumor vasculature. 12 Activated CD4+ T cells—particularly Th1 cells— localize near vascular endothelial cells by secreting the characteristic cytokine IFN-γ. 13 Based on this, it is worth studying whether moxibustion enhances the anti-tumor effect of chemotherapy drugs by regulating IFN-γ. Thus, this study focused on the tumor immune microenvironment and vascular normalization to further investigate the mechanisms by which cisplatin and/or moxibustion inhibit tumor growth, and verify the key role of IFN-γ. The aim was to provide evidence that moxibustion enhances the antitumor effects of chemotherapeutic drugs, support its clinical application in tumor treatment, and explore better strategies for the treatment of lung cancer.
Materials and Methods
Animals
C57BL/6J male mice (6 weeks old, body weight 18-24 g) were purchased from Beijing Wei Tong Li Hua Laboratory Animal Technology Co., Ltd. (Beijing, China; license number: SCXK (Beijing) 2016-0006, RRID: IMSR_JAX: 000664). All mice were housed with free access to food and water under controlled conditions: constant temperature (24°C), constant humidity (40%-50%), and a 12-hour light/dark cycle. All procedures were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals, issued by the Ministry of Science and Technology of China. The experiments were approved by the Animal Care and Use Committee of Tianjin University of Traditional Chinese Medicine (Permit Number: TCM-LAEC2019057).
In our study, LLC tumor-bearing mice were first ranked by tumor volume on day 7, then allocated to respective groups using a snake randomization method (alternate serpentine assignment). This approach ensured that the baseline tumor volumes were well-balanced across all groups, minimizing potential confounding biases arising from uneven distribution of tumor sizes. The study design, implementation, and reporting were conducted in accordance with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) 2.0 guidelines.
Cell Culture
The LLC cell line was purchased from Shanghai Fuheng Chemical Technology Co. Ltd. (ATCC Cat# CRL-1642, RRID: CVCL_4358). The LLC cell line has been verified by STR analysis, confirming the uniqueness of the cell line identity, with no cross-contamination or human-derived contamination detected. LLC Cells were cultured as described in our previous study. 14
Reagents
Cisplatin was administered according to the method described in our previous study. 12 In Vivo Mab anti-mouse IFN-γ antibody XMG1.2 (Bio X Cell Cat# BE0055, RRID: AB_110769) was administered intratumorally at a concentration of 0.6 mg/mL in 50 μL of In Vivo Pure pH 8.0 Dilution Buffer (Bio X cell Cat# IPT080), 3 times per week for 2 weeks. The reagents required for flow cytometry are as follows: eBioscience™ 10X RBC Lysis Buffer (Thermo Fisher Scientific Cat# 2652782). Brilliant Violet 711™ anti-mouse CD45 Antibody (BioLegend Cat# 103147, RRID: AB_2564383), PE/Cyanine5 anti-mouse CD4 Antibody (BioLegend Cat# 100409, RRID:AB_312694), Brilliant Violet 421™ anti-mouse CD8a Antibody (BioLegend Cat# 100753, RRID:AB_2562558), Brilliant Violet 605™ anti-mouse CCR6 Antibody (BioLegend Cat# 129819, RRID:AB_2562513), Mouse/Rat CCR10 APC-conjugated Antibody (R and D Systems Cat# FAB2815A, RRID: AB_1151964), PE anti-mouse CXCR3 Antibody (BioLegend Cat# 126505, RRID:AB_1027656), CD11b Monoclonal Antibody, Super Bright™ 600, eBioscience™ (Thermo Fisher Scientific Cat# 63-0112-82, RRID: AB_2637408), PE/Cyanine7 anti-mouse CD86 Antibody (BioLegend Cat# 105013, RRID:AB_439782), Alexa Fluor® 700 anti-mouse CD206 Antibody (BioLegend Cat# 141733, RRID:AB_2629636), PerCP/Cyanine5.5 anti-mouse CD11c Antibody (BioLegend Cat# 117327, RRID:AB_2129642)
Model and Intervention
After a 1-week acclimation period, 1 × 106 LLC cells were subcutaneously inoculated into the right groin of C57BL/6J mice on day 0. Moxibustion was applied at the Zusanli acupoint (ST36, located 2 mm lateral to the anterior tubercle of the tibia in the anterior tibial muscle and 4 mm distal to the lower point of the knee joint) for 15 minutes, 5 times per week for 2 weeks starting on day 7 (Figure 1A). In the sham moxibustion group, moxibustion was applied to the tail. Prior to treatment, fur over the moxibustion site was shaved to expose bilateral ST36. Mice were immobilized using a moxibustion fixation device (mouse fixator, Chengdu University of Chinese Medicine, Patent No. ZL201220746188). The moxibustion protocol followed the method used in our previous study. 12 To investigate the role of IFN-γ in the local tumor microenvironment, tumors in the TCM + XMG1.2 group were administered an intratumoral injection of the IFN-γ neutralizing antibody XMG1.2, while tumors in the TCM group received the corresponding antibody diluent as a vehicle control.
Moxibustion combined with cisplatin inhibits tumor growth. (A) Flowchart of experimental interventions. T: tumor group, TC: tumor + cisplatin group, TM: tumor + moxibustion group, TCM: tumor + cisplatin + moxibustion group, TCSM: tumor + cisplatin + sham moxibustion group, n = 9 to 10, LLC cells were transplanted into all mice on day 0. The time point for cisplatin was on days 7, 10, 14, and 17. Moxibustion at Zusanli (ST36) was on days 7 to 11, 14 to 18 (once a day), and sham moxibustion was performed on the mouse tails in the same time. The tumor volume was measured on days 7, 10, 14, 17, and 21, and mice were sacrificed on day 21 for sample collection. (B) Images of the dissected tumors from each group. The tumor was harvested on day 21, rinsed with PBS, and placed in a culture dish. During photography, a coordinate paper with a grid spacing of 0.5 cm was placed beneath the culture dish to provide a scale reference. (C) Box plot of tumor volume in each group on day 21, *P < .05. (D) Tumor growth curve. Tumor volume analysis of the LLC-bearing mice in each group at the corresponding time points. Data were expressed as mean ± SEM, “-” indicates that the comparison between the 2 groups at the corresponding time point shows P > .05, “+” indicates that the comparison between the 2 groups at the corresponding time point shows P < .05.
