Synergistic Anti-Tumor and Immunomodulatory Effects of Sporoderm-Removed Ganoderma Lucidum Spore Powder and Anti-PD-L1 Antibody in Lung Cancer Mice

Synergistic Anti-Tumor and Immunomodulatory Effects of Sporoderm-Removed Ganoderma Lucidum Spore Powder and Anti-PD-L1 Antibody in Lung Cancer Mice | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Synergistic Anti-Tumor and Immunomodulatory Effects of Sporoderm-Removed Ganoderma Lucidum Spore Powder and Anti-PD-L1 Antibody in Lung Cancer Mice Qiong Wang, Xiaorong Zheng, Zengyu Zhang, Min Hao, Jianwei Jiang, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7088435/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 12 You are reading this latest preprint version Abstract Background The therapeutic efficacy of PD-1/PD-L1 inhibitors in lung cancer management remains suboptimal. Sporoderm-removed Ganoderma Lucidum Spore Powder (RGLS), an immunostimulant, has been shown to amplify the anti-tumor effects of anti-PD-L1 antibodies (αPD-L1). However, the detailed molecular underpinnings of this enhancement are not yet fully elucidated. This study aimed to elucidate the antitumor mechanism and immunomodulatory effect of RGLS in synergistic relationship with αPD-L1 in lung cancer. Methods Employing network pharmacology approaches, this research analyzed the multifaceted target pathways of RGLS in lung cancer. High-Performance Liquid Chromatography (HPLC) facilitated the identification of RGLS constituents. In the established Lewis lung cancer tumor-bearing mouse model, the effects of RGLS, αPD-L1, and their combination were investigated, focusing on tumor growth, T cell responses, and the dynamics of myeloid-derived suppressor cells (MDSCs) within tumor microenvironments. Results Our network analysis revealed 26 bioactive components of RGLS and identified 227 potential lung cancer targets, among which PD-L1 had a significant response effect with 20 core targets. HPLC identified 14 triterpenoid components. Notably, the combination of RGLS and αPD-L1 significantly inhibited tumor growth, optimized the CD4 + /CD8 + T cell ratio in tumor tissue, increased IL-2 and IFN-γ levels, enhanced cytotoxic T lymphocyte (CTL) activity, and inhibited MDSC recruitment. Conclusion The synergistic application of RGLS and αPD-L1 in lung cancer treatment significantly improves immune responsiveness and reduces tumor-associated MDSCs, surpassing the efficacy of sole αPD-L1 application. This study underscores RGLS's burgeoning potential in bolstering lung cancer immunotherapy, paving the way for its clinical applications. Cancer immunotherapy Myeloid-Derived Suppressor Cell Ganoderma Lucidum Spore Powder PD-L1 Lung Cancer CD4+/CD8+ T cell ratio Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 INTRODUCTION As the leading cause of death for cancer patients, lung cancer is a malignant disease with a high morbidity and mortality rate that accounts for 18% of all fatalities globally[ 1 ]. Immunotherapy has been used in clinical trials in recent years to help people with lung cancer have better prognoses. It is known that the most effective treatment for lung cancer is to inhibit immunological checkpoints, specifically the programmed cell death-1 (PD-1)/programmed cell death ligand-1 (PD-L1) axis[ 2 , 3 ]. Through their inhibition of T cell activation, cytokine generation, and cytolytic action, PD-1 and its ligands contribute significantly to anti-tumor immune responses. Anti-PD-L1 antibodies (αPD-L1) can restore T cell responsiveness and anti-tumor effector function by blocking the interaction between ligands on cancer cells and inhibitory receptors on T cells[ 4 , 5 ]. Several studies have found new ways to combine inhibitors of the PD-1/PD-L1 pathway with immunomodulatory medications or other therapies to improve anti-tumor responses[ 6 ]. Polyporaceae. G. lucidum is a traditional Chinese medicine that has been used to cure a variety of illnesses, such as liver, breast, and lung cancer. It does this by lowering oxidative damage, modifying immunological processes, and preventing tumor growth. The reproductive units of Ganoderma lucidum are called spores (GLS), and they are the portion of the plant that is frequently employed in medicine[ 7 , 8 ]. According to earlier research, GLS might considerably raise the percentage of CD4 + cells, produce cytokines such as IL-4, IL-6, and IFN-γ, suppress Treg cell activity, lessen immunosuppression, and drastically lower the amount of CTLA-4 on the surface of immune cells that infiltrate tumors[ 9 ]. Furthermore, the Ganoderma lucidum spore water extract can improve the effectiveness of PD-L1 antibody and lessen the side effects of PD-L1 monoclonal antibody in osteosarcoma[ 10 ]. Nevertheless, no research has been done on how GLS and αPD-L1 interact to affect lung cancer. We selected sporoderm-removed Ganoderma lucidum spores (RGLS) for further investigations since prior research has demonstrated that RGLS have more and higher quantities of triterpenoids than GLS, as well as more pronounced anti-lung cancer properties and immunomodulatory activities[ 11 ]. This study aims to explore the synergistic anti-tumor and immunomodulatory effects of RGLS and αPD-L1 on lung cancer mice. METHODS Collection of targets corresponding to RGLS active ingredients The components of RGLS were examined utilizing the TCMSP online database ( https://old.tcmsp-e.com/tcmsp.php ). The screening criteria included oral bioavailability (OB) ≥ 30% and drug similarity (DL) ≥ 0.18. Finally, the structures of probable active components were validated using the PubChem (nih.gov) database. The targets associated with the possible active compounds examined above were then predicted using the Swiss-Prot database. Lung cancer related target screening The GeneCards and DisGeNET databases were used to search the potential disease targets of lung cancer. The two databases were screened by search “lung cancer” and the duplicate protein targets were removed to obtain the potential lung cancer protein targets. Protein–protein interaction (PPI) network construction A Venn diagram was conducted using Venny 2.1.0 to obtain the common protein targets between the proteins correlated with RGLS and lung cancer. After that, the common protein targets were guided into the STRING database. Homo sapiens were selected as the study species and then the protein target interaction analysis was performed to obtain the PPI network. GO and KEGG enrichment analyses The DAVID database was applied for GO function and KEGG pathway enrichment analysis. The top 20 pathways with the highest P values were sorted and selected and GO functional enrichment analysis, a BP, CC, MF trihistogram, and a KEGG pathway enrichment analysis bubble map was constructed using bioinformatics ( www.bioinformatics.com ). Construction of a drug–target–disease network The Cytoscape3.9.1 software was applied to establish the drug-target-disease network of RGLS and to analyze its network relationship. High-performance liquid chromatography analysis Referencing the method established by Shi Yuejiao et al. for the determination of ganoderic triterpenes[ 12 ], the content of 14 ganoderic triterpenes including Ganoderic acid I, Ganoderic acid C2, Ganoderic acid C6, Ganoderic acid G, Ganodermic acid B, Ganoderic acid N, Ganoderic acid B, Ganodermic acid A, Ganoderic acid A, Ganoderic acid H, Ganoderic acid D2, Ganodermic acid D, Ganoderic acid C1, Ganoderic acid F in the spore powder of de-walled Ganoderma lucidum was measured. All the above were purchased from Chengdu Pusi Biotechnology Co., Ltd., and RGLS was purchased from Zhejiang Shouxiangu Pharmaceutical Co., Ltd. Cell lines, mice and tumor models The Lewis cell line was obtained from the Shanghai Institutes of Biological Sciences, Chinese Academy of Sciences (Shanghai). Cell lines were grown in DMEM media (Beyotime, Shanghai, China) containing 10% FBS in an incubator at 37°C and 5% CO 2 . Shanghai Slack Laboratory Animal Co., Ltd. (Shanghai, China) provided 24 male C57BL/6 mice aged 5 weeks and weighing 20 ± 2 g. This work was authorized by the Zhejiang Cancer Hospital Animal Experiment Ethics Committee (approval number: SYKX 2017-0012). The Animal Research and Teaching Use Committee at the National Institutes of Health (NIH) approved all experimental protocols. To establish a tumor model, C57BL/6 mice were subcutaneously inoculated with Lewis cells (1×10 6 ). After cell injection, the mice were randomly divided into four groups, and treatment was initiated after the tumor grew to about 100 mm 3 . The mice were divided into the following groups: control group (0.2 mL 0.9% sodium chloride solution, intragastric administration, once a day), αPD-L1 group (200 µg αPD-L1, intraperitoneal injection, once every 4 days), RGLS group (0.3 g/kg RGLS, intragastric administration, once a day), and RGLS-αPD-L1 group (200 µg αPD-L1, intraperitoneal injection, once every 4 days, 0.3 g/kg RGLS, intragastric administration, once a day). The treatment of the four groups lasted for 12 days. On the 12th day of drug treatment, animals were euthanized by intraperitoneal injection of 150 mg/kg sodium pentobarbital. Tumor volume and weight were immediately measured and dissected. The calculation formula for spleen and