Glycyrrhiza uralensis polysaccharides as a DC-based Vaccine Adjuvant: Enhanced Immunotherapy Combined with PD-1 Blockade

Glycyrrhiza uralensis polysaccharides as a DC-based Vaccine Adjuvant: Enhanced Immunotherapy Combined with PD-1 Blockade | 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 Article Glycyrrhiza uralensis polysaccharides as a DC-based Vaccine Adjuvant: Enhanced Immunotherapy Combined with PD-1 Blockade Patanmu Aili, Shanshan Cai, Bei Chen, Nuerbiye Aobulikasimu, Lili Han, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8390778/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version Abstract The activation of naive CD8 + T cells by mature dendritic cells (DCs) through antigen presentation is essential for initiating effective anti-tumor immunity. In our previous study, Glycyrrhiza uralensis polysaccharides (GUPS) were prepared and found that they significantly promoted the maturation and cytokine secretion of human Mo-DC and murine BM-DC through TLR4 signaling pathways. In this study, HPV-DC vaccine prepared with GUPS (GUPS-DC + HPV) significantly suppressed the tumor growth and improved the survival of tumor mice, which was correlated with the induction of HPV-16-specific cellular response and the increased infiltration of CD8 + T cells in tumors. Although HPV-DC vaccine can induce CTL response and inhibit tumor growth, the late treatment effect needs to be further improved. Therefore, the combination treatment of HPV-DC vaccine and anti-PD-1 Ab could further improve the therapeutic efficacy, improve the survival rate of tumor mice, generated memory immune response and suppressed lung metastasis of tumors. These findings suggest GUPS as a potent adjuvant that promotes DC maturation, and offering a combination strategy for the treatment of advanced tumors. Biological sciences/Cancer Biological sciences/Immunology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Cancer immunotherapy is currently recognized as a pillar of treatment alongside traditional modalities such as surgery, radiation, and chemotherapy, and it is becoming one of the most exciting and rapidly expanding fields[1-2]. Various forms of immunotherapy, including adoptive cell transfer (ACT), immune checkpoint inhibitors (ICIs), cancer vaccines, oncolytic virus therapy (OVT), have demonstrated significant efficacy and promise[3]. These methods primarily involve inducing CD8+ T cell responses to enhance anti-tumor immunity or restoring the function of exhausted T cells. The presence of anti-tumor cytotoxic T lymphocytes (CTLs) and their infiltration within the tumor microenvironment are prerequisites for treatment efficacy[4-6]. Thus, CD8+ T cells are needed to be primed or back toward effector CTLs for making durable and efficient antitumor immune responses[7-8]. At the forefront of the cancer-immunity cycle, CD8+ T cells priming is essentially a corroborative process involving antigen presenting cells (APCs), including dendritic cells (DCs)[9]. Major histocompatibility complex I (MHC-I) molecules on the surface of DCs are identified by CD8+T cells, which attach to it and scan the surface by crawling over it[10-12]. Furthermore, the upregulation of costimulatory molecules, such as CD40 and CD86, and the release of cytokines during DCs maturation are crucial for establishing stable and enduring interactions with T cells at the immunological synapse (IS). These interactions are vital for T cell expansion and differentiation into memory and effector T cells, thereby underpinning robust cellular immune responses[13-14]. Given the central role of DCs in antitumor immunity, investigators have theorized that DC vaccine would serve as an ideal vaccine platform that can lead to the effective eradication of tumors. The safety and immunogenicity through vaccination potentially constitute a powerful anticancer strategy but clinical benefits have been largely disappointing[15]. New information on the contribution of DCs to tumor immunity should improve DC’s maturity in vitro, using adjuvants to optimize their performance in therapeutic applications. Currently approved adjuvants, such as alum and oil, deviate from the purpose for supporting tumor immunotherapy, because they mainly enhance humoral (Th2) immunity and are not without side effects[16]. The safety of DC vaccines has been fully demonstrated in numerous phase I and II clinical trials, with most DC vaccination regimens exhibiting minimal side effects [17-19]. Studies by Ralph Steinman and Michel Nussenzweig revealed that in vivo targeting of DCs through the conjugation of antigens to antibodies directed against DC surface receptors (such as DEC205 or DCIR) enables efficient antigen delivery and capture by DCs[20]. However, in the absence of adjuvants, targeting DEC205+ DCs in vivo can induce antigen-specific immune tolerance [21]. As observed in the original DEC205 studies, the presence or absence of adjuvants profoundly impacts immune responses. For instance, antigen delivery via CLEC9A-targeting antibodies without adjuvants triggers robust antibody responses associated with Tfh cell differentiation[22], while simultaneously inducing Treg differentiation[23]. To date, only a limited number of cytokines or TLR ligands have been successfully utilized as DC vaccine adjuvants in clinical applications, highlighting the need to identify novel, safe, and potent adjuvants to enhance DC maturation and improve vaccine immunogenicity and clinical efficacy. Consequently, adjuvants play a critical role in enabling DC vaccines to effectively activate T cell immune responses. In recent years, polysaccharides have emerged as promising adjuvant candidates due to their immunomodulatory activities and safety profiles[24-25]. Accumulating evidence demonstrates that polysaccharides can promote dendritic cell maturation, characterized by upregulation of co-stimulatory molecules and cytokines including IL-12[26]. Our previous studies reported the isolation and characterization of Glycyrrhiza polysaccharide (GUPS), which exhibits potent immunostimulatory activity in dendritic cells through TLR4 and downstream p38, JNK, and NF-κB signaling pathways [27-28]. In this study, we adopted GUPS as an adjuvant for human papillomavirus (HPV)-DC vaccine (GUPS-DC+HPV) and evaluated the antitumor response and efficacy of combination therapy with PD-1/PD-L1 inhibitors. The GUPS-DC+HPV effectively inhibited the growth of tumors, resulting in improved the initiation of CTLs, enhanced CD8+ T cell infiltration into tumor sites, and reduced production of regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs). The GUPS-DC+HPV also prolonged the survival rate of mice and suppressed the tumor metastasis. In combination with anti-PD-1, GUPS-DC+HPV further enhanced the antitumor efficacy. Throughout the therapy, GUPS elicited efficient adjuvant activity. 2. Materials and methods 2.1 Materials G.uralensis polysaccharides (GUPS) were obtained from the Xinjiang Key Laboratory of Biological Resources and Genetic Engineering, Xinjiang University (Urumqi, Xinjiang, China). The purified GUPS had a polysaccharide content of 93% and a molecular weight of 29.1 kDa.Fetal bovine serum (FBS) and Penicillin-streptomycin were purchased from MRC (Changzhou, China). Medium RPMI-1640 medium and phosphate-buffered saline (PBS) were purchased from Gibco (Grand Island, NY, USA). Trypsin was sourced from MRC (China). Granulocyte-macrophage colony-stimulating factor (GM-CSF) was purchased from PeproTech (Rocky Hill, NJ, USA). HPV-16 E6/E7 peptides, including E643–57 (QLLRREVYDFAFRDL), E653–62 (AFRDLCIVYR), E711–20 (YMLDLQPETT), E744–62 (QAEPDRAHYNIVTFCCKCD) and E781–94 (DLLMGTLGIVCPIC), were synthesized by Shanghai Science Peptide Biological Technology (China). These peptides were dissolved in DMSO at 20 mg/mL, filtered through a 0.22 µm filter membrane, and stored at -80℃. Trizol was purchased from Thermo Fisher Scientific (USA), and reverse transcription reagents were obtained from TAKARA Biotechnology. The Cytofix/Cytoperm kit and Golgi stop (Monensin) were purchased from BD Biosciences (USA). 4% Formaldehyde Fixation Solution was sourced from Biosharp (China). Anti-mouse monoclonal antibodies against PD-1(BE0033-2) and PD-L1 (BE0033-2) were purchased from BioXCell (USA). The antibodies for flow cytometry, including anti-CD25-APC, anti-CD49b-FITC, anti-CD11b-PE, anti-Gr-1-ER780, anti-CD19-APC, anti-CD4-ER780, anti-CD44-PE, anti-Foxp3-PE, anti-CD4-FITC, anti-IFN-γ-APC, were purchased from Elabscience (Wuhan, China). Furthermore, anti-MHC-I-FITC, anti-CD3-APC700, anti-CD8-PE-CY7 and anti-MHC-II-PE, were obtained from BD Biosciences. 2.2 Animals and ethics statement Female C57BL/6 mice (6-8 weeks old, weighing approximately 22 g) were acquired from the Beijing Laboratory Animal Research Center (Beijing, China) and housed at the temperature‑controlled (25˚C) and light‑cycled (12-hour light/12-hour dark) in Animal Facility of Xinjiang University (Urumqi, China). All animal experiments in this study were approved by the Ethics Committee on Animal Experiments at the Xinjiang Key Laboratory of Biological Resources and Genetic Engineering (approval number: BRGE-AE001) and were carried out in strict adherence to the guidelines set forth by the Animal Care and Use Committee of the College of Life Science and Technology, Xinjiang University. 