Tumor Volume Measurement
Tumor size was monitored using digital calipers on days 7, 10, 14, 17, and 21. Tumor volume was calculated using the formula: V = (a ×b2)/2, where V = tumor volume, a = maximum tumor diameter, and b = minimum tumor diameter. Blinded methods were used during tumor measurement, and the specific procedures are detailed in Supplemental Figure S1. On day 21, the LLC-bearing mice were weighed and deeply anesthetized with 4% isoflurane in oxygen (RWD Life Technology Cat# R510-22) prior to sacrifice, we harvested the tumor tissues for subsequent research.
RNA Sequencing and Data Analysis
RNA sequencing (RNA-seq) was used to investigate the molecular signaling pathways involved in moxibustion and/or cisplatin treatment. The raw data were submitted to the NCBI Gene Expression Omnibus (GEO) database (GSE306979). Tumor samples were excised from tumor-bearing mice treated with moxibustion and/or cisplatin. RNA-seq was performed by Shanghai Genechem Co., Ltd. (China). Total RNA was isolated from tumor tissues in the tumor group (T), tumor + cisplatin group (TC), tumor + moxibustion group (TM), and tumor + cisplatin + moxibustion group (TCM), n = 5. Samples were processed as previously described, and the RNA was extracted and purified following standard operating procedures. The quantity and purity of total RNA were assessed before constructing mRNA sequencing libraries. Differential gene expression analysis between samples was performed using edgeR. The parameter settings for differentially expressed genes (DEGs) are set as fold change ≥1.5 and adjusted P-value < .05. DEGs were input into the enrichment tool (http://enrich.shbio.com) for KEGG pathway enrichment analysis, and the top 30 KEGG pathways were identified. In addition, DEGs were submitted to the STRING database (https://www.string-db.org/) for protein-protein interaction (PPI) and network construction.
RT-qPCR
The real-time quantitative polymerase chain reaction (RT-qPCR) protocol followed that of our previous study. 12 The genes analyzed are listed in Table 1. β-actin was used as an endogenous reference. Relative mRNA expression levels of Il1β and Il2 were calculated using the 2−ΔΔCT method. The expression levels of Ifn-γ, Pf4, Vegfa, Flt1, and Angpt2 were calculated using the double-standard curve method.
Gene Sequence.
Flow Cytometric Staining and Analysis
Sample processing and single-cell suspension preparation were performed as described in our previous studies. 12 After suspension preparation, a drop from each sample was randomly applied to a slide and examined under a microscope to assess cell dispersion. If the cells remained aggregated, they were incubated for an additional 5 minutes on a shaker before being re-evaluated. Once adequately dispersed, the singlets suspension was filtered through a 70 μm mesh cell strainer. Lysis buffer was used to remove red blood cells. Cells were stained with the following antibodies: CD45 - Brilliant Violet 711™, CD4 - PE/Cyanine5, CD8 - Brilliant Violet 421™, CCR10 - APC, CCR6 - Brilliant Violet 605™, CD11b - Super Bright™ 600, CD86 - PE/Cyanine7, CD206 - Alexa Fluor® 700, CD11c - PerCP/Cyanine5.5. After staining, the cells were washed, resuspended, and analyzed using an Attune NxT flow cytometer (Thermo Fisher Scientific, Waltham, MA, USA). During the data acquisition phase for T lymphocyte subsetting, we encountered technical challenges with suboptimal resolution of CD3 antibody staining within this experimental system. To ensure a consistent analytical standard across all samples, subsequent analysis was performed on the population that exhibited clear morphological characteristics (FSC/SSC) and was double-positive for CD45 and CD4. The specific gating strategy is shown in Supplemental Figure S2. All data were analyzed as follows: positive cell events (%) = (target gate/total cell count) × 100%.
Liquid Phase Chip
Tumor tissues stored at −80°C were thawed and cut into pieces. Plus RIPA Lysis Buffer (Boster Biological Technology, Catalog #AR0102-100) was added to the chopped tissue, which was then ultrasonically homogenized on ice. Total protein was extracted, and the protein concentration was measured and normalized using a BCA kit (Boster Biological Technology, Catalog #AR0146). We followed the protocol of the manufacturer. Tumor samples were processed using Luminex-xMAP technology and analyzed with a Bio-Plex Pro Mouse Cytokine panel (M60009RDPD, Bio-Rad) to detect CXCL1, IL-13, CCL3, and CCL4.
Statistical Analysis
The Shapiro-Wilk test was used to assess the normality of the data. For comparisons between 2 groups, the Student’s t-test was used for normally distributed data; otherwise, the non-parametric Mann-Whitney U test was applied. For comparisons across multiple groups, one-way analysis of variance (ANOVA) was used if data were normally distributed. If the assumption of homogeneity of variance was met, post-hoc comparisons were performed using the Least Significant Difference (LSD) test; in cases of unequal variances, the Dunnett’s T3 test was used. For data that did not follow a normal distribution, the non-parametric Kruskal-Wallis test was employed. Since most of the tumor volume data did not conform to a normal distribution, a base-10 logarithmic transformation (log10) was applied to the data. Statistical analysis was conducted using SPSS (Version 21.0; IBM Corp., Armonk, NY, USA). Statistical significance was set at P < .05. Figures were generated using GraphPad Prism (Version 8.0; GraphPad software, San Diego, CA, USA).