thymus index is: organ index (%) = organ weight (g)/body weight (g) × 100%. All experimental procedures were strictly performed in adherence to the ARRIVE Guidelines (Animal Research: Reporting of In Vivo Experiments) and the Guide for the Care and Use of Laboratory Animals issued by the National Institutes of Health (NIH, USA), ensuring alignment with internationally recognized standards for laboratory animal research and ethical requirements. Immunohistochemistry Immunohistochemistry was used to detect the expression of Ki-67, CD3 + , CD11b + and Ly6G + in mouse cancer tissues. Paraffin sections were dewaxed with xylene, rehydrated with gradient ethanol, incubated with the corresponding primary antibody (abcom) at 4°C overnight, and incubated with the secondary antibody (abcom) for half an hour. Staining was performed with peroxidase 3,30-diaminobenzidine (DAB) substrate and counterstained with hematoxylin. TUNEL analysis The paraffin sections of tumor tissue were processed as above. Afterward, the TUNEL Kit detection was conducted according to instruction. The proportion of apoptosis cells /total cells was calculated by Image-pro Plus 6.0 software according to the AOD. Tumor-specific cytotoxic T lymphocyte response assay Mouse spleen lymphocytes were isolated according to the previous research method[ 13 ]. Spleen lymphocytes from each group of mice were cultured in 96-well plates for 24 h until adherence. Spleen lymphocytes were used as effector cells and Lewis cells were used as target cells. Specific concentrations of effector cells and target cells were inoculated in 96-well plates and incubated for 20 h, and then 20 µL of MTT solution (5 mg/mL) was added and incubated at 37°C for 4 h. The culture medium was then aspirated and 200 µL of DMSO was added. The optical density (OD) value at 490 nm was detected using a microplate reader (Varioskan Flash, Thermo Company). After background subtraction, the tumor-specific cytotoxic activity was calculated as follows: 100% × (OD target group − (OD experimental group − OD effector group)/OD target group). Flow cytometric analysis The peripheral blood of mice was collected. The erythrocytes were removed with erythrocyte pyrolysis liquid and were incubated with APC anti-CD4 antibody and PE anti-CD8 antibody to measure CD4 + and CD8 + T cells. The cells were stained for 1 h at 4°C and washed. After that, they were centrifuged (380 g for 5 min) and resuspended in 200 µL PBS for flow cytometric analysis. The ratio of positive stained cells was detected by the FACS can flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) and analyzed with FlowJo software. Cytokine quantification by enzyme-linked immunoassay The peripheral blood of mice was centrifuged at 3,000 rpm/min for 10 min to collect the supernatant serum. The contents of IL-2 and IFN-γ in the serum were detected using an enzyme-linked immunoassay (ELISA, MultiSciences [Lianke] Biotech) kit. Statistical analysis The Student’s t-test and one-way ANOVA (GraphPad Prism 8.0 software) were used for statistical analysis. All data were exhibited as mean ± SD. p < 0.05 were representing statistical significance and significant difference, respectively. RESULTS Network pharmacology analysis of RGLS in the treatment of lung cancer After deduplication from the TCMSP database, a total of 26 RGLS active ingredients and 443 related protein targets were obtained; in the GeneCards database and DisGeNet database, the keyword "lung cancer" was searched, and a total of 4,045 related genes were retained after the two databases were merged and deduplication was removed. After screening, RGLS-related genes and disease-related genes were introduced into the VENNY graph, and finally 227 cross-targets related to RGLS treatment of lung cancer were obtained (Fig. 1 A). Using the STRING database, a PPI network was constructed (Fig. 1 B). The network has 227 nodes and 801 edges, indicating that there is a high correlation between protein targets. The network was constructed using Cytoscape 3.9.1 software, and the PPI network was sorted using the Degree program (Fig. 1 C). The Centiscape 2.2 plug-in was used to screen core targets based on topological structure and biological characteristics. The interaction between PD-L1 (Fig. 1 D) and 20 core targets (Fig. 1 E), among which JAK1-3, STAT1, and EGFR targets had significant interaction effects with PD-L1. In the constructed PPI network, Degree reflects the central attribute of the node, and the darker the color, the higher the value. The enrichment analysis of the GO function resulted in 859 biological processes (BP), including the cellular positive regulation of the Notch signaling pathway, cell proliferation, negative regulation of NF-kappaB transcription factor activity, and regulation of the cell cycle, etc. There were also 93 cellular components (CC) obtained, including the inner membrane of the nucleus, spindle microtubules, the overall composition of the postsynaptic membrane. GO enrichment also resulted in 201 molecular functions (MF), involving retinal dehydrogenase activity, NADP binding activity, aspartate-type endopeptidase activity, GABA-gated chloride channel activity (Fig. 1 F). The longer the bar graph length, the more significant the enrichment. KEGG pathway enrichment analysis resulted in 214 pathways, whose common targets were mainly enriched in metabolic pathways, bile secretion, Wnt signaling, and adrenergic signaling in cardiomyocytes (Fig. 1 G). Composition analysis of RGLS Ganoderma lucidum has significant anti-cancer effects, and triterpenoids are considered to be its main anti-cancer components, so we used HPLC to identify the RGLS components. We determined the presence of 14 triterpenoids in Ganoderma lucidum, including ganoderic acid I, ganoderic acid C2, ganoderic acid C6, ganoderic acid G, ganoderic acid B, ganoderic acid N, ganoderic acid B, ganoderic acid A, ganoderic acid A, ganoderic acid H, ganoderic acid D2, ganoderic acid D, ganoderic acid C1, and ganoderic acid F in de-walled Ganoderma lucidum spore powder (Fig. 2 A, B). RGLS synergizes with αPD-L1 to inhibit tumor growth After 12 days of administration of RGLS, αPD-L1, and RGLS combined with αPD-L1 (Fig. 3 A), the tumors in the control group grew rapidly, while the tumor growth in the RGLS, αPD-L1, and RGLS-αPD-L1 groups was delayed (Fig. 3 B, C). The tumor volume and mass in the RGLS-αPD-L1 group were significantly lower than those in the single-drug group (Fig. 3 B, C). In addition, RGLS increased the spleen index and thymus index, but combined administration with αPD-L1 did not further increase the spleen index and thymus index (Fig. 3 D, E). RGLS synergizes with αPD-L1 to inhibit cancer cell proliferation and promote apoptosis Immunohistochemistry (IHC) staining and TUNEL staining were used to detect the expression of Ki-67 (a protein marker for cell proliferation) and apoptosis in tumors. Compared with the control group, Ki-67 in tumors of the αPD-L1, RGLS, and RGLS-αPD-L1 groups was significantly decreased. Compared with the αPD-L1 group, Ki-67 in the RGLS-αPD-L1 group was significantly decreased (Fig. 4 A). In addition, compared with the control group, the number of TUNEL-positive cells in the αPD-L1 and RGLS groups increased significantly, but there was no significant difference between the RGLS-αPD-L1 group and the single-drug group (Fig. 4 B). RGLS synergizes with αPD-L1 to promote tumor-specific CTL activity of splenic lymphocytes CTL plays an important role in inhibiting tumor cells. We detected the enhanced tumor-specific cytotoxic activity of spleen lymphocytes against Lewis cells. The results showed that compared with the control group, the tumor-specific cytotoxic activity of spleen lymphocytes in the mouse RGLS, αPD-L1 and RGLS-αPD-L1 groups increased when the ratio of effector cells to target cells (E/T ratio) in the co-culture wells was 10:1, 20:1 and 40:1, respectively. The tumor-specific cytotoxic activity of spleen lymphocytes in the RGLS-αPD-L1 group was significantly higher than that in the single-drug group (Fig. 5 ). RGLS synergizes with αPD-L1 to promote immune system activity To test whether the anti-tumor effect of RGLS combined with αPD-L1 is related to its immune-enhancing effect in lung cancer mice, we measured the CD4 + /CD8 + T cell ratio and IFN-γ and IL-2 levels in the peripheral blood of mice. Compared with the control group, the CD4+/CD8 + T cell ratios in the peripheral blood of the αPD-L1, RGLS, and RGLS-αPD-L1 groups were significantly increased. The CD4 + /CD8 + T cell ratio in the RGLS-αPD-L1 group was significantly higher than that in the αPD-L1 group (Fig. 6 A). In addition, we used IHC to detect the infiltration level of CD3 + cells in tumor tissues and found that compared with the control group, the expression of CD3 + cells in the αPD-L1, RGLS, and RGLS-αPD-L1 groups was increased, but there was no significant difference between the monotherapy group and the RGLS-αPD-L1 group (Fig. 6 B). In addition, the levels of IL-2 and IFN-γ in the peripheral blood of tumor mice were lower than those in normal mice, while the αPD-L1 group and RGLS group could increase the levels of IL-2 and IFN-γ, and the RGLS-αPD-L1 group could increase the effect of αPD-L1 (Fig. 6 C). These