2.3 Cell line The mouse TC-1 cell line was obtained from the Xinjiang Key Laboratory of Biological Resources and Genetic Engineering, Xinjiang University (Urumqi, Xinjiang, China). These cells were cultured in RPMI-1640 medium (Gibco), supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, and 10% heat-inactivated fetal bovine serum (Gibco). The cells were maintained at 37°C in a humidified incubator with a 5% CO2 atmosphere to ensure optimal growth conditions. 2.4 Vaccine preparation DCs were collected and cultured for 7 days before being seeded into 60 mm cell culture dishes at a density of 1×10 6 cells/mL, and added GUPS (20 μg/mL) for treatment. After 12 h of incubation, the cells were collected by centrifugation at 1200 rpm for 7 min. Then, the cells were resuspended in fresh medium (1×10 6 cells/mL) and incubation with HPV-16 E6/E7 polypeptide at a final concentration of 10 μg/mL for 2 h, centrifuged (1200 r/min, 7min) to collect, and washed once with PBS. The DCs vaccine prepared by PBS (1×10 6 cells /50 μL) were named GUPS-DC+HPV, and the GUPS-treated DCs were not incubated with HPV-16 E6/E7 polypeptide (GUPS-DC) as a control. 2.5 TC-1 Tumor model and treatment TC-1 cells, which stably express HPV-16 E6 and E7, were collected during log-phase growth and washed with PBS. TC-1 cells were re-suspended in PBS (1×10 5 cells/mL) and 100 μL of this suspension were subcutaneously injected into the right flank of C57BL/6 mice. The tumor-bearing mice were randomly divided into three groups (6 mice/group) and intradermally injected with 50 μL PBS (control group) or immunized with 1×10 6 cells (GUPS-DC or GUPS-DC+HPV) in 50 μL PBS on day 3 and day 10. Tumor size were measured every other day and tumor volumes were calculated using the formula: tumor volume (mm3)=(length×width 2 )/2. At the same time, measuring the weight of the mice every other day. At the end of study, the tumors were isolated, weighted and processed for further analysis. The tumors were fixed, sliced and subjected to hematoxylin and eosin (H&E) staining to evaluate histological changes. Splenocytes were used to detect the immune responses by flow cytometry. 2.6 Survival rate of TC-1 tumor mice On the 3rd day after the successful establishment of the TC-1 tumor model, the mice were randomly divided into three groups(10 mice/group). On the 3 th and 10 th days, the mice in the treatment groups were given intradermal injection of GUPS-DC or GUPS-DC+HPV, and the untreated group served as the Control group to monitor the survival rate of the tumor mice. 2.7 Flow cytometry To characterize the phenotype characterization of DCs and T cells, flow cytometry was performed on the isolated splenocytes following staining with specific mAbs. For the analysis of Tregs, the cells were first surface staining with anti-CD4 and anti-CD25 mAbs. Subsequently the cells were fixed and permeabilized using a Foxp3 Staining Buffer Set (eBioscience) and stained with Foxp3 mAbs. To assess antigen-specific CD8 + T cell responses, splenocytes (1×10 6 cells/mL) were stimulated with HPV-16 E6/E7 peptides and cultured overnight in the presence of Golgi stop (BD Biosciences). After stimulation, the cells were stained with an anti-CD8 mAbs to identify CD8 + T cell. Following surface staining, the cells were fixed and permeabilized using Cytofix/CytopermTM (BD Biosciences) and then stained with IFN-γ mAbs to detect intracellular IFN-γ production. All samples were collected on FACSCalibur (BD Biosciences) and analyzed using the FlowJo platform (Tree Star, Inc, Ashland, OR). 2.8 Hematoxylin and eosin(H&E) staining Mouse organs were fixed in 4% formaldehyde and then paraffin sections were performed. Sections were stained. Dip the slices into a coconut milk jar filled with mayerhematoxylin and stir for 30 s. Rinse the slide with water for 1 min. Dip the slices into 1% eosin Y solution and stir for 10-30 s. The section was dehydrated in 95% alcohol and 100% alcohol repeatedly, twice for each solution, 30 s each time. Ethanol is removed with two different xylene compounds. Add one to two drops of medium and cover with mulch. The stained sections were observed with an inverted microscope and photographed. 2.9 RNA-Seq Analysis Tumor tissues harvested on day 38 were homogenized in TRIzol reagent, and total RNA was extracted according to the manufacturer’s instructions. A Bioanalyzer (Agilent) was used to validate the quality and quantity of the purified RNA. Library construction for RNA-seq was performed using the TruSeq Stranded mRNA LT Sample Prep Kit (Illumina), following manufacturer’s instructions. The resulting library was then sequenced on a HISeq 2500 platform using the 100 bp single-end protocol (Illumina). Multiple mapped reads were segregated and distributed in a random manner using an in-house script. Reads mapping to protein-coding genes were counted using HTSeq with the GTF file, considering only the protein-coding feature. Differentially expressed genes were examined by DESeq. Genes of interest on pathways annotated in KEGG were selected if their expression levels were significantly different (adjusted p <0.05 and ±2-fold change) between the GUPS-DC+HPV and control groups. Read counts were normalized by total number of mapped reads and fit to a standard normal distribution for the depiction of heatmaps. 2.10 Immunohistochemistry For immunohistochemistry, the tissue sections were deparaffinized, rehydrated, and subsequently incubated in citrate buffer antigen repair buffer (pH 8.0) and 3% H 2 O 2 , respectively. After blocking with blocking solution for 1 h at room temperature, the sections were incubated for overnight at 4℃ in primary antibody. Following the washes, the sections were then incubated sequentially with the specific secondary antibodies and HRP-conjugated streptavidin. Finally, the sections were stained with 3,3’-diaminobenzidine (DAB) for color development and counterstained with haematoxylinthe. The results were observed and photographed under a fluorescence microscope. 2.11 Combined GUPS-DC+HPV with PD-1/PD-L1 antibody therapy On the 10th day after the successful establishment of the TC-1 tumor model, the mice were randomly divided into 6 groups(7 mice/group). The untreated group served as the Control group. On the 10th day, intradermal injection of GUPS-DC+HPV was used as the GUPS-DC+HPV group. The mAbPD-L1 (100 μg/mouse) or mAbPD-1 (100 μg/mouse) were intraperitoneally injected into the mAbPD-L1 group and mAbPD-1 group on the 10th, 13th, 16th, 19th and 22nd days, respectively. The combination of GUPS-DC+HPV with mAbPD-1or mAbPD-L1 was divided into the GUPS-DC+HPV+mAbPD-1 or GUPS-DC+HPV+mAbPD-L1 group, and the tumor size was measured every other day, mouse survival rate and immune memory response. To further test the effect of late treatment, On the 10 th day after the establishment of the TC-1 tumor model, the mice were randomly divided into 4 groups (6 mice/group). The untreated group served as the control group. Starting on the 16th day, intradermal injection of GUPS-DC+HPV was administered to the GUPS-DC+HPV group, with additional injections on the 25th day. The mAbPD-1 group received intraperitoneally injected of mAbPD-1 (100 μg/mouse) on the 16th, 19th, 22nd, 25th and 28th days, respectively. The mice receiving the combination of GUPS-DC+HPV with mAbPD-1 were designated as the GUPS-DC+HPV+mAbPD-1 group, and the tumor size in all groups was measured every other day，At the same time, measuring the weight of the mice every other day. To detect the effect of DC vaccine on tumor recurrences, on day 58, inoculation of TC-1 cells (1×10 5 cells/mouse) from 3 mice in the GUPS-DC+HPV group and GUPS-DC+HPV+mAbPD-1 group. At the same time, naïve mice were inoculated 1×10 5 cells selected as control. The growth of the mouse tumors in the above three groups was examined. 2.12 Lung metastasis models C57BL/6 mice were injected intravenously with TC-1 cells (1×10 5 cells) on day 0 and were randomly divided into 4 groups(5 mice/group). The untreated group served as control group. On the 3 day, the GUPS-DC+HPV group received an intradermal injection of GUPS-DC+HPV alone. The mAbPD-1 group was intraperitoneally injected with mAbPD-1 (100 μg/mouse) on the 6th, 9th, 12th，15th, 18th, and 21st days, respectively. The mice receiving the combination of GUPS-DC+HPV with mAbPD-1 were designated as the GUPS-DC+HPV+mAbPD-1 group, and the tumor size was measured every other day. On day 21, the mice were euthanized (n=5) and the spleens and lungs were collected for further analysis. The number of pulmonary tumor metastases was then counted. 2.13 Statistical analysis Data were reported as mean ± standard error of the mean (SEM). One-way analysis of variance (ANOVA), paired or unpaired t-test were used to analyze the statistical significance by Prism5 GraphPad software (GraphPad Software, La Jolla, CA). A value of < 0.05 was considered to be statistically significant. 