Results
Moxibustion Combined With Cisplatin Inhibits Tumor Growth
To evaluate whether moxibustion synergistically enhances the anti-tumor effect of cisplatin in NSCLC, we analyzed the tumor growth curves of LLC mice in the T, TC, TM, TCM, and tumor + cisplatin + sham moxibustion group (TCSM), n = 9-10. For the specific reasons leading to group differences of n, refer to the Supplemental Materials. The experimental process is illustrated in Figure 1A. The tumor was harvested on day 21; the tumor images and volume analysis are presented in Figure 1B and C. As shown in Figure 1D, there was no differences in tumor volume between the groups before the intervention (day 7 after tumor formation). On day 10, the tumor size did not differ between the groups. On day 14, the tumor volume in the TCM group was lower than that in the T group (P = .01, TCM vs T, n = 9, one-way ANOVA, LSD). At this time point, the tumor volume in the TCM group was smaller than that in the TM group (P = .032, TCM vs TM, n = 9-10, one-way ANOVA, LSD), indirectly reflecting the anti-tumor effect of cisplatin. On day 17, the anti-tumor effect of moxibustion combined with cisplatin persisted (P = .032, TCM vs T, n = 9, one-way ANOVA, Dunnett’s T3). On day 21, the tumor volume in the TCM and T group was smaller than that in the T group (P = .018, TCM vs T, Kruskal-Wallis test). No significant difference was found in the tumor volume between the TCSM and T groups, suggesting that moxibustion enhanced the anti-tumor effect of cisplatin, whereas sham moxibustion had no effect. In general, moxibustion combined with cisplatin had a significant inhibitory effect on tumor growth from day 14. Cisplatin or moxibustion alone were not as effective as moxibustion combined with cisplatin. The therapeutic effect observed in the TCSM group was similar to that in the TC group, without any increase in the therapeutic effect.
Possible Mechanisms of Moxibustion or Cisplatin in the Treatment of Tumor
The anti-tumor effects of moxibustion or cisplatin alone were not obvious, but according to the tumor growth curve, they tended to inhibit tumor growth. Therefore, we conducted a preliminary exploration of the mechanisms underlying the effects of moxibustion and cisplatin. The tumor tissues were sequenced, and the results showed that the samples between the groups were homogeneous (Figure 2A). We compared the TC and T groups and found 455 DEGs between the 2 groups, of which 211 were upregulated and 244 were downregulated (Figure 2B). To further elucidate the potential regulatory pathways through which different interventions inhibit tumor growth, KEGG enrichment analysis was performed. DEGs were mainly enriched in signaling pathways, such as the “Toll-like receptor signaling pathway” and “NK cell-mediated cytotoxicity” (Figure 2C). We compared differential gene expression levels between the 2 groups according to the KEGG pathway list for gene sequencing (Table 2). SH2D1A, which positively regulates NK cell function, and NFATC4, which is involved in T cell activation and induces the anti-inflammatory cytokines IL2 and IL4, were upregulated by cisplatin.
Heatmap, volcano plot and KEGG pathways of DEGs. (A) Heat map (TC vs T), n = 5. (B) Volcano plot of DEGs (TC vs T). (C) KEGG pathway enrichment analysis (TC vs T); (D) Heat map (TM vs T). (E) Volcano plot of DEGs (TM vs T). (F) KEGG pathway enrichment analysis (TM vs T).
Key Pathways of TC Versus T.
Next, we compared the TM and T groups and found that the samples between the groups were homogeneous (Figure 2D). A total of 1187 DEGs were differentially expressed between the 2 groups, of which 831 were upregulated and 356 were downregulated (Figure 2E). KEGG enrichment analysis showed that DEGs were mainly enriched in the “VEGF signaling pathway,” “Toll-like receptor signaling pathway,” “T cell receptor signaling pathway,” “Natural killer cell-mediated cytotoxicity,” “Cytokine and cytokine receptor interaction,” “Chemokine signaling pathway,” and “Cell adhesion molecule” (Figure 2F). Using the KEGG pathway list, we screened out “Antigen presentation” pathways in addition to those overlapping with the top 30 KEGG pathways based on enrich factors (Table 3). In these pathways, the comparison of DEGs between the 2 groups showed that moxibustion upregulated the expression levels of Cd8a, Cd8b1, Irf5, and Tlr5, which promote immunity, and downregulated the expression levels of Flt3, which promotes tumor proliferation, and Vegfa, which promotes angiogenesis.
Key Pathways of TM Versus T.
Moxibustion Combined With Cisplatin Improves Tumor Immunosuppression and Vascular Normalization
In animal experiments, the antitumor effects of moxibustion combined with cisplatin were better than those of moxibustion or cisplatin alone. Therefore, we explored the anti-tumor mechanism of moxibustion combination with cisplatin. Gene sequencing results showed that the samples between the groups were homogeneous (Figure 3A). TCM group had 893 DEGs compared with the T group. Among these, 618 were upregulated and 275 were downregulated (Figure 3B). KEGG enrichment analysis showed that DEGs were mainly enriched in “Toll-like receptor signaling pathway,” “T cell receptor signaling pathway,” “Natural killer cell-mediated cytotoxicity,” “Cytokine-cytokine receptor interaction,” and “Chemokine signaling pathway” (Figure 3C). We also examined the KEGG gene sequencing list based on the enrichment factors. In addition to pathways that overlapped with the top 30 KEGG pathways, “Antigen presentation” and “Cell adhesion molecules” were among other pathways of interest (Table 4). The results of the KEGG enrichment analysis showed that the mechanism by which moxibustion combined with cisplatin inhibits tumor growth involves regulation of the tumor immune microenvironment and normalization of blood vessels.