data indicate that RGLS can increase the ratio of CD4 + /CD8 + T lymphocytes in lung cancer mice treated with αPD-L1, thereby causing them to secrete IFN-γ and IL-2 and enhance immune activity. RGLS synergizes with αPD-L1 to inhibit the accumulation of myeloid-derived suppressor cells (MDSC) PD-L1 expression may inhibit the anti-tumor immune activity of T lymphocytes due to the deposition of MDSC in tumor tissues. In mice, the definition of MDSC is based on the surface markers CD11b + and LY6G + . Therefore, we evaluated the expression of CD11b + and LY6G + by immunohistochemical staining to analyze MDSC in tumor tissues. Compared with the control group, the aggregation of MDSC in the αPD-L1 group, RGLS group, and RGLS-αPD-L1 group was significantly reduced, and the aggregation of MDSC in the RGLS-αPD-L1 group was significantly lower than that in the αPD-L1 group (Fig. 7 ). Our results show that RGLS can reduce the aggregation of MDSC in tumor tissues, thereby enhancing the anti-tumor effect of αPD-L1. DISCUSSION Lung cancer is the most common malignant tumor seen in clinical practice and the main cause of cancer-related death worldwide. As anti-tumor immunity research advances, immune checkpoint inhibitors such as PD-1/PD-L1 have emerged as a viable treatment for lung cancer[ 14 ]. However, the low response rate (30%) of αPD-L1 medicines remains a significant impediment to treating lung cancer. Due to the complexity of immune control in the body, immunotherapy is thought to have substantial limits. Combined immunotherapy, which relies on various immune regulatory systems, has emerged as a new medicinal technology[ 15 ]. According to earlier research, GLS can drastically suppress the expression of PD-1 and CTLA-4 in the spleen of the tumor group, indicating that it affects the PD-1/PD-L1 pathway[ 10 ]. GLS powder has significant in vivo anti-tumor activity in tumor mice, and its mechanism is to stimulate NK cells, T cells, and macrophages, enhancing the host's immune response[ 16 ]. Compound-target-pathway disease network analysis showed that RGLS contained 61 active ingredients and 227 related targets, which are potential targets for lung cancer treatment. KEGG pathway enrichment analysis showed that bile secretion, Wnt signaling, and adrenaline signaling in cardiomyocytes may be potential mechanisms for RGLS to exert anti-tumor effects. Using the Centiscape 2.2 plug-in, core targets were screened based on topological structure and biological characteristics. PD-L1 had a significant response effect with 20 core targets. Drawing on insights from network pharmacology, we speculate that RGLS may have a synergistic effect with anti-tumor immunosuppressants, which may enhance their efficacy. Mouse experiments showed that RGLS synergistically with αPD-L1 significantly inhibited tumor growth in the lung cancer mouse model compared with single treatment, as shown by a decrease in tumor volume and tumor weight. Previous studies have reported that although the direct antitumor effects of GLS and αPD-L1 are extremely limited, they can significantly enhance antitumor immune activity[ 17 ]. Therefore, based on the in vivo antitumor activity and improved immune function, we continued to investigate the potential mechanisms associated with the combination of RGLS and αPD-L1. T cells play a critical role in producing and regulating immune responses to tumor antigens. T lymphocytes can be further classified into two types: CD4 + T cells and CD8 + T cells[ 18 ]. CD4 + and CD8 + T cells contribute to and coordinate antitumor immune responses. Their levels reflect the body's anti-tumor immunological response. Among these, the CD4+/CD8 + ratio is a direct reflection of the body's anti-tumor immunity[ 19 ]. Our research indicates that RLGS can increase the proportion of CD4 + /CD8 + T cells in the peripheral blood of lung cancer mice treated with alpha PD-L1. Subsequently, we further validated the synergistic anti-tumor effect of RGLS combined with α PD-L1 through tumor specific CTL activity assays. The results showed that mouse spleen lymphocytes treated with RGLS combined with αPD-L1 exhibited stronger tumor specific cytotoxic activity than model mice treated with αPD-L1. CD4 + T cells release cytokines like IL-2 and IFN-γ, which can boost the development of CD8 + cytotoxic T cells. IL-2 promotes T cell proliferation and differentiation into mature CD4 + and CD8 + T cells[ 20 ]. Activated CD8 + T lymphocytes can aggregate and release cytokines such as IFN - γ in the tumor microenvironment. The expression level of IFN - γ in CD8 + T cells is closely related to tumor specific cytotoxic activity[ 21 ]. In our study, ELISA showed that the combination of RGLS and alpha PD-L1 resulted in higher levels of IL-2 and IFN - γ production by T cells in lung cancer mice compared to treatment with αPD-L1 alone. MDSCs mainly exist in bone marrow, spleen, and tumor tissues, playing a key role in the tumor immune microenvironment. When MDSCs infiltrate into tumor tissue, they reduce the proliferation and function of T cells and NK cells, while inducing Treg cells[ 22 ]. In previous studies, αPD-L1 reduced the number of MDSCs in tumor tissue, and GLS also had a similar effect[ 23 , 24 ]. Our experiments showed that RGLS combined with αPD-L1 reduced the number of MDSCs in tumor tissues of lung cancer mice compared with either treatment alone. The change in MDSCs is the theoretical basis for the combined treatment of lung cancer with RGLS and αPD-L1. However, our research still has some limitations. This study was only based on the Lewis lung cancer mouse model and did not include patient-derived xenograft (PDX) models of human lung cancer. Furthermore, the specific target differences of the 14 triterpenoid components in RGLS were not explored in depth; future studies should clarify key active components through component-specific intervention experiments. We will achieve them one by one in future plans. The results of this study highlight the great potential of combining traditional Chinese medicine with modern immunotherapy in the treatment of lung cancer. The synergistic effects of RGLS and αPD-L1 observed in mouse models suggest that this combination therapy is expected to become a valuable supplement to current lung cancer treatments. Future clinical trials should explore the efficacy and safety of this combination therapy in human patients, especially those with limited response to αPD-L1 monotherapy. In addition, further research on the specific mechanisms of RGLS immunomodulatory effects may reveal new therapeutic targets and biomarkers to guide personalized treatment strategies. CONCLUSION Our study confirmed that RGLS combined with αPD-L1 can effectively reduce the infiltration of MDSCs in tumor tissues and enhance the activity of the immune system, thereby inhibiting the development of lung cancer, compared with the single-agent group. Essentially, these findings provides preclinical evidence from animal experiments for subsequent clinical research on RGLS combined immunotherapy for lung cancer. Declarations Acknowledgements Not applicable. Funding This work was supported by Zhejiang Anti-Cancer Association (Grant No. 201902) , Zhejiang Traditional Chinese Medicine Administration (Grant No. 2021ZZ006), Zhejiang Province Traditional Chinese Medicine Science and Technology Plan Project (Grant No. 2024ZL300). Competing interests The authors confirm that there are no conflicts of interest. Ethics approval 1.Statement on the maximal tumor size/burden permitted by the ethics committee : "The Zhejiang Cancer Hospital Animal Experiment Ethics Committee (Approval No.: SYKX 2017-0012) established explicit limits for tumor burden in Lewis lung cancer-bearing C57BL/6 mice in this study: a maximum permitted tumor volume of 2000 mm³, and a maximum permitted tumor weight not exceeding 10% of the mouse’s body weight." 2.Statement confirming no exceedance of the maximal tumor size/burden : "Throughout the 12-day experimental period, tumor burden in mice from all treatment groups (control group, αPD-L1 group, RGLS group, and RGLS-αPD-L1 group) was monitored every 2 days. Tumor volume was calculated as (length × width²)/2 via caliper measurements of tumor length and width, and tumor weight was measured at the experimental endpoint. Results showed that the maximum tumor volume across all groups was 1666 ± 298 mm³ (control group on day 12), and the maximum tumor weight was 1.68±0.23 g (control group on day 12)-neither exceeding the maximal tumor size/burden limits set by the ethics committee." Data availability The data that support the findings of this study are available from the corresponding author upon reasonable request. Contribution of authors Conceptualization, Qiong Wang , Xiaogrong Zheng , Zengyu Zhang, Jianwei Jiang Junfei Li, and Hongyan Zhang; methodology, Qiong Wang , Junfei Li, Jianwei Jiang and Xiaorong Zheng; investigation,Qiong Wang , Junfei Li, Jianwei Jiang and Hao Min; Data curation, Hao Min; writing-original draft preparation, Qiong Wang , Zengyu Zhang , Xiaorong-Zheng and Kao Shi; writing-review and editing, Qiong Wang , Junfei Li, Hao Min; supervision, Hongyan Zhang and Junfei Li; funding acquisition, Hong-Yan Zhang, Jun-Fei Li. 