3. Results 3.1 GUPS improved the antitumor efficacy of HPV-DC vaccine The adjuvant effect of GUPS on the HPV-DC vaccine was detected in TC-1 tumor mouse model. Tumor mice were treated with GUPS-DC+HPV on day 3 and 10 after injection of TC-1 cells (Fig.1A). As shown in Fig.1B&C, GUPS-DC+HPV markedly inhibited tumor growth compared with control and showed complete tumor regression in two mice. The H&E staining of tumors showed that the number of tumor cells was dramatically reduced and the necrotic areas were increased in GUPS-DC+HPV group compared with control group (Fig.1D). The effect of GUPS-DC+HPV on survival rate of tumor mice was further evaluated in another tumor mouse study. Tumor mice were treated with GUPS-DC+HPV on day 3 and 10 after injection of TC-1 cells. Similarly, GUPS-DC+HPV also inhibited tumor growth compared with control (data not shown). On day 54, all mice in the control group had died, and 1 out of 10 mice survived in the GUPS-DC group. Five out of 10 mice were survival in GUPS-DC+HPV group, suggesting that GUPS-DC+HPV greatly improved the survival rate of tumor mice (Fig.1E). Fig. 1. Tumor growth and tumor mouse survival. 3.2 GUPS-DC+HPV induced the antigen-specific CD8 + T cell immune responsese In light of the excellent anti-tumor activity of the GUPS-DC+HPV, the correlation of antitumor effect and immune responses generated by GUPS-DC+HPV were carried out to explore the infiltration level of myeloid-derived suppressor cells (MDSCs, CD11b + Gr-1 + ), regulatory T cells (iTregs, CD4 + CD25 - Foxp3 + and nTregs, CD4 + CD25 + Foxp3 + ) and CD8 + T (CD8 + CD44 + ) cells. As expected, Compared to control group, the frequencies of MDSCs and induced Tregs were significantly decreased (Fig.2A&B), and the frequencies of CD8 + T cells and CD8 + CD44 + T cells were notably increased in GUPS-DC+HPV group (Fig. 2C&D). After HPV-16 E6/E7 peptides treatment, HPV-specific cellular responses were detected. As shown in Fig.2E, the frequencies of HPV-specific CD8 + IFN-γ + T cells markedly increased in GUPS-DC+HPV group compared with control group. Moreover, GUPS-DC+HPV enhanced the infiltration of CD8 + T cells in tumors. These results indicated that both the reduction of inhibitory cells and induction of antigen-specific CD8 + T cell response contributed to suppressing tumor growth in GUPS-DC+HPV group. Fig. 2. The immune responses in TC-1 tumor mice after treatment with GUPS-DC+HPV. In order to elucidate the molecular mechanisms underlying the inhibition of tumor growth by GUPS-DC+HPV, we undertook a comprehensive transcriptomic analysis. Our findings revealed that 2326 differentially expressed genes were identified in the GUPS-DC+HPV group compared to the Control group, with 1810 genes upregulated and 516 genes downregulated (|log2 fold change| > 0, p < 0.001). Genes with elevated expression were depicted in red, while those with reduced expression were shown in blue (Fig. 3A). From the results of GO enrichment analysis, the most significant 10 Term plots are displayed, and the size and color of points correspond to the number of genes annotated to GO Term and the significance (Fig. 3B). Concomitantly, numerous pathways related to anti-tumor immunity were upregulated in the GUPS-DC+HPV (Fig. 3C). In particular, cytokine-cytokine receptor interaction, chemokine signaling, cell adhesion molecules and nature killer cell mediated cytotoxicity (Fig.3C). Fig.3. RNA-seq analysis of the expression profiles of differentially expressed genes in immune-related pathways. In nature killer cell mediated cytotoxicity pathway，cytotoxicity associated genes，including Fas , G zmB , Prf1 , CD244 , were significantly increased in response to GUPS-DC+HPV(Fig. 4A). Immunohistochemical data also showed GzmB and Fas upregulated in tumor tissue in the GUPS-DC+HPV treatment group. On the other hand, BCL-2 was decreased with the GUPS-DC+HPV therapy (Fig. 4C). In addition, numerous genes related to cytokine-cytokine receptor interaction were upregulated, such as Ifng, Ccl10, Ccl14, Il15, and Il17, were significantly increased in response to GUPS (Fig. 4B). The expression of TNF-α was increased compared with the control group. Taken together, these results strongly suggest that GUPS-DC+HPV therapy elicits Th1-type anti-tumor immunity with essential cytokine induction. Fig.4. Perform RNA sequencing analysis on the expression profiles of differentially expressed genes in immune-related pathways. However, it was worth noting that the high expression levels of CD274 was observed, which might express on the surface of tumor cells and activated APCs (Fig. 4A). Next, we devised a combination therapy with GUPS-DC+HPV and anti-PD-L1 Ab or anti-PD-1 Ab against TC-1 tumors. 3.3 GUPS-DC+HPV combined with anti-PD-1/PD-L1 Ab therapy further inhibited tumor growth and improved memory immune response and survival in tumor mice Although the mechanism of action of DC vaccine is clear, the tumor immune microenvironment and tumor immunosuppressive mechanisms greatly limit the effect of DC tumor vaccine. Therefore, we established a TC-1 tumor mouse model and evaluated the anti-tumor effect of GUPS-DC+HPV vaccine combined with PD-1 and PD-L1 blocking antibodies in the treatment of advanced tumor mice. The mice were given GUPS-DC+HPV vaccine on the 10 th day after inoculation of TC-1 cells, and the mice were given 100 μg of mAbPD-1 or mAbPD-L1 on the 10, 13, 16, 19 and 22 th days (Fig. 5A). Tumor size was measured every other day. At the same time, measuring the weight of the mice every other day. It is found that GUPS-DC+HPV, mAbPD-1 and mAbPD-L1 significantly inhibited tumor growth and had no significant effect on the weight of mice, with no significant side effects on the mice (Fig. 5B). When all the mice in the control group died, the survival rate of the GUPS-DC+HPV group was 75% (Fig. 5C). The survival rate of the GUPS-DC+HPV+mAbPD-1 group was 87.5% (Fig. 5C). The survival rate of the GUPS-DC+HPV+mAbPD-L1 group was only 50%. In the mAbPD-1 group, only one mouse survived, and in the mAbPD-L1 group, only two mice survived. Compared with the control group, GUPS-DC+HPV+mAbPD-1 significantly improved the survival rate (Fig. 5C). These results indicated that GUPS-DC+HPV combined with mAbPD-1 could significantly improve the probability of survivability in mice. On day 58, mice whose tumors had completely disappeared in the GUPS-DC+HPV group and the GUPS-DC+mAbPD-1 combination group were selected for the second inoculation with TC-1 cells. Naive mice of the same age were also inoculated with TC-1 cells as controls. The results showed that there were no tumors in GUPS-DC+HPV group and GUPS-DC+HPV+mAbPD-1 vaccine combination group, indicating that the vaccine and combination treatment induced memory immune response in mice (Fig.5D). The above results indicated that the combination of HPV-DC vaccine and PD-1 blocking antibody significantly inhibited the growth of TC-1 tumors, produced a strong memory immune response, improved the survival rate of mice. At present, HPV-DC vaccine research and development is becoming more mature. With the increases of HPV vaccine coverage, the incidence of a disease related to HPV is gradually reduced. However, in the late stage of cancer, advanced cancer cells have spread throughout the body, and conventional treatments were ineffective, so we established a late treatment model to evaluate its anti-tumor effect, 10 days after the establishment of TC-1 tumor model, patients were randomly divided into 4 groups, 5 cases in each group. The untreated group was used as control group. On day 16, GUPS-DC+HPV was injected intraderally as GUPS-DC+HPV group. The mAbPD-1 group received intraperitoneal injection of mAbPD-1 (100 μg/animal) at 16, 19, 22, 25, and 28 th days respectively. The GUPS-DC+HPV+mAbPD-1 combination treatment was divided into the GUPS-DC+ HPV+mAbPD-1 group, and tumor size was measured every other day (Fig. 5E). The results showed that HPV-DC+mAbPD-1 treatment induced durable, objective tumor regression and immune response in patients (Fig. 5F). The above results suggest that the combination therapy of HPV-DC and PD-1/PD-L1 is a safe, effective and feasible treatment strategy, which will bring new hope for patients with HPV infection. Fig.5. Treatment of tumor mice by GUPS-DC+HPV combined with mAbPD-1 or mAbPD-L1. 