Moxibustion combined with cisplatin improves tumor immunosuppression and vascular normalization. (A) Heat map (TCM vs T), n = 5. (B) Volcano plot of DEGs (TCM vs T). (C) KEGG pathway enrichment analysis (TCM vs T). (D) PPI network analyses of DEGs (TCM vs T). (E) Gene expression levels of factors related to immune. n = 7 for T and 5 for TCM. (F) Protein expression levels of factors related to immune, n = 7. Data were expressed as mean ± SEM. *P < .05, **P < .01.
Key Pathways of TCM Versus T.
To further understand the effects of moxibustion combined with cisplatin on the tumor immune microenvironment and vascular normalization, PPI analysis was used to screen for high-node genes. According to the PPI network diagram (Figure 3D), the relevant data were derived from the analysis of high-node genes (≥10 counts) using the String website. Among these, Cd247, Pdcd1, Cxcl10, CxCl12, Cxcl13, Lck, Stat1, Cd3d, Cd3e, Cd3g, Lat, Itk, Cd8a, Cd8b1, Ccl5, and Prkcq were closely related to immunity (Table 5). These results indicate that the inhibitory effect of moxibustion combined with cisplatin on tumor growth was mainly achieved by improving the immune microenvironment of the tumor. The results of RT-qPCR in tumor tissues showed that TCM could reduce Il1β (P = .006), and increase Il2 (P = .004) at the mRNA level (TCM vs T, n = 7 for T and 5 for TCM) (Figure 3E). For the specific reasons leading to the group differences of n, refer to the Supplemental Materials. TCM also could downregulate CXCL1 (P = .013), IL-13 (P = .032), CCL3 (P = .015), and CCL4 (P = .009) at the protein level (TCM vs T, n = 7) (Figure 3F).
Genes With High Nodes Count in PPI Network of TCM Versus T.
Molecular Mechanism of Moxibustion Combined With Cisplatin in Regulating Tumor Immune Microenvironment
To further study the mechanism by which moxibustion combined with cisplatin improves the tumor immune microenvironment at the cellular level, we repeated the anti-tumor effect of moxibustion combined with cisplatin (Figure 4A). The tumor was harvested on day 21, the tumor images are presented in Figure 4B. As shown in Figure 4C and D, moxibustion combined with cisplatin inhibited tumor growth on day 21 (P = .008, TCM vs T, n = 9, Student’s t-test), further confirming the anti-tumor effects of moxibustion combined with cisplatin. Fresh tumor samples were subjected to flow cytometry (Figure 4E and F). The results showed that moxibustion combined with cisplatin enhanced the tumor immune response, as evidenced by increased infiltration of CD4⁺ T cells (P = .04, Mann-Whitney U test) and Th1 cells (P = .002, Student’s t-test), along with an elevated Th1/Th2 ratio (P = .005, Student’s t-test) (Figure 4G). Moreover, it increased the M1/M2 ratio (P = .019, Student’s t-test) and the proportion of myeloid dendritic cells (mDCs) (P = .005, Student’s t-test), while reducing the proportion of M2-type cells (P = .050, Student’s t-test) (Figure 4H). (TCM vs T, n = 9). Although increases were observed in the proportions of CD8+ T cells and M1 macrophages, neither of these changes reached statistical significance. Cell marker phenotypes are presented in Table 6. At the cellular level, the combined application of moxibustion and cisplatin promoted the infiltration of CD4+ T cells and mDCs. Moreover, it elevated the M1/M2 and Th1/Th2 ratios along with enhanced Th1 cell polarization, thereby positively modulating the immune microenvironment.
Molecular mechanism of moxibustion combined with cisplatin in regulating tumor immune microenvironment. (A) Flowchart of experimental interventions. LLC cells were transplanted into mice on day 0. T: tumor group, TCM: tumor + cisplatin + moxibustion group, n = 9. Mice in the TCM were treated with cisplatin on days 7, 10, 14, and 17, and moxibustion at Zusanli (ST36) on days 7 to 11, 14 to 18 (once a day). The tumor volume was measured on days 7, 10, 14, 17, and 21, and mice were sacrificed on day 21 for sample collection. (B) Images of the dissected tumors from each group. The tumor was harvested on day 21, rinsed with PBS, and placed in a culture dish. During photography, a coordinate paper with a grid spacing of 0.5 cm was placed beneath the culture dish to provide a scale reference. (C) Box plot of tumor volume in each group on day 21. ** P < .01. (D) Tumor growth curve. Tumor volume analysis of the LLC-bearing mice in each group at the corresponding time points. (E) Gating strategy to identify T cells. (F) Gating strategy to myeloid cells in tumor. (G) Quantification of T cells in tumor by flow cytometry. (H) Quantification of myeloid cells in tumor by flow cytometry. TCM versus T, n = 9. Data were expressed as mean ± SEM.
Cell Subpopulation Markers.