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Zeng Y, Li B, Liang Y, Reeves PM, Qu X, Ran C, et al. Dual blockade of CXCL12-CXCR4 and PD-1-PD-L1 pathways prolongs survival of ovarian tumor-bearing mice by prevention of immunosuppression in the tumor microenvironment. FASEB J. 2019;33(5):6596–608. 10.1096/fj.201802067RR . Epub 2019/02/26. Wang Y, Fan X, Wu X. Ganoderma lucidum polysaccharide (GLP) enhances antitumor immune response by regulating differentiation and inhibition of MDSCs via a CARD9-NF-kappaB-IDO pathway. Biosci Rep. 2020;40(6). Epub 2020/06/13. 10.1042/BSR20201170 . PubMed PMID: 32530032; PubMed Central PMCID: PMCPMC7313449. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 29 Sep, 2025 Reviews received at journal 18 Sep, 2025 Reviewers agreed at journal 15 Sep, 2025 Reviews received at journal 04 Sep, 2025 Reviewers agreed at journal 02 Sep, 2025 Reviews received at journal 02 Sep, 2025 Reviewers agreed at journal 01 Sep, 2025 Reviewers agreed at journal 29 Aug, 2025 Reviewers invited by journal 27 Aug, 2025 Editor assigned by journal 17 Aug, 2025 Submission checks completed at journal 14 Aug, 2025 First submitted to journal 14 Aug, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7088435","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":509401564,"identity":"968e38cc-386a-4128-8e04-fdd976bff183","order_by":0,"name":"Qiong Wang","email":"","orcid":"","institution":"Zhejiang Cancer Hospital","correspondingAuthor":false,"prefix":"","firstName":"Qiong","middleName":"","lastName":"Wang","suffix":""},{"id":509401565,"identity":"361f005e-c381-44e4-849d-2bc2e7d6c54c","order_by":1,"name":"Xiaorong Zheng","email":"","orcid":"","institution":"Zhejiang Cancer Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xiaorong","middleName":"","lastName":"Zheng","suffix":""},{"id":509401566,"identity":"65f8f613-1d64-4975-a65c-1bd408ea70ec","order_by":2,"name":"Zengyu Zhang","email":"","orcid":"","institution":"ICU, Zhejiang Cancer Hospital","correspondingAuthor":false,"prefix":"","firstName":"Zengyu","middleName":"","lastName":"Zhang","suffix":""},{"id":509401567,"identity":"327e1255-493f-4d1c-a0c3-1799893ebe4b","order_by":3,"name":"Min Hao","email":"","orcid":"","institution":"Zhejiang Chinese Medical University","correspondingAuthor":false,"prefix":"","firstName":"Min","middleName":"","lastName":"Hao","suffix":""},{"id":509401568,"identity":"8ad95615-e118-4319-abd6-39cca43e6d7a","order_by":4,"name":"Jianwei Jiang","email":"","orcid":"","institution":"Zhejiang Cancer Hospital","correspondingAuthor":false,"prefix":"","firstName":"Jianwei","middleName":"","lastName":"Jiang","suffix":""},{"id":509401569,"identity":"42dcccaa-8842-4df0-ac70-d177590930bc","order_by":5,"name":"Junfei Li","email":"","orcid":"","institution":"Zhejiang Cancer Hospital","correspondingAuthor":false,"prefix":"","firstName":"Junfei","middleName":"","lastName":"Li","suffix":""},{"id":509401570,"identity":"692780b0-d6d7-46b4-8f2c-b2c90ab6b6fe","order_by":6,"name":"Hongyan Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA30lEQVRIiWNgGAWjYDACCSBmbGCQY2A4QKIWY9K1JDYQ7S752c3PHn7dcTh9fuPhhx8YKu7ZNbCfxW8f45xj5sayZw7nNjYcM5ZgOFOc3MCTl4BXC7NEgpm0ZNvh3GaGM2wMjG0JyQwSPAZ4tbBJpH8DaUlnI1oLj0SOmeTHtsMJPFAtdgS1SEjklEkztqUbzmAA+iXhTEICG08Ofi3yM9K3Sf5ss5aXnwEMsQ8VCfb87GfwawEBZh6GZqB9BxgYEhgYEtsIqgcCxh8MdQwM/A1gjj0xOkbBKBgFo2BkAQCgsEGLNQDc+AAAAABJRU5ErkJggg==","orcid":"","institution":"ICU, Zhejiang Cancer Hospital","correspondingAuthor":true,"prefix":"","firstName":"Hongyan","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2025-07-10 03:23:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7088435/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7088435/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":90618686,"identity":"09bdeaee-8f67-4d02-bca8-ed7e7485fe27","added_by":"auto","created_at":"2025-09-04 19:28:16","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":883378,"visible":true,"origin":"","legend":"\u003cp\u003eNetwork pharmacology Analysis. (A) VENNY plot of the intersection of targets related to RGLS composition and lung cancer. (B) PPI network diagram of RGLS for lung cancer treatment. (C) Active ingredient-target network of RGLS for treating lung cancer. (D) Network map of the top 20 core targets. (E) The correlation between 20 core targets and PD-L1. (F) Bar chart of GO enrichment analysis of RGLS in lung cancer. (G) KEGG pathway enrichment analyses of potential targets.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7088435/v1/82029d11ee43c38e580281c8.png"},{"id":90619837,"identity":"0ad380fb-ba35-4cd5-a73a-7a305bc3250d","added_by":"auto","created_at":"2025-09-04 19:52:16","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":54767,"visible":true,"origin":"","legend":"\u003cp\u003eHigh-Performance Liquid Chromatography Chromatogram of RGLS. A: Reference solution; B: Test solution. 1: Ganoderic acid I; 2: Ganoderic acid C2; 3: Ganoderic acid C6; 4: Ganoderic acid G; 5: Ganodermic acid B; 6: Ganoderic acid N; 7: Ganoderic acid B; 8: Ganodermic acid A; 9: Ganoderic acid A; 10: Ganoderic acid H; 11: Ganoderic acid D2;12: Ganodermic acid D; 13: Ganoderic acid C1; 14: Ganoderic acid F.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7088435/v1/916ba2c0754bb7827c8c80a5.png"},{"id":90619689,"identity":"0204ce3c-4c72-4028-80c2-4dd2f17e8540","added_by":"auto","created_at":"2025-09-04 19:44:16","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":127228,"visible":true,"origin":"","legend":"\u003cp\u003eThe anti-tumor effect of RGLS combined with αPD-L1 in Lewis lung carcinoma tumor-bearing mice. (A) The time points of drug administration throughout the experiment. (B) RGLS combined with αPD-L1 showed significant inhibition of tumor volume (n = 10). (C) RGLS combined with αPD-L1 showed significant inhibition of tumor weight. (D) Spleen index. (E) Thymus index. Data are presented as the mean ± SD. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 compared with the control group; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 compared with the αPD-L1 group.\u003csup\u003e ++\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 compared with the RGLS group. n = 10.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7088435/v1/f270d85799be2e539e40d298.png"},{"id":90618692,"identity":"0b6ab00b-032a-4161-95d3-34eaecdacd07","added_by":"auto","created_at":"2025-09-04 19:28:16","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1117443,"visible":true,"origin":"","legend":"\u003cp\u003eRGLS combined with αPD-L1 inhibited proliferation and triggered apoptosis in Lewis tumor-bearing mice in vivo. (A) Immunostaining indicated that RGLS combined with αPD-L1 treatment markedly decreased the expression of Ki-67, and the AOD was quantified. (B) TUNEL staining showed that RGLS combined with αPD-L1 treatment increased the number of apoptotic cells in the tumor compared to the control group, and the AOD was quantified. Data are presented as the mean ± SD. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 compared with the control group; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 compared with the αPD-L1 group. n = 3.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7088435/v1/ac1457ecf87de7edf9f97488.png"},{"id":90619048,"identity":"2c9fd823-abd8-4eda-af02-af2040facf90","added_by":"auto","created_at":"2025-09-04 19:36:16","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":118502,"visible":true,"origin":"","legend":"\u003cp\u003eRGLS combined with αPD-L1 on the tumor-specific cytotoxic activity of splenocytes in tumor-bearing mice, and the CTL activity was quantified. Data are presented as the mean ± SD. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 compared with the control group; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 compared with the αPD-L1 group. \u003csup\u003e+\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e++\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 compared with the RGLS group. n=10.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7088435/v1/1a2d1b02fe24578685631bb5.png"},{"id":90619054,"identity":"a4ab1fc8-5b56-4164-9b20-ce28a06d16da","added_by":"auto","created_at":"2025-09-04 19:36:16","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":504799,"visible":true,"origin":"","legend":"\u003cp\u003eRGLS combined with αPD-L1 treatment on T cell subsets from peripheral blood or tissue in Lewis tumor-bearing mice. (A) Representative cytometric dot plots of CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e T cells in peripheral blood and the proportions of CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e T cells in peripheral blood in the different treatment groups (n = 3). (B) The expression of CD3\u003csup\u003e+\u003c/sup\u003e in tumor tissue and the AOD were quantified (n = 3). (C) Effect of RGLS combined with αPD-L1 on the level of IFN-γ and IL-2 in the peripheral blood of mice (n = 10). Data are presented as the mean ± SD. *p \u0026lt; 0.05 compared with the control group; *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 compared with the control group; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 compared with the αPD-L1 group; \u003csup\u003e\u0026amp;\u0026amp;\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 compared with the normal group.