3.4 The role of GUPS-DC+HPV combined with PD-1 antibody in the treatment of lung metastases The lungs are the main site of metastatic spread. Due to their extensive vascularization and large surface area, which is essential for normal lung function, tumors have many opportunities to stop, extravasate, and colonize this organ. The poor prognosis of patients with recurrent and metastatic cervical cancer has always been a daunting challenge for clinicians. Thus, we developed a tumor lung metastasis model. The untreated group was used as control group. On the 3 th day, GUPS-DC+HPV was given intradermal injection alone was the GUPS-DC+HPV group. mAbPD-1 (100 μg/animal) were intraperitoneally injected into the mAbPD-1 group, on the 6, 9, 12，15, 18, and 21th days respectively (Fig. 6A). It was found that after the tumor cells were injected through the tail vein, they reached the lungs of the mice through the blood vessels and formed lung metastases. Compared with the control group, HPV-DC, mAbPD-1 alone and HPV-DC combined with mAbPD-1 significantly reduced the number of lung metastases, and the HPV-DC combined with mAbPD-1 had the best effect (Fig. 6B). MDSCs and Tregs contribute to tumor growth and the inhibition of antitumor immune responses [2 9- 32] . On day 21, the spleens were collected to detect the immune cells，and found that the GUPS-DC+HPV, mAbPD-1 and GUPS-DC+HPV+mAbPD-1 groups significantly reduced the proportion of Tregs cells and significantly increased the proportion of activated CD4+ and CD8 + T cells compared with the control group(Fig. 6C). The combination of HPV-DC to PD-1 blocking antibodies provides an inhibitory environment for the tumor microenvironment, which regulate immunosuppression and activates immune cells. Therefore, the reduction of inhibitory cells and induction of CD8 + T cell response together inhibited tumor growth in GUPS-DC+HPV+mAbPD-1 group, and proposing a new therapeutic strategy for the treatment of cervical cancer. Fig.6. GUPS-DC+HPV combined with mAbPD-1 or mAbPD-L1 to treat lung metastatic tumor mice. 4. Conclusion Although a large number of clinical trials have proved that DC vaccine has the advantages of high safety and induced antigen-specific immune response, its clinical efficacy needs to be improved, especially the insufficient maturity of DC in vitro, resulting in the limited degree of immune response [33-35] . Therefore, this study used GUPS as an adjuvant of HPV-DC vaccine to improve the immunogenicity of the vaccine. Following vaccination of normal C57BL/6 mice and stimulating HPV-DC prepared from mature DC with GUPS, the vaccine significantly enhanced CD8 + T cell activation and antigen-specific CTL response compared with the HPV vaccine prepared from immature DC, which not only inhibited tumor growth but also increased survival of tumor mice. Transcriptome data revealed that compared to the control group, the GM-DC+HPV group exhibited upregulation of numerous anti-tumor immune-related pathways, particularly cytokine-cytokine receptor interactions, chemokine signaling, cell adhesion molecules, and natural killer (NK) cell-mediated cytotoxicity. Notably, genes associated with cytokine-cytokine receptor interactions—including Ifng, Ccl10, Ccl14, Il15, and Il17 —were significantly upregulated. Within the NK cell-mediated cytotoxicity pathway, cytotoxic-related genes such as Fas, GZMB, Prf1, and CD244 also showed increased expression. These findings strongly suggest that GM-DC+HPV therapy induces a Th1-polarized anti-tumor immune response by activating key cytokines. However, further functional gene analysis revealed elevated expression of CD274 (PD-L1) in the GM-DC+HPV group, indicating persistent immunosuppressive signals within the tumor microenvironment (TME). Traditionally, cancer was attributed to the accumulation of oncogenic and tumor suppressor gene mutations. However, recent paradigm shifts have established the TME as a critical driver of tumor progression. The TME comprises diverse cellular components—lymphocytes, immune cells, cancer-associated fibroblasts, endothelial cells, pericytes, and tissue-resident cells—These host cells were once considered bystanders in tumorigenesis, but they are now known to play a critical role in cancer progression [36] . Tumor cells evade immune surveillance through multiple mechanisms: loss of tumor antigen expression or downregulation of MHC-I molecules, secretion of immunosuppressive cytokines ( IL-10, TGF- β ), expansion of regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs), activation of immune-inhibitory pathways, and overexpression of immune checkpoint molecules such as PD-1, BTLA , and CTLA-4 [37] . Overcoming TME-induced immune tolerance/suppression thus demands novel therapeutic strategies. Tumor cells frequently overexpress PD-L1 to suppress T-cell cytotoxicity and evade CTL-mediated killing [38-40] , making immune checkpoint inhibitors a focal point in cancer immunotherapy. FDA-approved CTLA-4 and PD-1/PD-L1 inhibitors have demonstrated clinical efficacy, while next-generation checkpoint inhibitors (IDO inhibitors) [41] are under clinical investigation. Rigo et al [42] . reported that combined anti-PD-L1/PD-1 and anti-CD4 immunotherapy achieved cure in syngeneic disseminated neuroblastoma. In non-small cell lung cancer, radiotherapy synergized with anti-PD-L1 antibodies to enhance antitumor responses [43] . Ren et al [44] . further demonstrated that imiquimod potentiates the therapeutic effect of exogenous BM-DC vaccines in murine melanoma.Massarelli et al [45] . reported that ISA101, an HPV-16 synthetic long peptide vaccine combined with nivolumab, a PD-1 immune checkpoint inhibitor, exhibited promising efficacy outcomes in patients with recurrent HPV-16–positive cancer. the statistical power was reduced to77.6%. with anORR of 33% and responses were durable with63%. Liuet al [46] . reported that Combinatorial therapy with ADEC205-SC-E7 vaccine and anti-PD-L1 blockade showed significantly enhanced therapeutic effects. Penget al [47] . reported that intratumoral injection of TA-CIN vaccine can induce a strong E7-specific CD8+T cell response both locally and systemically. When combining the intratumoral TA-CIN vaccine with PD-1 blockade, combination therapy has much stronger antitumor effects compared to either TA-CIN vaccination or anti-PD-1 antibody administration alone. We designed a combination therapy integrating GM-DC+HPV with anti-PD-L1 or anti-PD-1 antibodies for TC-1 tumor treatment. As we expected, GUPS-DC+HPV, mAbPD-1 and mAbPD-L1 significantly inhibited tumor growth and had no significant effect on the weight of mice, with no significant side effects on the mice. The survival rate of the GUPS-DC+HPV+mAbPD-1 group was 87.5% and produced a strong memory immune response. Consistently, we also observed that Compared with the control group, HPV-DC, mAbPD-1 alone and HPV-DC combined with mAbPD-1 significantly reduced the number of lung metastases. GUPS demonstrate significant potential in immunomodulation, antitumor activity, and chronic disease management. However, current clinical applications of GUPS are predominantly limited to adjuvant therapy, such as alleviating cancer-related fatigue and chemotherapy-induced side effects. Their potential as standalone therapeutic agents still requires validation through large-scale clinical trials. Future efforts must focus on advancing clinical translational research, overcoming technical bottlenecks, and facilitating its transition from laboratory research to clinical practice. As an adjuvant for human papillomavirus (HPV)-dendritic cell (DC) vaccines, GUPS enhances antigen-specific immune responses, promotes CD8 + T cell infiltration into tumor tissues, suppresses tumor growth, and improves survival rates in tumor-bearing mice. The combination of mAbPD-1 and GUPS-DC+HPV further enhanced anti-tumor efficacy and increased the survival rate of mice . These findings provide a critical research foundation for developing therapeutic vaccines and combination strategies, potentially benefiting patients with advanced, recurrent, or metastatic cervical cancer and offering robust data support for clinical investigations. Abbreviations BM, bone marrow; CTL , cytotoxic T lymphocytes; DC, dendritic cell; GUPS， Glycyrrhiza uralensis polysaccharides; HPV, human papillomavirus; LN, lymph node; Th cell, T helper cell. Declarations Conflict of Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Ethics Statement Approval of the research protocol by an Institutional Review Board: Ethics Committee-approved protocols (XJUAE-2023-030). Author Contributions PA: Writing–original draft, Conceptualization, Data curation, Formal Analysis, Methodology, Validation. SC: Writing–original draft, Data curation，Methodology, Validation. BC: Writing–review & editing, Conceptualization. NA: Writing–original draft, Conceptualization, Visualization. LH: Validation, Supervision, Funding acquisition, Conceptualization. AA: Writing–review & editing, Validation, Supervision, Funding acquisition, Conceptualization. Funding This work was supported by the Key research and development program in Xinjiang Uygur Autonomous Region (2022B03018-5) and the Natural Science Foundation of Xinjiang Uygur Autonomous Region (2022D01C97), Fudan University Ministry of Education / National Health Commission / Academy of Medical Sciences Key Laboratory of Molecular Virology Open Projects (FDMV-2024003) and the Jiayin Hospital Horizontal Project (No. 202306140004) . Data Availability Statement The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding authors. References Igarashi Y, Sasada T. Cancer Vaccines: Toward the Next Breakthrough in Cancer Immunotherapy[J]. J Immunol Res. 2020 Nov 17;2020:5825401. Riley RS, June CH, Langer R. Delivery technologies for cancer immunotherapy[J]. Nat Rev Drug Discov. 2019 Mar;18(3):175-196. Zhang Y, Zhang Z. 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Cancer Immunol Immunother. 2021 Apr;70(4):1049-1062. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 12 Feb, 2026 Reviews received at journal 10 Feb, 2026 Reviews received at journal 28 Jan, 2026 Reviews received at journal 21 Jan, 2026 Reviewers agreed at journal 20 Jan, 2026 Reviewers agreed at journal 20 Jan, 2026 Reviewers agreed at journal 20 Jan, 2026 Reviewers invited by journal 20 Jan, 2026 Editor assigned by journal 14 Jan, 2026 Submission checks completed at journal 25 Dec, 2025 First submitted to journal 17 Dec, 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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08:03:17","extension":"png","order_by":27,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":63869,"visible":true,"origin":"","legend":"","description":"","filename":"OnlinegraphicalAbstract.png","url":"https://assets-eu.researchsquare.com/files/rs-8390778/v1/fef2c6974dceea831009cd12.png"},{"id":101203105,"identity":"31a5b9fa-7684-406f-8011-1776a77641e5","added_by":"auto","created_at":"2026-01-27 09:38:45","extension":"xml","order_by":28,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":112642,"visible":true,"origin":"","legend":"","description":"","filename":"4eb130f49c86421dbc8b57d43e4490a91structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8390778/v1/02cd74d503914c21b2b9a0d2.xml"},{"id":101202711,"identity":"441054a2-d0fc-4843-8b1c-18afefec526d","added_by":"auto","created_at":"2026-01-27 09:37:19","extension":"html","order_by":29,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":121691,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8390778/v1/daae672f14dbe944568abe2a.html"},{"id":101942809,"identity":"c724f3bc-0b86-4520-8c3f-49f250327a21","added_by":"auto","created_at":"2026-02-05 09:38:29","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":95804,"visible":true,"origin":"","legend":"\u003cp\u003eTumor growth and tumor mouse survival.BM-DC were treated with GUPS for 12 h, then pulsed with HPV-16 E6/E7 peptides (GUPS-DC+HPV). Tumor mice were treated with GUPS-DC+HPV on day 3 and 10 after injection of TC-1 cells (A). Tumor growth curves reflected by the average tumor volume during the period of immunization (B). Tumors were isolated and weighted on day 38. The tumor photo and weight are shown in upper and lower panels, respectively. The p value was indicated (unpaired t-test) (C). H\u0026amp;E staining images of tumor tissues (D). The survival of tumor mice after GUPS-DC+HPV treatment. Tumor mice were treated with GUPS-DC+HPV on day 3 and 10, then the survival of tumor mice was monitored (E).\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8390778/v1/0f72f28b231456e4426738a2.jpg"},{"id":100958495,"identity":"1672078a-3c06-4958-8098-ccb175ec3255","added_by":"auto","created_at":"2026-01-23 08:03:16","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":88754,"visible":true,"origin":"","legend":"\u003cp\u003eThe immune responses in TC-1 tumor mice after treatment with GUPS-DC+HPV. On day 38, splenocytes were isolated to analyze the frequencies of MDSCs (A), iTregs and nTregs (B), CD8\u003csup\u003e+\u003c/sup\u003eT cells (C) and CD8\u003csup\u003e+\u003c/sup\u003eCD44\u003csup\u003e+\u003c/sup\u003e T cells (D). Splenocytes were stimulated with HPV-16 E6/E7 peptides overnight in the presence of Golgi stop. The frequency of antigen specific CD8\u003csup\u003e+\u003c/sup\u003eIFN-γ\u003csup\u003e+\u003c/sup\u003eT cells was analyzed by flow cytometry (E).(*\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 versus the control group).\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8390778/v1/73a65113b0c8320202ee6740.jpg"},{"id":101202938,"identity":"d6fdd934-5a76-489f-a053-d4be0ccec632","added_by":"auto","created_at":"2026-01-27 09:38:11","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":109455,"visible":true,"origin":"","legend":"\u003cp\u003eRNA-seq analysis of the expression profiles of differentially expressed genes in immune-related pathways. differential gene volcano plot, Compared with the Control group, a total of 2326 differential genes were detected, of which 1,810 were upadjusted genes and 516 were down-adjusted genes(A). Histogram of Go enrichment of all differential genes (B). The most significant 30 Term scatter plots with GO enrichment in GUPS-DC+HPV vs Control groups (C).\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8390778/v1/7a2fbf8cfe0a165d759a9fec.jpg"},{"id":101202733,"identity":"02883dd9-328f-48de-9b3a-707e690f60c7","added_by":"auto","created_at":"2026-01-27 09:37:24","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":80717,"visible":true,"origin":"","legend":"\u003cp\u003ePerform RNA sequencing analysis on the expression profiles of differentially expressed genes in immune-related pathways. Cluster analysis of the expression profiles of differentially expressed genes in the pathway related to natural killer cell-mediated cytotoxicity (A). Cluster analysis of the expression profiles of differentially expressed genes in the pathway related to cytokine-cytokine receptor interaction (B). \u0026nbsp;Immunohistochemical staining images of \u003cem\u003eBCL-2, TNF-α, GzmB and Fas \u003c/em\u003ein mouse tumor tissue array (magnification of 200) (4C).(*\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 versus the control group).\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8390778/v1/1bb2f39dbb13f287316a5ea8.jpg"},{"id":101202605,"identity":"c9c417cf-bcde-48ea-b66e-473ed8a39436","added_by":"auto","created_at":"2026-01-27 09:36:40","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":95874,"visible":true,"origin":"","legend":"\u003cp\u003eTreatment of tumor mice by GUPS-DC+HPV combined with mAbPD-1 or mAbPD-L1.The therapeutic strategy for mice (A) .Tumor mice were treated with GUPS-DC+HPV on day 10 and treated with mAbPD-1or mAbPD-L1 on days 10, 13, 16, 19, 22, then Tumor size and body weight was measured every other day,the survival of tumor mice was monitored.Tumor growth curves reflected by the average tumor volume during the period of immunization(B). The survival of tumor mice after GUPS-DC+HPV treatment (C). Tumor volumes were measured of the memory immune responses in mice (D). To evaluate the effect of GUPS-DC+ HPV combined with PD-1 antibody treatment in the advanced treatment of tumor mice. On the 10 th day after the establishment of TC-1 tumor model, On the 16th day, intradermal injection of GUPS-DC+HPV was used as the GUPS-DC+HPV group. mAbPD-1 (100 μg/ animal) were intraperitoneally injected into the mAbPD-1 group on the 16th, 19th, 22th, 25th and 28th days (E).Tumor growth curves reflected by the average tumor volume during the period of immunization (F). (*\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001 versus the control group).\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8390778/v1/f30c7bedfbb0c649a62f2178.jpg"},{"id":100958499,"identity":"45e76ba5-bdc3-4c4f-984b-02302fef6aa6","added_by":"auto","created_at":"2026-01-23 08:03:16","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":63253,"visible":true,"origin":"","legend":"\u003cp\u003eGUPS-DC+HPV combined with mAbPD-1 or mAbPD-L1 to treat lung metastatic tumor mice. Tumor mice were treated with GUPS-DC+HPV on day 3 and treated with mAbPD-1 or mAbPD-L1 on days 6, 9, 12, 15, 19, 21, then body weight was measured every other day(A). Pictures of cervical cancer, tail vein injection and metastasis to the lungs (B) .Statistics on the number of lung metastases (C). The immune responses in TC-1 tumor mice after treatment with GUPS-DC+HPV+mAbPD1, The frequencies of immune cells in spleens. The effect of GUPS-DC+HPV+mAbPD1 on Treg cells CD4\u003csup\u003e+\u003c/sup\u003eCD25\u003csup\u003e+\u003c/sup\u003eFoxp3\u003csup\u003e+\u003c/sup\u003e cells (D). Proportion of activated CD4\u003csup\u003e+\u003c/sup\u003eT cells(CD19b\u003csup\u003e-\u003c/sup\u003eCD3\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003eCD44\u003csup\u003e+\u003c/sup\u003e) and activated CD8\u003csup\u003e+\u003c/sup\u003eT cells (CD19b\u003csup\u003e-\u003c/sup\u003eCD3\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eCD44\u003csup\u003e+\u003c/sup\u003e). (*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 versus the control group).\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8390778/v1/0707af1f8841e2a785128bce.jpg"},{"id":102397161,"identity":"7781f050-9339-49e9-9ba3-644ac751cc1c","added_by":"auto","created_at":"2026-02-11 10:05:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1337501,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8390778/v1/4752848e-9b45-4990-a6e8-3405a6af3d66.