Mechanism of Moxibustion in the Treatment of Tumor With Moxibustion Combined With Cisplatin
According to the gene sequencing results, the samples within each group were homogeneous (Figure 5A), consistent with the comparison between the TCM and T groups (n = 5). TCM and TC groups had 893 DEGs, of which 590 were upregulated and 251 were downregulated (Figure 5B). KEGG enrichment analysis showed that DEGs were mainly enriched in the “VEGF signaling pathway,” “Toll-like receptor signaling pathway,” “Natural killer cell-mediated cytotoxicity,” “Cytokine-cytokine receptor interaction,” and “Chemokine signaling pathway.” These signaling pathways are closely related to immunity and vasculature (Figure 5C). Simultaneously, we screened the KEGG list based on enrichment factors, and in addition to the pathways overlapping with the top 30 KEGG pathways, the “HIF-1 signaling pathway” was related to blood vessels (Table 7). Based on a comparison between the TCM and TC groups, it can be inferred that moxibustion plays a crucial role in regulating tumor blood vessels. To further elucidate the mechanism by which moxibustion and combined chemotherapy influence the tumor immune microenvironment and vascular normalization, PPI analysis was used to identify high-node genes, and some factors were consistent with the key genes in the KEGG enrichment analysis. Vegfa, Edn1, Pf4, Ccr2, Ccr5, Cxcl5, Cxcr6, and Ccl20 were closely related to immunity and blood vessels (Table 8) according to the PPI network diagram (Figure 5D) and related data from STRING website analysis of gene high node number (≥10 counts). In addition, these genes overlapped with those in major KEGG pathways. These findings prompted us to validate gene expression in tumor immunity and blood vessels.
Action mechanism of moxibustion in the treatment of tumor with moxibustion combined with cisplatin. (A) Heat map (TCM vs TC), n = 5. (B) Volcano plot of DEGs (TCM vs TC). (C) KEGG pathway enrichment analysis (TCM vs TC). (D) PPI network analyses of DEGs (TCM vs TC). (E) Gene expression levels of factors related to tumor immune and angiogenesis. (TCM vs TC, n = 5 for Ifnγ, n = 6 for Pf4, Vegfa, Flt and Aanpt2). Data were expressed as mean ± SEM.
Key Pathways of TCM Versus TC.
Genes With High Nodes Count in PPI Network of TCM Versus TC.
To investigate the effects of moxibustion alone or in combination with chemotherapy on tumor growth, tumor immune expression, and vascular normalization-related genes, we detected the immune cytokine Ifnγ and its chemokine cxcl11 and tumor vascular normalization factors, such as anti-angiogenic Pf4 and pro-angiogenic Vegfa, Flt1, and Angpt2 using RT-qPCR. The results showed that compared with the cisplatin group, moxibustion combined with cisplatin could increase Ifn-γ (P = .025, TCM vs TC, n = 5) and Pf4 (P = .017, TCM vs TC, n = 6), and reduce Vegfa (P < .001, TCM vs TC, n = 6), Flt1 (P < .001, TCM vs TC, n = 6), and there was a tendency to reduce Angpt2 (P = .057, TCM vs TC, n = 6) (Student’s t-test) (Figure 5E), which leads to abnormal angiogenesis, thus promoting the dynamic balance of anti-angiogenic and pro-angiogenic factors and promoting vascular normalization. These results further confirmed that the inhibition of tumor growth by moxibustion combined with cisplatin was related to the regulation of tumor immunity and vascular normalization; moxibustion was particularly important for the positive regulation of local blood vessels in the tumor. Meanwhile, IFN-γ, the key factor of tumor immunity, can also inhibit angiogenesis. Based on the results of the bioinformatics analysis and validation at the cellular and molecular levels, this study highlights the important role of IFN-γ. Therefore, we used IFN-γ neutralizing antibody XMG1.2 to study whether IFN-γ mediates tumor-inhibitory effect of moxibustion combined with cisplatin in LLC mice.
Role of IFN-γ in Inhibiting Tumor Growth by Moxibustion Combined With Cisplatin
LLC mice were randomly divided into T, TC, TCM, and moxibustion combined with cisplatin and IFN-γ neutralizing antibody XMG1.2 group (TCM + XMG1.2), n = 9 to 11. For the specific reasons leading to group differences of n, refer to the Supplemental Materials. The experimental process is illustrated in Figure 6A. The tumor was harvested on day 21, the tumor images and volume analysis are presented in Figure 6B and C. As shown in Figure 6D, the tumor volume in the TC group was smaller than that in the T group on days 17 (P = .005) and 21 (P = .041) (TC vs T), indicating that cisplatin inhibited tumor growth in mice with LLC. The tumor volumes of mice in the moxibustion combined with cisplatin group were lower than those in the tumor group on days 10 (P = .042) and 21 (P = .001) (TCM vs T). The tumor volume in the cisplatin group was smaller than that in the TCM + XMG1.2 group on days 17 (P < .001) and 21 (P = .046) (TCM + XMG1.2 vs TC). The tumor volume in the TCM group was smaller than that in the TCM + XMG1.2 group on days 10 (P = .006), 14 (P = .03), 17 (P = .008) and 21 (P = .001) (TCM + XMG1.2 vs TCM), suggesting that IFN-γ neutralizing antibody antagonized the anti-tumor effect of moxibustion combined with cisplatin. The statistical analysis of the above results was performed using one-way ANOVA, with post hoc pairwise comparisons conducted by the LSD method. These results suggest that moxibustion combined with cisplatin enhances the anti-tumor effect of cisplatin, and IFN-γ plays a key role in mediating this effect.