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7088435/v1/4ac1c98b272821ad8d46c2ce.png"},{"id":90619692,"identity":"f7355eae-3fc1-4aec-8d1c-318ac07b5fa1","added_by":"auto","created_at":"2025-09-04 19:44:16","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":837115,"visible":true,"origin":"","legend":"\u003cp\u003eRGLS combined with αPD-L1 on MDSCs and PD-L1 in tumor tissue. Immunostaining indicated that RGLS combined with αPD-L1 treatment markedly decreased the accumulation of MDSCs. The AOD was then quantified. Data are presented as the mean ± SD. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 compared with the control group; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 compared with the αPD-L1 group. n=3.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7088435/v1/cd969e1c68d02e288b93dfce.png"},{"id":90620184,"identity":"3a55a3ec-8724-466e-893f-2b42f1352f88","added_by":"auto","created_at":"2025-09-04 20:00:18","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4461898,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7088435/v1/ba55358b-04e1-4652-b94d-34a86bd95ddd.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Synergistic Anti-Tumor and Immunomodulatory Effects of Sporoderm-Removed Ganoderma Lucidum Spore Powder and Anti-PD-L1 Antibody in Lung Cancer Mice","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eAs the leading cause of death for cancer patients, lung cancer is a malignant disease with a high morbidity and mortality rate that accounts for 18% of all fatalities globally[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Immunotherapy has been used in clinical trials in recent years to help people with lung cancer have better prognoses. It is known that the most effective treatment for lung cancer is to inhibit immunological checkpoints, specifically the programmed cell death-1 (PD-1)/programmed cell death ligand-1 (PD-L1) axis[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Through their inhibition of T cell activation, cytokine generation, and cytolytic action, PD-1 and its ligands contribute significantly to anti-tumor immune responses. Anti-PD-L1 antibodies (αPD-L1) can restore T cell responsiveness and anti-tumor effector function by blocking the interaction between ligands on cancer cells and inhibitory receptors on T cells[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Several studies have found new ways to combine inhibitors of the PD-1/PD-L1 pathway with immunomodulatory medications or other therapies to improve anti-tumor responses[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cem\u003ePolyporaceae. G. lucidum\u003c/em\u003e is a traditional Chinese medicine that has been used to cure a variety of illnesses, such as liver, breast, and lung cancer. It does this by lowering oxidative damage, modifying immunological processes, and preventing tumor growth. The reproductive units of Ganoderma lucidum are called spores (GLS), and they are the portion of the plant that is frequently employed in medicine[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. According to earlier research, GLS might considerably raise the percentage of CD4\u003csup\u003e+\u003c/sup\u003e cells, produce cytokines such as IL-4, IL-6, and IFN-γ, suppress Treg cell activity, lessen immunosuppression, and drastically lower the amount of CTLA-4 on the surface of immune cells that infiltrate tumors[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Furthermore, the Ganoderma lucidum spore water extract can improve the effectiveness of PD-L1 antibody and lessen the side effects of PD-L1 monoclonal antibody in osteosarcoma[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Nevertheless, no research has been done on how GLS and αPD-L1 interact to affect lung cancer. We selected sporoderm-removed Ganoderma lucidum spores (RGLS) for further investigations since prior research has demonstrated that RGLS have more and higher quantities of triterpenoids than GLS, as well as more pronounced anti-lung cancer properties and immunomodulatory activities[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. This study aims to explore the synergistic anti-tumor and immunomodulatory effects of RGLS and αPD-L1 on lung cancer mice.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eCollection of targets corresponding to RGLS active ingredients\u003c/h2\u003e\u003cp\u003eThe components of RGLS were examined utilizing the TCMSP online database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://old.tcmsp-e.com/tcmsp.php\u003c/span\u003e\u003cspan address=\"https://old.tcmsp-e.com/tcmsp.php\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The screening criteria included oral bioavailability (OB)\u0026thinsp;\u0026ge;\u0026thinsp;30% and drug similarity (DL)\u0026thinsp;\u0026ge;\u0026thinsp;0.18. Finally, the structures of probable active components were validated using the PubChem (nih.gov) database. The targets associated with the possible active compounds examined above were then predicted using the Swiss-Prot database.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eLung cancer related target screening\u003c/h3\u003e\n\u003cp\u003eThe GeneCards and DisGeNET databases were used to search the potential disease targets of lung cancer. The two databases were screened by search \u0026ldquo;lung cancer\u0026rdquo; and the duplicate protein targets were removed to obtain the potential lung cancer protein targets.\u003c/p\u003e\n\u003ch3\u003eProtein–protein interaction (PPI) network construction\u003c/h3\u003e\n\u003cp\u003eA Venn diagram was conducted using Venny 2.1.0 to obtain the common protein targets between the proteins correlated with RGLS and lung cancer. After that, the common protein targets were guided into the STRING database. Homo sapiens were selected as the study species and then the protein target interaction analysis was performed to obtain the PPI network.\u003c/p\u003e\n\u003ch3\u003eGO and KEGG enrichment analyses\u003c/h3\u003e\n\u003cp\u003eThe DAVID database was applied for GO function and KEGG pathway enrichment analysis. The top 20 pathways with the highest P values were sorted and selected and GO functional enrichment analysis, a BP, CC, MF trihistogram, and a KEGG pathway enrichment analysis bubble map was constructed using bioinformatics (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e\u003ca href=\"https://old.tcmsp-e.com/tcmsp.php\" target=\"_blank\"\u003ewww.bioinformatics.com\u003c/a\u003e\u003c/span\u003e\u003cspan address=\"http://www.bioinformatics.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eConstruction of a drug–target–disease network\u003c/h3\u003e\n\u003cp\u003eThe Cytoscape3.9.1 software was applied to establish the drug-target-disease network of RGLS and to analyze its network relationship.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eHigh-performance liquid chromatography analysis\u003c/h2\u003e\u003cp\u003eReferencing the method established by Shi Yuejiao et al. for the determination of ganoderic triterpenes[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], the content of 14 ganoderic triterpenes including Ganoderic acid I, Ganoderic acid C2, Ganoderic acid C6, Ganoderic acid G, Ganodermic acid B, Ganoderic acid N, Ganoderic acid B, Ganodermic acid A, Ganoderic acid A, Ganoderic acid H, Ganoderic acid D2, Ganodermic acid D, Ganoderic acid C1, Ganoderic acid F in the spore powder of de-walled Ganoderma lucidum was measured. All the above were purchased from Chengdu Pusi Biotechnology Co., Ltd., and RGLS was purchased from Zhejiang Shouxiangu Pharmaceutical Co., Ltd.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eCell lines, mice and tumor models\u003c/h3\u003e\n\u003cp\u003eThe Lewis cell line was obtained from the Shanghai Institutes of Biological Sciences, Chinese Academy of Sciences (Shanghai). Cell lines were grown in DMEM media (Beyotime, Shanghai, China) containing 10% FBS in an incubator at 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\u003cp\u003e Shanghai Slack Laboratory Animal Co., Ltd. (Shanghai, China) provided 24 male C57BL/6 mice aged 5 weeks and weighing 20\u0026thinsp;\u0026plusmn;\u0026thinsp;2 g. This work was authorized by the Zhejiang Cancer Hospital Animal Experiment Ethics Committee (approval number: SYKX 2017-0012). The Animal Research and Teaching Use Committee at the National Institutes of Health (NIH) approved all experimental protocols.\u003c/p\u003e\u003cp\u003eTo establish a tumor model, C57BL/6 mice were subcutaneously inoculated with Lewis cells (1\u0026times;10\u003csup\u003e6\u003c/sup\u003e). After cell injection, the mice were randomly divided into four groups, and treatment was initiated after the tumor grew to about 100 mm\u003csup\u003e3\u003c/sup\u003e. The mice were divided into the following groups: control group (0.2 mL 0.9% sodium chloride solution, intragastric administration, once a day), αPD-L1 group (200 \u0026micro;g αPD-L1, intraperitoneal injection, once every 4 days), RGLS group (0.3 g/kg RGLS, intragastric administration, once a day), and RGLS-αPD-L1 group (200 \u0026micro;g αPD-L1, intraperitoneal injection, once every 4 days, 0.3 g/kg RGLS, intragastric administration, once a day). The treatment of the four groups lasted for 12 days. On the 12th day of drug treatment, animals were euthanized by intraperitoneal injection of 150 mg/kg sodium pentobarbital. Tumor volume and weight were immediately measured and dissected. The calculation formula for spleen and thymus index is: organ index (%)\u0026thinsp;=\u0026thinsp;organ weight (g)/body weight (g) \u0026times; 100%.