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Glycyrrhiza uralensis polysaccharides as a DC-based Vaccine Adjuvant: Enhanced Immunotherapy Combined with PD-1 Blockade","fulltext":[{"header":"1. Introduction","content":"Cancer immunotherapy is currently recognized as a pillar of treatment alongside traditional modalities such as surgery, radiation, and chemotherapy, and it is becoming one of the most exciting and rapidly expanding fields[1-2]. Various forms of immunotherapy, including adoptive cell transfer (ACT), immune checkpoint inhibitors (ICIs), cancer vaccines, oncolytic virus therapy (OVT), have demonstrated significant efficacy and promise[3]. These methods primarily involve inducing CD8+ T cell responses to enhance anti-tumor immunity or restoring the function of exhausted T cells. The presence of anti-tumor cytotoxic T lymphocytes (CTLs) and their infiltration within the tumor microenvironment are prerequisites for treatment efficacy[4-6]. Thus, CD8+ T cells are needed to be primed or back toward effector CTLs for making durable and efficient antitumor immune responses[7-8]. At the forefront of the cancer-immunity cycle, CD8+ T cells priming is essentially a corroborative process involving antigen presenting cells (APCs), including dendritic cells (DCs)[9]. Major histocompatibility complex I (MHC-I) molecules on the surface of DCs are identified by CD8+T cells, which attach to it and scan the surface by crawling over it[10-12]. Furthermore, the upregulation of costimulatory molecules, such as CD40 and CD86, and the release of cytokines during DCs maturation are crucial for establishing stable and enduring interactions with T cells at the immunological synapse (IS). These interactions are vital for T cell expansion and differentiation into memory and effector T cells, thereby underpinning robust cellular immune responses[13-14]. \nGiven the central role of DCs in antitumor immunity, investigators have theorized that DC vaccine would serve as an ideal vaccine platform that can lead to the effective eradication of tumors. The safety and immunogenicity through vaccination potentially constitute a powerful anticancer strategy but clinical benefits have been largely disappointing[15]. New information on the contribution of DCs to tumor immunity should improve DC’s maturity in vitro, using adjuvants to optimize their performance in therapeutic applications. Currently approved adjuvants, such as alum and oil, deviate from the purpose for supporting tumor immunotherapy, because they mainly enhance humoral (Th2) immunity and are not without side effects[16].\nThe safety of DC vaccines has been fully demonstrated in numerous phase I and II clinical trials, with most DC vaccination regimens exhibiting minimal side effects [17-19]. Studies by Ralph Steinman and Michel Nussenzweig revealed that in vivo targeting of DCs through the conjugation of antigens to antibodies directed against DC surface receptors (such as DEC205 or DCIR) enables efficient antigen delivery and capture by DCs[20]. However, in the absence of adjuvants, targeting DEC205+ DCs in vivo can induce antigen-specific immune tolerance [21]. As observed in the original DEC205 studies, the presence or absence of adjuvants profoundly impacts immune responses. For instance, antigen delivery via CLEC9A-targeting antibodies without adjuvants triggers robust antibody responses associated with Tfh cell differentiation[22], while simultaneously inducing Treg differentiation[23]. To date, only a limited number of cytokines or TLR ligands have been successfully utilized as DC vaccine adjuvants in clinical applications, highlighting the need to identify novel, safe, and potent adjuvants to enhance DC maturation and improve vaccine immunogenicity and clinical efficacy. Consequently, adjuvants play a critical role in enabling DC vaccines to effectively activate T cell immune responses. In recent years, polysaccharides have emerged as promising adjuvant candidates due to their immunomodulatory activities and safety profiles[24-25]. Accumulating evidence demonstrates that polysaccharides can promote dendritic cell maturation, characterized by upregulation of co-stimulatory molecules and cytokines including IL-12[26]. Our previous studies reported the isolation and characterization of Glycyrrhiza polysaccharide (GUPS), which exhibits potent immunostimulatory activity in dendritic cells through TLR4 and downstream p38, JNK, and NF-κB signaling pathways [27-28].\nIn this study, we adopted GUPS as an adjuvant for human papillomavirus (HPV)-DC vaccine (GUPS-DC+HPV) and evaluated the antitumor response and efficacy of combination therapy with PD-1/PD-L1 inhibitors. The GUPS-DC+HPV effectively inhibited the growth of tumors, resulting in improved the initiation of CTLs, enhanced CD8+ T cell infiltration into tumor sites, and reduced production of regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs). The GUPS-DC+HPV also prolonged the survival rate of mice and suppressed the tumor metastasis. In combination with anti-PD-1, GUPS-DC+HPV further enhanced the antitumor efficacy. Throughout the therapy, GUPS elicited efficient adjuvant activity. \n"},{"header":"2. Materials and methods","content":"\u003ch2\u003e2.1 Materials\u003c/h2\u003e\n\u003cp\u003e\u003cem\u003eG.uralensis\u003c/em\u003e polysaccharides (GUPS) were obtained from the Xinjiang Key Laboratory of Biological Resources and Genetic Engineering, Xinjiang University (Urumqi, Xinjiang, China). The purified GUPS had a polysaccharide content of 93% and a molecular weight of 29.1 kDa.Fetal bovine serum (FBS) and Penicillin-streptomycin were purchased from MRC (Changzhou, China). Medium RPMI-1640 medium and phosphate-buffered saline (PBS) were purchased from Gibco (Grand Island, NY, USA). Trypsin was sourced from MRC (China). Granulocyte-macrophage colony-stimulating factor (GM-CSF) was purchased from PeproTech (Rocky Hill, NJ, USA). HPV-16 E6/E7 peptides, including E643\u0026ndash;57 (QLLRREVYDFAFRDL), E653\u0026ndash;62 (AFRDLCIVYR), E711\u0026ndash;20 (YMLDLQPETT), E744\u0026ndash;62 (QAEPDRAHYNIVTFCCKCD) and E781\u0026ndash;94 (DLLMGTLGIVCPIC), were synthesized by Shanghai Science Peptide Biological Technology (China). These peptides were dissolved in DMSO at 20 mg/mL, filtered through a 0.22 \u0026micro;m filter membrane, and stored at -80℃. Trizol was purchased from Thermo Fisher Scientific (USA), and reverse transcription reagents were obtained from TAKARA Biotechnology. The Cytofix/Cytoperm kit and Golgi stop (Monensin) were purchased from BD Biosciences (USA). 4% Formaldehyde Fixation Solution was sourced from Biosharp (China). Anti-mouse monoclonal antibodies against PD-1(BE0033-2) and PD-L1 (BE0033-2) were purchased from BioXCell (USA). The antibodies for flow cytometry, including anti-CD25-APC, anti-CD49b-FITC, anti-CD11b-PE, anti-Gr-1-ER780, anti-CD19-APC, anti-CD4-ER780, anti-CD44-PE, anti-Foxp3-PE, anti-CD4-FITC, anti-IFN-\u0026gamma;-APC, were purchased from Elabscience (Wuhan, China). Furthermore, anti-MHC-I-FITC, anti-CD3-APC700, anti-CD8-PE-CY7 and anti-MHC-II-PE, \u0026nbsp;were obtained from BD Biosciences.\u003c/p\u003e\n\u003ch2\u003e2.2 Animals and ethics statement\u003c/h2\u003e\n\u003cp\u003eFemale C57BL/6 mice (6-8 weeks old, weighing approximately 22 g) were acquired from the Beijing Laboratory Animal Research Center (Beijing, China) and housed at the temperature‑controlled (25˚C) and light‑cycled (12-hour light/12-hour dark) in Animal Facility of Xinjiang University (Urumqi, China). All animal experiments in this study were approved by the Ethics Committee on Animal Experiments at the Xinjiang Key Laboratory of Biological Resources and Genetic Engineering (approval number: BRGE-AE001) and were carried out in strict adherence to the guidelines set forth by the Animal Care and Use Committee of the College of Life Science and Technology, Xinjiang University.\u003c/p\u003e\n\u003ch2\u003e2.3 Cell line\u003c/h2\u003e\n\u003cp\u003eThe mouse TC-1 cell line was obtained from the Xinjiang Key Laboratory of Biological Resources and Genetic Engineering, Xinjiang University (Urumqi, Xinjiang, China). These cells were cultured in RPMI-1640 medium (Gibco), supplemented with 100 U/ml penicillin, 100 \u0026mu;g/ml streptomycin, and 10% heat-inactivated fetal bovine serum (Gibco). The cells were maintained at 37\u0026deg;C in a humidified incubator with a 5% CO2 atmosphere to ensure optimal growth conditions.\u003c/p\u003e\n\u003ch2\u003e2.4 Vaccine preparation\u003c/h2\u003e\n\u003cp\u003eDCs were collected and cultured for 7 days before being seeded into 60 mm cell culture dishes at a density of 1\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells/mL, and added GUPS (20 \u0026mu;g/mL) for treatment. After 12 h of incubation, the cells were collected by centrifugation at 1200 rpm for 7 min. Then, the cells were resuspended in fresh medium (1\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells/mL) and incubation with HPV-16 E6/E7 polypeptide at a final concentration of 10 \u0026mu;g/mL for 2 h, centrifuged (1200 r/min, 7min) to collect, and washed once with PBS. The DCs vaccine prepared by PBS (1\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells /50 \u0026mu;L) were named GUPS-DC+HPV, and the GUPS-treated DCs were not incubated with HPV-16 E6/E7 polypeptide (GUPS-DC) as a control.\u003c/p\u003e\n\u003ch2\u003e2.5 TC-1 Tumor model and treatment\u003c/h2\u003e\n\u003cp\u003eTC-1 cells, which stably express HPV-16 E6 and E7, were collected during log-phase growth and washed with PBS. TC-1 cells were re-suspended in PBS (1\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/mL) and 100 \u0026mu;L of this suspension were subcutaneously injected into the right flank of C57BL/6 mice. The tumor-bearing mice were randomly divided into three groups (6 mice/group) and intradermally injected with 50 \u0026mu;L PBS (control group) or immunized with 1\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells (GUPS-DC or GUPS-DC+HPV) in 50 \u0026mu;L PBS on day 3 and day 10. Tumor size were measured every other day and tumor volumes were calculated using the formula: tumor volume (mm3)=(length\u0026times;width\u003csup\u003e2\u003c/sup\u003e)/2. \u0026nbsp;At the same time, measuring the weight of the mice every other day. At the end of study, the tumors were isolated, weighted and processed for further analysis. The tumors were fixed, sliced and subjected to hematoxylin and eosin (H\u0026amp;E) staining to evaluate histological changes. Splenocytes were used to detect the immune responses by flow cytometry.