Role of IFN-γ in inhibiting tumor growth by moxibustion combined with cisplatin. (A) Flowchart of experimental interventions. T: tumor group, TC: tumor + cisplatin group, TM: tumor + moxibustion group, TCM: tumor + cisplatin + moxibustion group, TCM + XMG1.2: tumor + cisplatin + moxibustion + IFN-γ neutralizing antibody XMG1.2 group. n = 9 to 11. LLC cells were transplanted into mice on day 0. Mice in TC, TCM, and TCM + XMG1.2 were treated with cisplatin on days 7, 10, 14, and 17. Mice in the TCM and TCM + XMG1.2 were treated with moxibustion at Zusanli (ST36) on days 7 to 11, 14 to 18 (once a day), and in TCM + XMG1.2, XMG1.2 was injected into the tumor on days 7, 9, 11, 14, 16, and 18. The tumor volume was measured on days 7, 10, 14, 17, and 21, and mice were sacrificed on day 21 for sample collection. (B) Images of the dissected tumors from each group. The tumor was harvested on day 21, rinsed with PBS, and placed in a culture dish. During photography, a coordinate paper with a grid spacing of 0.5 cm was placed beneath the culture dish to provide a scale reference. (C) Box plot of tumor volume in each group on day 21. *P < .05, ** P < .01. (D) Tumor growth curve. Tumor volume analysis of the LLC-bearing mice in each group. at the corresponding time points. Data were expressed as mean ± SEM, “-” indicates that the comparison between the 2 groups at the corresponding time point shows P > .05; + indicates that the comparison between the 2 groups at the corresponding time point shows P < .05, “+” an “++” indicate that the comparison between the 2 groups at the corresponding time point shows P < .05 and P < .01, respectively.
Discussion
In our study, bioinformatics analysis revealed that the tumor-suppressive effects of both moxibustion combined with chemotherapy and moxibustion alone were associated with regulation of tumor immune microenvironment and vascular normalization. At the cellular level, moxibustion combined with cisplatin promoted the infiltration of CD4+ T cells and mDCs. Moreover, it elevated the M1/M2 and Th1/Th2 ratios along with enhanced Th1 cell polarization, thereby positively modulating the immune microenvironment. DC are a key determinant of the anti-tumor immune response and can significantly improve immune function by recognizing and infiltrating tumors, 15 “Antigen presentation” signaling pathway is also highlighted in the RNA-Seq analysis. Moxibustion combined with cisplatin modulated immune-related molecules through upregulation of Il2 and downregulation of Il1β at the mRNA level, accompanied by decreased protein expression of CXCL1, IL-13, CCL3, and CCL4. Furthermore, compared with cisplatin alone, moxibustion combined with cisplatin upregulated the mRNA levels of Ifnγ and Pf4 and downregulated Vegfa and Flt1. These results suggest a molecular mechanism by which moxibustion regulates the balance of tumor pro-angiogenic and anti-angiogenic factors and improves the immune microenvironment. IFN-γ is a major regulator of tumor immune microenvironment, capable of modulating various immune cell functions. It can promote the recruitment of immune cells in the tumor microenvironment. 16 IFN-γ is primarily used in combination therapy strategies in clinical setting. It enhances the anti-tumor effect when used in alongside anti-angiogenic drugs and increases the sensitivity of immunotherapy when combined with immune checkpoint inhibitors. 17 In this study, the upregulation of IFN-γ improved the tumor immune-vascular microenvironment during treatment with moxibustion combined with cisplatin.
Tumor vasculature exhibits abnormal structural and functional features, 18 which contribute to increased tumor aggressiveness and chemotherapy resistance. 19 Thus, normalization of the tumor vasculature can reduce vascular leakage, improve chemotherapy delivery, reduce the number of immunosuppressive cells, 20 restore normal maturation and function of DCs, reshape the anti-tumor immune microenvironment, and trigger other phenotypic changes in tumor cells—the regulation of immunosuppressive molecules, enhancement of antigen presentation function, and increased secretion of inflammatory factors. Immune activation can also synergistically enhance the efficacy of anti-angiogenic therapy. 21 In the tumor microenvironment, immune activation and vascular normalization form an interdependent and mutually reinforcing positive feedback loop 22 RNA-seq analysis revealed that, compared to the T group, both the TM and TCM groups exhibited increased levels of factors associated with vascular normalization, T-cell activity, NK-cell activity, cytokine production, and antigen presentation. We propose that moxibustion increases the number of mDCs and elevates the M1/M2 ratio, thereby enhancing antigen-presenting function, which in turn promotes the proliferation and polarization of antitumor immune subtypes such as Th1 and CD8+ T cells. Consequently, IFN-γ activity is also increased. However, the upstream-downstream relationship between immune activation and vascular normalization requires further validation, which will be a focus of our future research.
In our previous study, 12 we examined the myeloid cell populations and observed an increase in the number of Treg cells in the TCM group. This suggests that the immunomodulatory effect of moxibustion combined with cisplatin is characterized by complexity and holism, rather than a simple unidirectional “enhancement” or “suppression.” Instead, it may represent a multidimensional and dynamically balanced immune remodeling process. The increase in Treg cells could be a key mechanism for maintaining immune homeostasis within this process. Meanwhile, no significant differences were detected in MDSC proportions among the groups, indicating that this combined therapy may not broadly affect all myeloid cells, and its effects likely exhibit selectivity depending on cell type and functional state. Therefore, these findings require further validation in subsequent studies. It should be noted that the current flow cytometry data reflect only cellular proportions and do not assess functional suppressive capacity. Additionally, all reported proportions and phenotypes of T lymphocyte and myeloid cell subsets were derived from analyses of locally infiltrating immune cells in tumor tissues. Future work could further validate and explore key findings using peripheral blood samples.