\u003c/p\u003e\u003cp\u003e All experimental procedures were strictly performed in adherence to the ARRIVE Guidelines (Animal Research: Reporting of In Vivo Experiments) and the Guide for the Care and Use of Laboratory Animals issued by the National Institutes of Health (NIH, USA), ensuring alignment with internationally recognized standards for laboratory animal research and ethical requirements.\u003c/p\u003e\n\u003ch3\u003eImmunohistochemistry\u003c/h3\u003e\n\u003cp\u003eImmunohistochemistry was used to detect the expression of Ki-67, CD3\u003csup\u003e+\u003c/sup\u003e, CD11b\u003csup\u003e+\u003c/sup\u003e and Ly6G\u003csup\u003e+\u003c/sup\u003e in mouse cancer tissues. Paraffin sections were dewaxed with xylene, rehydrated with gradient ethanol, incubated with the corresponding primary antibody (abcom) at 4\u0026deg;C overnight, and incubated with the secondary antibody (abcom) for half an hour. Staining was performed with peroxidase 3,30-diaminobenzidine (DAB) substrate and counterstained with hematoxylin.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eTUNEL analysis\u003c/h2\u003e\u003cp\u003eThe paraffin sections of tumor tissue were processed as above. Afterward, the TUNEL Kit detection was conducted according to instruction. The proportion of apoptosis cells /total cells was calculated by Image-pro Plus 6.0 software according to the AOD.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eTumor-specific cytotoxic T lymphocyte response assay\u003c/h2\u003e\u003cp\u003eMouse spleen lymphocytes were isolated according to the previous research method[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Spleen lymphocytes from each group of mice were cultured in 96-well plates for 24 h until adherence. Spleen lymphocytes were used as effector cells and Lewis cells were used as target cells. Specific concentrations of effector cells and target cells were inoculated in 96-well plates and incubated for 20 h, and then 20 \u0026micro;L of MTT solution (5 mg/mL) was added and incubated at 37\u0026deg;C for 4 h. The culture medium was then aspirated and 200 \u0026micro;L of DMSO was added. The optical density (OD) value at 490 nm was detected using a microplate reader (Varioskan Flash, Thermo Company). After background subtraction, the tumor-specific cytotoxic activity was calculated as follows: 100% \u0026times; (OD target group \u0026minus; (OD experimental group\u0026thinsp;\u0026minus;\u0026thinsp;OD effector group)/OD target group).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eFlow cytometric analysis\u003c/h2\u003e\u003cp\u003eThe peripheral blood of mice was collected. The erythrocytes were removed with erythrocyte pyrolysis liquid and were incubated with APC anti-CD4 antibody and PE anti-CD8 antibody to measure CD4\u0026thinsp;+\u0026thinsp;and CD8\u0026thinsp;+\u0026thinsp;T cells. The cells were stained for 1 h at 4\u0026deg;C and washed. After that, they were centrifuged (380 g for 5 min) and resuspended in 200 \u0026micro;L PBS for flow cytometric analysis. The ratio of positive stained cells was detected by the FACS can flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) and analyzed with FlowJo software.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eCytokine quantification by enzyme-linked immunoassay\u003c/h2\u003e\u003cp\u003eThe peripheral blood of mice was centrifuged at 3,000 rpm/min for 10 min to collect the supernatant serum. The contents of IL-2 and IFN-γ in the serum were detected using an enzyme-linked immunoassay (ELISA, MultiSciences [Lianke] Biotech) kit.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eThe Student\u0026rsquo;s t-test and one-way ANOVA (GraphPad Prism 8.0 software) were used for statistical analysis. All data were exhibited as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were representing statistical significance and significant difference, respectively.\u003c/p\u003e\u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eNetwork pharmacology analysis of RGLS in the treatment of lung cancer\u003c/h2\u003e\u003cp\u003eAfter deduplication from the TCMSP database, a total of 26 RGLS active ingredients and 443 related protein targets were obtained; in the GeneCards database and DisGeNet database, the keyword \"lung cancer\" was searched, and a total of 4,045 related genes were retained after the two databases were merged and deduplication was removed. After screening, RGLS-related genes and disease-related genes were introduced into the VENNY graph, and finally 227 cross-targets related to RGLS treatment of lung cancer were obtained (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Using the STRING database, a PPI network was constructed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). The network has 227 nodes and 801 edges, indicating that there is a high correlation between protein targets. The network was constructed using Cytoscape 3.9.1 software, and the PPI network was sorted using the Degree program (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). The Centiscape 2.2 plug-in was used to screen core targets based on topological structure and biological characteristics. The interaction between PD-L1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD) and 20 core targets (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE), among which JAK1-3, STAT1, and EGFR targets had significant interaction effects with PD-L1. In the constructed PPI network, Degree reflects the central attribute of the node, and the darker the color, the higher the value.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe enrichment analysis of the GO function resulted in 859 biological processes (BP), including the cellular positive regulation of the Notch signaling pathway, cell proliferation, negative regulation of NF-kappaB transcription factor activity, and regulation of the cell cycle, etc. There were also 93 cellular components (CC) obtained, including the inner membrane of the nucleus, spindle microtubules, the overall composition of the postsynaptic membrane. GO enrichment also resulted in 201 molecular functions (MF), involving retinal dehydrogenase activity, NADP binding activity, aspartate-type endopeptidase activity, GABA-gated chloride channel activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). The longer the bar graph length, the more significant the enrichment. KEGG pathway enrichment analysis resulted in 214 pathways, whose common targets were mainly enriched in metabolic pathways, bile secretion, Wnt signaling, and adrenergic signaling in cardiomyocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eComposition analysis of RGLS\u003c/h2\u003e\u003cp\u003eGanoderma lucidum has significant anti-cancer effects, and triterpenoids are considered to be its main anti-cancer components, so we used HPLC to identify the RGLS components. We determined the presence of 14 triterpenoids in Ganoderma lucidum, including ganoderic acid I, ganoderic acid C2, ganoderic acid C6, ganoderic acid G, ganoderic acid B, ganoderic acid N, ganoderic acid B, ganoderic acid A, ganoderic acid A, ganoderic acid H, ganoderic acid D2, ganoderic acid D, ganoderic acid C1, and ganoderic acid F in de-walled Ganoderma lucidum spore powder (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, B).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eRGLS synergizes with αPD-L1 to inhibit tumor growth\u003c/h2\u003e\u003cp\u003eAfter 12 days of administration of RGLS, αPD-L1, and RGLS combined with αPD-L1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), the tumors in the control group grew rapidly, while the tumor growth in the RGLS, αPD-L1, and RGLS-αPD-L1 groups was delayed (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, C). The tumor volume and mass in the RGLS-αPD-L1 group were significantly lower than those in the single-drug group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, C). In addition, RGLS increased the spleen index and thymus index, but combined administration with αPD-L1 did not further increase the spleen index and thymus index (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, E).