\u003c/p\u003e\n\u003ch2\u003e2.6 Survival rate of TC-1 tumor mice\u003c/h2\u003e\n\u003cp\u003eOn the 3rd day after the successful establishment of the TC-1 tumor model, the mice were randomly divided into three groups(10 mice/group). On the 3 th and 10 th days, the mice in the treatment groups were given intradermal injection of GUPS-DC or GUPS-DC+HPV, and the untreated group served as the Control group to monitor the survival rate of the tumor mice.\u003c/p\u003e\n\u003ch2\u003e2.7 Flow cytometry\u003c/h2\u003e\n\u003cp\u003eTo characterize the phenotype characterization of DCs and T cells, flow cytometry was performed on the isolated splenocytes following staining with specific mAbs. For the analysis of Tregs, the cells were first surface staining with anti-CD4 and anti-CD25 mAbs. Subsequently the cells were fixed and permeabilized using a Foxp3 Staining Buffer Set (eBioscience) and stained with Foxp3 mAbs. To assess antigen-specific CD8\u003csup\u003e+\u003c/sup\u003eT cell responses, splenocytes (1\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells/mL) were stimulated with HPV-16 E6/E7 peptides and cultured overnight in the presence of Golgi stop (BD Biosciences). After stimulation, the cells were stained with an anti-CD8 mAbs to identify CD8\u003csup\u003e+\u003c/sup\u003eT cell. Following surface staining, the cells were fixed and permeabilized using Cytofix/CytopermTM (BD Biosciences) and then stained with IFN-\u0026gamma; mAbs to detect intracellular IFN-\u0026gamma;\u0026nbsp;production. All samples were collected on FACSCalibur (BD Biosciences) and analyzed using the FlowJo platform (Tree Star, Inc, Ashland, OR).\u003c/p\u003e\n\u003ch2\u003e2.8 Hematoxylin and eosin(H\u0026amp;E) staining\u003c/h2\u003e\n\u003cp\u003eMouse organs were fixed in 4% formaldehyde and then paraffin sections were performed. Sections were stained. Dip the slices into a coconut milk jar filled with mayerhematoxylin and stir for 30 s. Rinse the slide with water for 1 min. Dip the slices into 1% eosin Y solution and stir for 10-30 s. The section was dehydrated in 95% alcohol and 100% alcohol repeatedly, twice for each solution, 30 s each time. Ethanol is removed with two different xylene compounds. Add one to two drops of medium and cover with mulch. The stained sections were observed with an inverted microscope and photographed.\u003c/p\u003e\n\u003ch2\u003e2.9 RNA-Seq Analysis\u003c/h2\u003e\n\u003cp\u003eTumor tissues harvested on day 38 were homogenized in TRIzol reagent, and total RNA was extracted according to the manufacturer\u0026rsquo;s instructions. A Bioanalyzer (Agilent) was used to validate the quality and quantity of the purified RNA. Library construction for RNA-seq was performed using the TruSeq Stranded mRNA LT Sample Prep Kit (Illumina), following manufacturer\u0026rsquo;s instructions. The resulting library was then sequenced on a HISeq 2500 platform using the 100 bp single-end protocol (Illumina). Multiple mapped reads were segregated and distributed in a random manner using an in-house script. Reads mapping to protein-coding genes were counted using HTSeq with the GTF file, considering only the protein-coding feature. Differentially expressed genes were examined by DESeq. Genes of interest on pathways annotated in KEGG were selected if their expression levels were significantly different (adjusted \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05 and \u0026plusmn;2-fold change) between the GUPS-DC+HPV and control groups. Read counts were normalized by total number of mapped reads and fit to a standard normal distribution for the depiction of heatmaps.\u003c/p\u003e\n\u003ch2\u003e2.10 Immunohistochemistry\u003c/h2\u003e\n\u003cp\u003eFor immunohistochemistry, the tissue sections were deparaffinized, rehydrated, and subsequently incubated in citrate buffer antigen repair buffer (pH 8.0) and 3% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, respectively. After blocking with blocking solution for 1 h at room temperature, the sections were incubated for overnight at 4℃ in primary antibody. Following the washes, the sections were then incubated sequentially with the specific secondary antibodies and HRP-conjugated streptavidin. Finally, the sections were stained with 3,3\u0026rsquo;-diaminobenzidine (DAB) for color development and counterstained with haematoxylinthe. The results were observed and photographed under a fluorescence microscope.\u003c/p\u003e\n\u003ch2\u003e2.11 Combined GUPS-DC+HPV with PD-1/PD-L1 antibody therapy\u003c/h2\u003e\n\u003cp\u003eOn the 10th day after the successful establishment of the TC-1 tumor model, the mice were randomly divided into 6 groups(7 mice/group). The untreated group served as the Control group. On the 10th day, intradermal injection of GUPS-DC+HPV was used as the GUPS-DC+HPV group. The mAbPD-L1 (100 \u0026mu;g/mouse) or mAbPD-1 (100 \u0026mu;g/mouse) were intraperitoneally injected into the mAbPD-L1 group and mAbPD-1 group on the 10th, 13th, 16th, 19th and 22nd days, respectively. The combination of GUPS-DC+HPV with mAbPD-1or mAbPD-L1 was divided into the GUPS-DC+HPV+mAbPD-1 or GUPS-DC+HPV+mAbPD-L1 group, and the tumor size was measured every other day, \u0026nbsp;mouse survival rate and immune memory response.\u003c/p\u003e\n\u003cp\u003eTo further test the effect of late treatment, On the 10 th day after the establishment of the TC-1 tumor model, the mice were randomly divided into 4 groups\u0026nbsp;(6 mice/group). The untreated group served as the control group. Starting on the 16th day, intradermal injection of GUPS-DC+HPV was administered to the GUPS-DC+HPV group, with additional injections on the 25th day. The mAbPD-1 group received intraperitoneally injected of mAbPD-1 (100 \u0026mu;g/mouse) on the 16th, 19th, 22nd, 25th and 28th days, respectively. The mice receiving the combination of GUPS-DC+HPV with mAbPD-1 were designated as the GUPS-DC+HPV+mAbPD-1 group, and the tumor size in all groups was measured every other day，At the same time, measuring the weight of the mice every other day.\u003c/p\u003e\n\u003cp\u003eTo detect the effect of DC vaccine on tumor recurrences, on day 58, inoculation of TC-1 cells (1\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/mouse) from 3 mice in the GUPS-DC+HPV group and GUPS-DC+HPV+mAbPD-1 group. At the same time, na\u0026iuml;ve mice were inoculated 1\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells selected as control. The growth of the mouse tumors in the above three groups was examined.\u003c/p\u003e\n\u003ch2\u003e2.12 Lung metastasis models\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eC57BL/6 mice were injected intravenously with TC-1 cells (1\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells) on day 0 and were randomly divided into 4 groups(5 mice/group). The untreated group served as control group. On the 3 day, the GUPS-DC+HPV group received an intradermal injection of GUPS-DC+HPV alone. The mAbPD-1 group was intraperitoneally injected with mAbPD-1 (100 \u0026mu;g/mouse) on the 6th, 9th, 12th，15th, 18th, and 21st days, respectively. The mice receiving the combination of GUPS-DC+HPV with mAbPD-1 were designated as the GUPS-DC+HPV+mAbPD-1 group, and the tumor size was measured every other day. On day 21, the mice were euthanized (n=5) and the spleens and lungs were collected for further analysis. The number of pulmonary tumor metastases was then counted.\u003c/p\u003e\n\u003ch2\u003e2.13 Statistical analysis\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eData were reported as mean \u0026plusmn; standard error of the mean (SEM). One-way analysis of variance (ANOVA), paired or unpaired t-test were used to analyze the statistical significance by Prism5 GraphPad software (GraphPad Software, La Jolla, CA). A value of \u0026lt; 0.05 was considered to be statistically significant.\u003c/p\u003e"},{"header":"3. Results","content":"\u003ch2\u003e3.1 GUPS improved the antitumor efficacy of HPV-DC vaccine\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eThe adjuvant effect of GUPS on the HPV-DC vaccine was detected in TC-1 tumor mouse model. Tumor mice were treated with GUPS-DC+HPV on day 3 and 10 after injection of TC-1 cells (Fig.1A). As shown in Fig.1B\u0026amp;C, GUPS-DC+HPV markedly inhibited tumor growth compared with control and showed complete tumor regression in two mice. The H\u0026amp;E staining of tumors showed that the number of tumor cells was dramatically reduced and the necrotic areas were increased in GUPS-DC+HPV group compared with control group (Fig.1D).