A core function of Th1 cells is to activate CTLs through the secretion of cytokines such as IFN-γ. CTLs are key effector cells responsible for directly killing tumor cells. Further analysis of the effect of moxibustion combined with chemotherapy on T cells, based on in-depth mining of RNA-seq data, revealed that the combination treatment significantly enriched genes associated with T cell activation, proliferation, and cytotoxic function, including Cd8a, CD8b1, CD3d, CD3g, CD3e, Tcf7, Tbx21, Prf1, Gzma, and Gzmb. Among these, Tbx21 encodes T-bet, which participates in the differentiation of Th1 cells and CTLs and drives IFN-γ production in CD8+ T cells. Prf1 encodes perforin, while Gzma and Gzmb encode granzyme A and granzyme B, respectively—all of which are involved in the cytotoxic function of CTLs and NK cells. Their simultaneous upregulation suggests that cytotoxic immune cells were successfully activated, infiltrated the tumor, and initiated immune-killing functions. These findings indicate that the activity of CTLs and NK cells is elevated under the combined treatment of moxibustion and cisplatin. Whether moxibustion plays a dominant role in this process requires further investigation. Future studies could employ multi-omics analysis of these molecules at both gene and protein levels to compare and validate the roles of individual and combined therapies in enhancing CTL and NK cell activity. Our current study lacks a comprehensive functional validation of Th1 cells, which also highlights the need to examine Th1-related chemokines (CXCL9, CXCL10, CXCL11) and the signature transcription factor T-bet in future work, thereby deepening the functional investigation of Th1 cells under the combined treatment of moxibustion and chemotherapy.
For the IFN-γ neutralization experiment, we selected intratumoral injection. This approach is designed to specifically block IFN-γ within the tumor without significantly affecting systemic immune status. Compared with systemic administration, intratumoral injection allows for a more precise verification of whether local IFN-γ is necessary for the synergistic antitumor effect produced by moxibustion combined with cisplatin. This helps avoid the complex off-target effects and interference with the overall immune system that may result from systemic inhibition of IFN-γ. However, systemic administration is indeed the more commonly used approach in clinical practice. In future studies, we will also incorporate this route into our experimental design to examine whether the molecule can accumulate in the tumor and exert its therapeutic effects. The IFN-γ neutralizing antibody employed in this study is well-established and has been utilized in numerous high-quality studies, which reduces the likelihood of non-specific effects. 23 Moreover, the same specialized in vivo dilution buffer was injected in the TCM group, thereby excluding potential effects attributable to the solvent itself. Nevertheless, the inclusion of an antibody-only control group remains essential to demonstrate that the antibody itself does not directly influence tumor growth and to strengthen the argument for the specific role of IFN-γ. This aspect warrants further attention in subsequent research. In our study, although IFN-γ neutralization abolished the observed effects, the downstream signaling pathways remain unexplored and warrant further investigation. Additionally, it would be valuable to test whether exogenous supplementation of IFN-γ alone can augment the efficacy of cisplatin.
While our study provides evidence that moxibustion combined with chemotherapy inhibits tumor growth, several limitations and future directions warrant consideration. First, subcutaneous implantation of LLC was used in this study. For subsequent research, human NSCLC cells should be used to establish an in situ carcinoma model, which can better simulate the growth and metastasis characteristics of human non-small cell lung cancer. Second, regarding the intervention parameters in animal experiments, the selection of moxibustion duration and frequency currently lacks a theoretical basis aligned with clinical protocols. Additionally, dose-response studies of cisplatin are warranted in future research to establish optimal dosing parameters, which will facilitate clinical translation. Third, this study has certain limitations in research methods. In terms of therapeutic efficacy assessment, there is currently a lack of intuitive histopathological indicators, such as observing changes in tumor cell density and necrosis via Hematoxylin and Eosin staining, which could provide more direct morphological evidence for evaluating treatment outcomes. Concerning vascular normalization, the current research only focused on relevant pathways and performed gene-level validation following RNA-seq analysis. Although preliminary experiments involving CD31/α-SMA double immunofluorescence staining of tumor tissues were conducted to assess pericyte coverage, with results indicating a higher α-SMA area density in the TCM group compared to the TC group, 12 further systematic validation remains necessary. Immunohistochemical findings suggest that moxibustion can suppress the protein expression of HIF-1α, VEGFA, and CD31 in tumor tissues. 24 In light of this background, the functional and morphological features of tumor vasculature require more in-depth investigation and verification. Therefore, subsequent studies will employ histopathological techniques to provide direct histopathological evidence of therapeutic effects and to systematically explore both functional and morphological aspects of vascular normalization. Additionally, to address the current lack of cross-validation in this study, future work will include comprehensive detection and analysis of key molecules within critical signaling pathways at both genetic and protein levels, emphasizing cross-validation through multiple technical approaches, with further confirmation using methods such as Western blot analysis. Fourth, samples were collected at the fixed time point of day 21, by which time differences in tumor volume had already emerged among the groups. Therefore, the potential influence of elevated intratumoral pressure on vascular architecture in larger tumors cannot be ruled out, which may introduce some confounding to the interpretation of causal relationships. Future studies could consider adopting a staggered sacrifice design based on tumor volume, which would help to clarify this issue more definitively. Finally, the mechanistic link between immune infiltration and tumor vascular normalization remains unclear, constituting another important direction for our future studies. Our subsequent work will systematically elucidate the regulatory relationship between the 2 processes and identify key mediating molecules in order to provide further mechanistic evidence and refine the study.
With the advancement of integrative oncology, cancer treatment has increasingly emphasized combining mainstream therapies and complementary approaches with the goal of efficacy enhancment and toxicity reduction, aimed at improving anticancer outcomes while fully preserving patients’ physical function, quality of life, and long-term health. Our research, through the intervention method of moxibustion combined with chemotherapy, together with recent research using multi-omics technology to clarify the anti-gastric cancer molecular mechanisms of piperine, an active component of Piper longum, 25 reflects the multidimensional landscape of integrative oncology development. Pan et al’s study evaluated the anticancer efficacy of Piper longum in gastric cancer, described therapy-induced changes in metabolic pathways and gut microbiota composition, and identified key molecular targets mediating its therapeutic effects. Our research employs moxibustion as an external treatment to synergize with chemotherapy and improve the tumor immune-vascular microenvironment. Future integrative oncology research should also aim to combine such mechanistically elucidated active components with integrative therapies that improve host status, thereby constructing a logically rigorous, novel cancer treatment strategy spanning from molecular to systemic levels.