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eRGLS synergizes with αPD-L1 to inhibit cancer cell proliferation and promote apoptosis\u003c/h2\u003e\u003cp\u003eImmunohistochemistry (IHC) staining and TUNEL staining were used to detect the expression of Ki-67 (a protein marker for cell proliferation) and apoptosis in tumors. Compared with the control group, Ki-67 in tumors of the αPD-L1, RGLS, and RGLS-αPD-L1 groups was significantly decreased. Compared with the αPD-L1 group, Ki-67 in the RGLS-αPD-L1 group was significantly decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). In addition, compared with the control group, the number of TUNEL-positive cells in the αPD-L1 and RGLS groups increased significantly, but there was no significant difference between the RGLS-αPD-L1 group and the single-drug group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003eRGLS synergizes with αPD-L1 to promote tumor-specific CTL activity of splenic lymphocytes\u003c/h2\u003e\u003cp\u003eCTL plays an important role in inhibiting tumor cells. We detected the enhanced tumor-specific cytotoxic activity of spleen lymphocytes against Lewis cells. The results showed that compared with the control group, the tumor-specific cytotoxic activity of spleen lymphocytes in the mouse RGLS, αPD-L1 and RGLS-αPD-L1 groups increased when the ratio of effector cells to target cells (E/T ratio) in the co-culture wells was 10:1, 20:1 and 40:1, respectively. The tumor-specific cytotoxic activity of spleen lymphocytes in the RGLS-αPD-L1 group was significantly higher than that in the single-drug group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003eRGLS synergizes with αPD-L1 to promote immune system activity\u003c/h2\u003e\u003cp\u003eTo test whether the anti-tumor effect of RGLS combined with αPD-L1 is related to its immune-enhancing effect in lung cancer mice, we measured the CD4\u003csup\u003e+\u003c/sup\u003e/CD8\u003csup\u003e+\u003c/sup\u003e T cell ratio and IFN-γ and IL-2 levels in the peripheral blood of mice. Compared with the control group, the CD4+/CD8\u0026thinsp;+\u0026thinsp;T cell ratios in the peripheral blood of the αPD-L1, RGLS, and RGLS-αPD-L1 groups were significantly increased. The CD4\u003csup\u003e+\u003c/sup\u003e/CD8\u003csup\u003e+\u003c/sup\u003eT cell ratio in the RGLS-αPD-L1 group was significantly higher than that in the αPD-L1 group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). In addition, we used IHC to detect the infiltration level of CD3\u003csup\u003e+\u003c/sup\u003e cells in tumor tissues and found that compared with the control group, the expression of CD3\u003csup\u003e+\u003c/sup\u003e cells in the αPD-L1, RGLS, and RGLS-αPD-L1 groups was increased, but there was no significant difference between the monotherapy group and the RGLS-αPD-L1 group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). In addition, the levels of IL-2 and IFN-γ in the peripheral blood of tumor mice were lower than those in normal mice, while the αPD-L1 group and RGLS group could increase the levels of IL-2 and IFN-γ, and the RGLS-αPD-L1 group could increase the effect of αPD-L1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). These data indicate that RGLS can increase the ratio of CD4\u003csup\u003e+\u003c/sup\u003e/CD8\u003csup\u003e+\u003c/sup\u003e T lymphocytes in lung cancer mice treated with αPD-L1, thereby causing them to secrete IFN-γ and IL-2 and enhance immune activity.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\u003ch2\u003eRGLS synergizes with αPD-L1 to inhibit the accumulation of myeloid-derived suppressor cells (MDSC)\u003c/h2\u003e\u003cp\u003ePD-L1 expression may inhibit the anti-tumor immune activity of T lymphocytes due to the deposition of MDSC in tumor tissues. In mice, the definition of MDSC is based on the surface markers CD11b\u003csup\u003e+\u003c/sup\u003e and LY6G\u003csup\u003e+\u003c/sup\u003e. Therefore, we evaluated the expression of CD11b\u003csup\u003e+\u003c/sup\u003e and LY6G\u003csup\u003e+\u003c/sup\u003e by immunohistochemical staining to analyze MDSC in tumor tissues. Compared with the control group, the aggregation of MDSC in the αPD-L1 group, RGLS group, and RGLS-αPD-L1 group was significantly reduced, and the aggregation of MDSC in the RGLS-αPD-L1 group was significantly lower than that in the αPD-L1 group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Our results show that RGLS can reduce the aggregation of MDSC in tumor tissues, thereby enhancing the anti-tumor effect of αPD-L1.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eLung cancer is the most common malignant tumor seen in clinical practice and the main cause of cancer-related death worldwide. As anti-tumor immunity research advances, immune checkpoint inhibitors such as PD-1/PD-L1 have emerged as a viable treatment for lung cancer[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. However, the low response rate (30%) of αPD-L1 medicines remains a significant impediment to treating lung cancer. Due to the complexity of immune control in the body, immunotherapy is thought to have substantial limits. Combined immunotherapy, which relies on various immune regulatory systems, has emerged as a new medicinal technology[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. According to earlier research, GLS can drastically suppress the expression of PD-1 and CTLA-4 in the spleen of the tumor group, indicating that it affects the PD-1/PD-L1 pathway[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. GLS powder has significant in vivo anti-tumor activity in tumor mice, and its mechanism is to stimulate NK cells, T cells, and macrophages, enhancing the host's immune response[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eCompound-target-pathway disease network analysis showed that RGLS contained 61 active ingredients and 227 related targets, which are potential targets for lung cancer treatment. KEGG pathway enrichment analysis showed that bile secretion, Wnt signaling, and adrenaline signaling in cardiomyocytes may be potential mechanisms for RGLS to exert anti-tumor effects. Using the Centiscape 2.2 plug-in, core targets were screened based on topological structure and biological characteristics. PD-L1 had a significant response effect with 20 core targets. Drawing on insights from network pharmacology, we speculate that RGLS may have a synergistic effect with anti-tumor immunosuppressants, which may enhance their efficacy. Mouse experiments showed that RGLS synergistically with αPD-L1 significantly inhibited tumor growth in the lung cancer mouse model compared with single treatment, as shown by a decrease in tumor volume and tumor weight. Previous studies have reported that although the direct antitumor effects of GLS and αPD-L1 are extremely limited, they can significantly enhance antitumor immune activity[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Therefore, based on the in vivo antitumor activity and improved immune function, we continued to investigate the potential mechanisms associated with the combination of RGLS and αPD-L1.\u003c/p\u003e\u003cp\u003eT cells play a critical role in producing and regulating immune responses to tumor antigens. T lymphocytes can be further classified into two types: CD4\u003csup\u003e+\u003c/sup\u003eT cells and CD8\u003csup\u003e+\u003c/sup\u003eT cells[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e T cells contribute to and coordinate antitumor immune responses. Their levels reflect the body's anti-tumor immunological response. Among these, the CD4+/CD8\u0026thinsp;+\u0026thinsp;ratio is a direct reflection of the body's anti-tumor immunity[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Our research indicates that RLGS can increase the proportion of CD4\u003csup\u003e+\u003c/sup\u003e/CD8\u003csup\u003e+\u003c/sup\u003eT cells in the peripheral blood of lung cancer mice treated with alpha PD-L1. Subsequently, we further validated the synergistic anti-tumor effect of RGLS combined with α PD-L1 through tumor specific CTL activity assays. The results showed that mouse spleen lymphocytes treated with RGLS combined with αPD-L1 exhibited stronger tumor specific cytotoxic activity than model mice treated with αPD-L1. CD4\u003csup\u003e+\u003c/sup\u003e T cells release cytokines like IL-2 and IFN-γ, which can boost the development of CD8\u003csup\u003e+\u003c/sup\u003e cytotoxic T cells. IL-2 promotes T cell proliferation and differentiation into mature CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e T cells[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Activated CD8\u0026thinsp;+\u0026thinsp;T lymphocytes can aggregate and release cytokines such as IFN - γ in the tumor microenvironment. The expression level of IFN - γ in CD8\u0026thinsp;+\u0026thinsp;T cells is closely related to tumor specific cytotoxic activity[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. In our study, ELISA showed that the combination of RGLS and alpha PD-L1 resulted in higher levels of IL-2 and IFN - γ production by T cells in lung cancer mice compared to treatment with αPD-L1 alone.\u003c/p\u003e\u003cp\u003eMDSCs mainly exist in bone marrow, spleen, and tumor tissues, playing a key role in the tumor immune microenvironment. When MDSCs infiltrate into tumor tissue, they reduce the proliferation and function of T cells and NK cells, while inducing Treg cells[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. In previous studies, αPD-L1 reduced the number of MDSCs in tumor tissue, and GLS also had a similar effect[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Our experiments showed that RGLS combined with αPD-L1 reduced the number of MDSCs in tumor tissues of lung cancer mice compared with either treatment alone. The change in MDSCs is the theoretical basis for the combined treatment of lung cancer with RGLS and αPD-L1.