Moxibustion can reduce the toxicity and side effects of radiotherapy and chemotherapy and enhance the anticancer effects of chemotherapeutic drugs to a certain extent. 8 Therefore, the anticancer effects of moxibustion combined with chemotherapeutic drugs need to be confirmed in large-scale clinical studies. In the initial phase of clinical research, the primary task is to systematically elucidate the principles of acupoint selection, establish safe operational protocols, and standardize treatment parameters. On this basis, rigorous clinical efficacy verification should be conducted to provide scientific evidence and research direction for further exploration of this therapy’s mechanisms of action. Meanwhile, research on the combined application of moxibustion with other therapeutic approaches should be actively advanced to promote the practice and development of integrative oncology.
Conclusion
This study demonstrated that moxibustion and cisplatin synergistically inhibit tumor growth, ameliorate the tumor immune microenvironment at the cellular and molecular levels, and induce tumor vascular normalization. Notably, we identified that upregulation of the immune cytokine IFN-γ may serve as a pivotal mediator of this process. These findings further elucidate the mechanisms by which moxibustion enhances the antitumor efficacy of chemotherapeutic drugs, supporting its clinical adoption as complementary therapy. Our study highlights the potential of moxibustion to synergize with conventional anticancer regimens, offering a simple and feasible adjunctive strategy for cancer treatment.
Supplemental Material
sj-docx-1-ict-10.1177_15347354261450974 – Supplemental material for Moxibustion Promotes Tumor Immune Responses and Vascular Normalization to Enhance Antitumor Effect of Cisplatin in Lewis Lung Carcinoma Mice
Supplemental material, sj-docx-1-ict-10.1177_15347354261450974 for Moxibustion Promotes Tumor Immune Responses and Vascular Normalization to Enhance Antitumor Effect of Cisplatin in Lewis Lung Carcinoma Mice by Shanshan Lu, Jiaqi Wang, Yue Pan, Yuanzhen Yang, Jin Huang, Shanshan Li, Bin Wang, Suhong Zhao, Ning Ma, Jiayue Zhao, Jing Huang, Yi Guo and Zhifang Xu in Integrative Cancer Therapies
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sj-jpg-2-ict-10.1177_15347354261450974 – Supplemental material for Moxibustion Promotes Tumor Immune Responses and Vascular Normalization to Enhance Antitumor Effect of Cisplatin in Lewis Lung Carcinoma Mice
Supplemental material, sj-jpg-2-ict-10.1177_15347354261450974 for Moxibustion Promotes Tumor Immune Responses and Vascular Normalization to Enhance Antitumor Effect of Cisplatin in Lewis Lung Carcinoma Mice by Shanshan Lu, Jiaqi Wang, Yue Pan, Yuanzhen Yang, Jin Huang, Shanshan Li, Bin Wang, Suhong Zhao, Ning Ma, Jiayue Zhao, Jing Huang, Yi Guo and Zhifang Xu in Integrative Cancer Therapies
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sj-jpg-3-ict-10.1177_15347354261450974 – Supplemental material for Moxibustion Promotes Tumor Immune Responses and Vascular Normalization to Enhance Antitumor Effect of Cisplatin in Lewis Lung Carcinoma Mice
Supplemental material, sj-jpg-3-ict-10.1177_15347354261450974 for Moxibustion Promotes Tumor Immune Responses and Vascular Normalization to Enhance Antitumor Effect of Cisplatin in Lewis Lung Carcinoma Mice by Shanshan Lu, Jiaqi Wang, Yue Pan, Yuanzhen Yang, Jin Huang, Shanshan Li, Bin Wang, Suhong Zhao, Ning Ma, Jiayue Zhao, Jing Huang, Yi Guo and Zhifang Xu in Integrative Cancer Therapies
Footnotes
Acknowledgements
The authors thank Editage for professional English editing services.
Abbreviations
LLC: Lewis lung carcinoma
Th: Helper T
mDCs: myeloid dendritic cells
NSCLC: Non-small cell lung cancer
NK: natural killer
CTLs: cytotoxic T lymphocytes
VEGF: Vascular endothelial growth factor
GEO: Gene Expression Omnibus
DEGs: differentially expressed genes
PPI: protein interaction
RT-qPCR: real-time quantitative polymerase chain reaction
Ethical Considerations
Approval from the Animal Care and Use Committee of Tianjin University of Traditional Chinese Medicine (Permit Number: TCM-LAEC2019057, approval date: May 13th, 2020).
Author Contributions
ZFX and YG contributed to the research design. SSL, JQW, YP, YZY, JH, and SSL performed the experiments. SSL, JQW, and YP performed the data curation, completed manuscript writing and preparation. YZY, JH, and SSL performed the formal analysis. BW, SHZ, and NM provided substantial revisions and oversaw the research program. JYZ and JH made figures and tables. All authors read and approved the final version of the manuscript.
Funding
The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by National Key Research and Development Program of China (NO. 2022YFC3500404), Beijing-Tianjin-Hebei Basic Research Cooperation Project (NO. 22JCZXJC00070), and Hebei Provincial Administration of Traditional Chinese Medicine Administration Management Plan Project (NO. T2025096, NO. T2026073).
Declaration of Conflicting Interests
The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Data Availability Statement
The data generated in the present study may be requested from the corresponding author. The RNA-seq raw data generated in the present study may be found in the NCBI GEO database (GSE306979).*
Supplemental Material
Supplemental material for this article is available online.
References
Supplementary Material
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