\u003c/p\u003e\u003cp\u003eHowever, our research still has some limitations. This study was only based on the Lewis lung cancer mouse model and did not include patient-derived xenograft (PDX) models of human lung cancer. Furthermore, the specific target differences of the 14 triterpenoid components in RGLS were not explored in depth; future studies should clarify key active components through component-specific intervention experiments. We will achieve them one by one in future plans.\u003c/p\u003e\u003cp\u003eThe results of this study highlight the great potential of combining traditional Chinese medicine with modern immunotherapy in the treatment of lung cancer. The synergistic effects of RGLS and αPD-L1 observed in mouse models suggest that this combination therapy is expected to become a valuable supplement to current lung cancer treatments. Future clinical trials should explore the efficacy and safety of this combination therapy in human patients, especially those with limited response to αPD-L1 monotherapy. In addition, further research on the specific mechanisms of RGLS immunomodulatory effects may reveal new therapeutic targets and biomarkers to guide personalized treatment strategies.\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eOur study confirmed that RGLS combined with αPD-L1 can effectively reduce the infiltration of MDSCs in tumor tissues and enhance the activity of the immune system, thereby inhibiting the development of lung cancer, compared with the single-agent group. Essentially, these findings provides preclinical evidence from animal experiments for subsequent clinical research on RGLS combined immunotherapy for lung cancer.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Zhejiang Anti-Cancer Association (Grant No. 201902) , Zhejiang Traditional Chinese Medicine Administration (Grant No. 2021ZZ006), Zhejiang Province Traditional Chinese Medicine Science and Technology Plan Project (Grant No. 2024ZL300).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors confirm that there are no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e1.Statement on the maximal tumor size/burden permitted by the ethics committee\u003c/strong\u003e:\u003c/p\u003e\n\u003cp\u003e\"The Zhejiang Cancer Hospital Animal Experiment Ethics Committee (Approval No.: SYKX 2017-0012) established explicit limits for tumor burden in Lewis lung cancer-bearing C57BL/6 mice in this study: a maximum permitted tumor volume of 2000 mm³, and a maximum permitted tumor weight not exceeding 10% of the mouse’s body weight.\"\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.Statement confirming no exceedance of the maximal tumor size/burden\u003c/strong\u003e:\u003c/p\u003e\n\u003cp\u003e\"Throughout the 12-day experimental period, tumor burden in mice from all treatment groups (control group, αPD-L1 group, RGLS group, and RGLS-αPD-L1 group) was monitored every 2 days. Tumor volume was calculated as (length × width²)/2 via caliper measurements of tumor length and width, and tumor weight was measured at the experimental endpoint. Results showed that the maximum tumor volume across all groups was 1666 ± 298 mm³ (control group on day 12), and the maximum tumor weight was 1.68±0.23 g (control group on day 12)-neither exceeding the maximal tumor size/burden limits set by the ethics committee.\"\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContribution of authors\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization, Qiong Wang , Xiaogrong Zheng , Zengyu Zhang, Jianwei Jiang Junfei Li, and Hongyan Zhang; methodology, Qiong Wang , Junfei Li, Jianwei Jiang and Xiaorong Zheng; investigation,Qiong Wang , Junfei Li, Jianwei Jiang and Hao Min; Data curation, Hao Min; writing-original draft preparation, Qiong Wang , Zengyu Zhang , Xiaorong-Zheng and Kao Shi; writing-review and editing, Qiong Wang , Junfei Li, Hao Min; supervision, Hongyan Zhang and Junfei Li; funding acquisition, Hong-Yan Zhang, Jun-Fei Li. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHarethardottir H, Jonsson S, Gunnarsson O, Hilmarsdottir B, Asmundsson J, Gudmundsdottir I, et al. [Advances in lung cancer diagnosis and treatment - a review]. Laeknabladid. 2022;108(1):17\u0026ndash;29. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.17992/lbl.2022.01.671\u003c/span\u003e\u003cspan address=\"10.17992/lbl.2022.01.671\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. Epub 2021/12/21.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLahiri A, Maji A, Potdar PD, Singh N, Parikh P, Bisht B, et al. Lung cancer immunotherapy: progress, pitfalls, and promises. 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PubMed PMID: 32530032; PubMed Central PMCID: PMCPMC7313449.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"discover-oncology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"dion","sideBox":"Learn more about [Discover Oncology](https://www.springer.com/12672)","snPcode":"","submissionUrl":"","title":"Discover Oncology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Cancer immunotherapy, Myeloid-Derived Suppressor Cell, Ganoderma Lucidum Spore Powder, PD-L1, Lung Cancer, CD4+/CD8+ T cell ratio","lastPublishedDoi":"10.21203/rs.3.rs-7088435/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7088435/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eThe therapeutic efficacy of PD-1/PD-L1 inhibitors in lung cancer management remains suboptimal. Sporoderm-removed Ganoderma Lucidum Spore Powder (RGLS), an immunostimulant, has been shown to amplify the anti-tumor effects of anti-PD-L1 antibodies (αPD-L1). However, the detailed molecular underpinnings of this enhancement are not yet fully elucidated. This study aimed to elucidate the antitumor mechanism and immunomodulatory effect of RGLS in synergistic relationship with αPD-L1 in lung cancer.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eEmploying network pharmacology approaches, this research analyzed the multifaceted target pathways of RGLS in lung cancer. High-Performance Liquid Chromatography (HPLC) facilitated the identification of RGLS constituents. In the established Lewis lung cancer tumor-bearing mouse model, the effects of RGLS, αPD-L1, and their combination were investigated, focusing on tumor growth, T cell responses, and the dynamics of myeloid-derived suppressor cells (MDSCs) within tumor microenvironments.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eOur network analysis revealed 26 bioactive components of RGLS and identified 227 potential lung cancer targets, among which PD-L1 had a significant response effect with 20 core targets. HPLC identified 14 triterpenoid components. Notably, the combination of RGLS and αPD-L1 significantly inhibited tumor growth, optimized the CD4\u003csup\u003e+\u003c/sup\u003e/CD8\u003csup\u003e+\u003c/sup\u003e T cell ratio in tumor tissue, increased IL-2 and IFN-γ levels, enhanced cytotoxic T lymphocyte (CTL) activity, and inhibited MDSC recruitment.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eThe synergistic application of RGLS and αPD-L1 in lung cancer treatment significantly improves immune responsiveness and reduces tumor-associated MDSCs, surpassing the efficacy of sole αPD-L1 application. This study underscores RGLS's burgeoning potential in bolstering lung cancer immunotherapy, paving the way for its clinical applications.\u003c/p\u003e","manuscriptTitle":"Synergistic Anti-Tumor and Immunomodulatory Effects of Sporoderm-Removed Ganoderma Lucidum Spore Powder and Anti-PD-L1 Antibody in Lung Cancer Mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-04 19:28:11","doi":"10.21203/rs.3.rs-7088435/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-29T11:17:27+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-18T16:14:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"23972791246422962048969372829244487900","date":"2025-09-15T12:27:58+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-05T02:07:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"147167036362904833627240983315141907532","date":"2025-09-03T03:52:57+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-02T13:55:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"215016656815965530839911917937089409967","date":"2025-09-02T01:17:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"271450507233827281592777800034301780738","date":"2025-08-30T02:55:38+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-27T18:13:26+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-17T11:06:04+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-15T03:17:35+00:00","index":"","fulltext":""},{"type":"submitted","content":"Discover Oncology","date":"2025-08-15T03:14:42+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"discover-oncology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"dion","sideBox":"Learn more about [Discover Oncology](https://www.springer.com/12672)","snPcode":"","submissionUrl":"","title":"Discover Oncology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5eecdb2b-c7f9-4aae-8d7c-efac0a74d9bd","owner":[],"postedDate":"September 4th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-03-13T08:56:40+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-04 19:28:11","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7088435","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7088435","identity":"rs-7088435","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}