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe effect of GUPS-DC+HPV on survival rate of tumor mice was further evaluated in another tumor mouse study. Tumor mice were treated with GUPS-DC+HPV on day 3 and 10 after injection of TC-1 cells. Similarly, GUPS-DC+HPV also inhibited tumor growth compared with control (data not shown). On day 54, all mice in the control group had died, and 1 out of 10 mice survived in the GUPS-DC group. Five out of 10 mice were survival in GUPS-DC+HPV group, suggesting that GUPS-DC+HPV greatly improved the survival rate of tumor mice (Fig.1E).\u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFig. 1.\u0026nbsp;Tumor growth and tumor mouse survival.\u003c/p\u003e\n\u003ch2\u003e3.2 GUPS-DC+HPV induced the antigen-specific CD8\u003csup\u003e+\u003c/sup\u003eT cell immune responsese\u003c/h2\u003e\n\u003cp\u003eIn light of the excellent anti-tumor activity of the GUPS-DC+HPV, the correlation of antitumor effect and immune responses generated by GUPS-DC+HPV were carried out to explore the infiltration level of myeloid-derived suppressor cells (MDSCs, CD11b\u003csup\u003e+\u003c/sup\u003eGr-1\u003csup\u003e+\u003c/sup\u003e), regulatory T cells (iTregs, CD4\u003csup\u003e+\u003c/sup\u003eCD25\u003csup\u003e-\u003c/sup\u003eFoxp3\u003csup\u003e+\u003c/sup\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003eand nTregs, CD4\u003csup\u003e+\u003c/sup\u003eCD25\u003csup\u003e+\u003c/sup\u003eFoxp3\u003csup\u003e+\u003c/sup\u003e) and CD8\u003csup\u003e+\u003c/sup\u003eT (CD8\u003csup\u003e+\u003c/sup\u003eCD44\u003csup\u003e+\u003c/sup\u003e) cells. As expected,\u0026nbsp;Compared to control group, the frequencies of MDSCs and induced Tregs were significantly decreased (Fig.2A\u0026amp;B), and the frequencies of CD8\u003csup\u003e+\u003c/sup\u003e T cells and CD8\u003csup\u003e+\u003c/sup\u003eCD44\u003csup\u003e+\u003c/sup\u003e T cells were notably increased in GUPS-DC+HPV group (Fig. 2C\u0026amp;D). After HPV-16 E6/E7 peptides treatment, HPV-specific cellular responses were detected. As shown in Fig.2E, the frequencies of HPV-specific CD8\u003csup\u003e+\u003c/sup\u003eIFN-\u0026gamma;\u003csup\u003e+\u003c/sup\u003e T cells markedly increased in GUPS-DC+HPV group compared with control group. Moreover, GUPS-DC+HPV enhanced the infiltration of CD8\u003csup\u003e+\u003c/sup\u003eT cells in tumors. These results indicated that both the reduction of inhibitory cells and induction of antigen-specific CD8\u003csup\u003e+\u003c/sup\u003eT cell response contributed to suppressing tumor growth in GUPS-DC+HPV group. \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFig. 2. The immune responses in TC-1 tumor mice after treatment with GUPS-DC+HPV.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn order to elucidate the molecular mechanisms underlying the inhibition of tumor growth by GUPS-DC+HPV, we undertook a comprehensive transcriptomic analysis. Our findings revealed that 2326 differentially expressed genes were identified in the GUPS-DC+HPV group compared to the Control group, with 1810 genes upregulated and 516 genes downregulated (|log2 fold change| \u0026gt; 0, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001). Genes with elevated expression were depicted in red, while those with reduced expression were shown in blue (Fig. 3A). From the results of GO enrichment analysis, the most significant 10 Term plots are displayed, and the size and color of points correspond to the number of genes annotated to GO Term and the significance (Fig. 3B). Concomitantly, numerous pathways related to anti-tumor immunity were upregulated in the GUPS-DC+HPV (Fig. 3C). In particular, cytokine-cytokine receptor interaction, chemokine signaling, cell adhesion molecules and nature killer cell mediated cytotoxicity (Fig.3C).\u003c/p\u003e\n\u003cp\u003eFig.3. RNA-seq analysis of the expression profiles of differentially expressed genes in immune-related pathways.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn nature killer cell mediated cytotoxicity pathway，cytotoxicity associated genes，including\u003cem\u003e\u0026nbsp;Fas\u003c/em\u003e, \u003cem\u003eG\u003c/em\u003e\u003cem\u003ezmB\u003c/em\u003e, \u003cem\u003ePrf1\u003c/em\u003e, \u003cem\u003eCD244\u003c/em\u003e,\u003cem\u003e\u0026nbsp;\u003c/em\u003ewere significantly increased in response to GUPS-DC+HPV(Fig. 4A). Immunohistochemical data also showed GzmB and Fas upregulated in tumor tissue in the GUPS-DC+HPV treatment group. On the other hand, BCL-2 was decreased with the GUPS-DC+HPV therapy (Fig. 4C). In addition, numerous genes related to cytokine-cytokine receptor interaction were upregulated, such as \u003cem\u003eIfng, Ccl10, Ccl14, Il15, and Il17,\u003c/em\u003e were significantly increased in response to GUPS (Fig. 4B). The expression of TNF-\u0026alpha; was increased compared with the control group.\u0026nbsp;Taken together, these results strongly suggest that GUPS-DC+HPV therapy elicits Th1-type anti-tumor immunity with essential cytokine induction.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFig.4. Perform RNA sequencing analysis on the expression profiles of differentially expressed genes in immune-related pathways.\u003c/p\u003e\n\u003cp\u003eHowever, it was worth noting that the high expression levels of CD274 was observed, which might express on the surface of tumor cells and activated APCs (Fig. 4A). Next, we devised a combination therapy with GUPS-DC+HPV and anti-PD-L1 Ab or anti-PD-1 Ab against TC-1 tumors.\u003c/p\u003e\n\u003ch2\u003e3.3 GUPS-DC+HPV combined with anti-PD-1/PD-L1 Ab therapy further inhibited tumor growth and improved memory immune response and survival in tumor mice\u003c/h2\u003e\n\u003cp\u003eAlthough the mechanism of action of DC vaccine is clear, the tumor immune microenvironment and tumor immunosuppressive mechanisms greatly limit the effect of DC tumor vaccine. Therefore, we established a TC-1 tumor mouse model and evaluated the anti-tumor effect of GUPS-DC+HPV vaccine combined with PD-1 and PD-L1 blocking antibodies in the treatment of advanced tumor mice. The mice were given GUPS-DC+HPV vaccine on the 10 th day after inoculation of TC-1 cells, and the mice were given 100 \u0026mu;g of mAbPD-1 or mAbPD-L1 on the 10, 13, 16, 19 and 22 th days (Fig. 5A). Tumor size was measured every other day. At the same time, measuring the weight of the mice every other day. It is found that GUPS-DC+HPV, mAbPD-1 and mAbPD-L1 significantly inhibited tumor growth and had no significant effect on the weight of mice, with no significant side effects on the mice (Fig. 5B). When all the mice in the control group died, the survival rate of the GUPS-DC+HPV group was 75% (Fig. 5C). The survival rate of the GUPS-DC+HPV+mAbPD-1 group was 87.5% (Fig. 5C). The survival rate of the GUPS-DC+HPV+mAbPD-L1 group was only 50%. In the mAbPD-1 group, only one mouse survived, and in the mAbPD-L1 group, only two mice survived. Compared with the control group, GUPS-DC+HPV+mAbPD-1 significantly improved the survival rate (Fig. 5C). These results indicated that GUPS-DC+HPV combined with mAbPD-1 could significantly improve the probability of survivability in mice. On day 58, mice whose tumors had completely disappeared in the GUPS-DC+HPV group and the GUPS-DC+mAbPD-1 combination group were selected for the second inoculation with TC-1 cells. Naive mice of the same age were also inoculated with TC-1 cells as controls. The results showed that there were no tumors in GUPS-DC+HPV group and GUPS-DC+HPV+mAbPD-1 vaccine combination group, indicating that the vaccine and combination treatment induced memory immune response in mice (Fig.5D). The above results indicated that the combination of HPV-DC vaccine and PD-1 blocking antibody significantly inhibited the growth of TC-1 tumors, produced a strong memory immune response, improved the survival rate of mice.\u003c/p\u003e\n\u003cp\u003eAt present, HPV-DC vaccine research and development is becoming more mature. With the increases of HPV vaccine coverage, the incidence of a disease related to HPV is gradually reduced. However, in the late stage of cancer, advanced cancer cells have spread throughout the body, and conventional treatments were ineffective, so we established a late treatment model to evaluate its anti-tumor effect, 10 days after the establishment of TC-1 tumor model, patients were randomly divided into 4 groups, 5 cases in each group. The untreated group was used as control group. On day 16, GUPS-DC+HPV was injected intraderally as GUPS-DC+HPV group. The mAbPD-1 group received intraperitoneal injection of mAbPD-1 (100 \u0026mu;g/animal) at 16, 19, 22, 25, and 28 th days respectively. The GUPS-DC+HPV+mAbPD-1 combination treatment was divided into the GUPS-DC+ HPV+mAbPD-1 group, and tumor size was measured every other day (Fig. 5E). The results showed that HPV-DC+mAbPD-1 treatment induced durable, objective tumor regression and immune response in patients (Fig. 5F). The above results suggest that the combination therapy of HPV-DC and PD-1/PD-L1 is a safe, effective and feasible treatment strategy, which will bring new hope for patients with HPV infection.\u003c/p\u003e\n\u003cp\u003eFig.5. Treatment of tumor mice by GUPS-DC+HPV combined with mAbPD-1 or mAbPD-L1.\u003c/p\u003e\n\u003ch2\u003e3.4 The role of GUPS-DC+HPV combined with PD-1 antibody in the treatment of lung metastases\u003c/h2\u003e\n\u003cp\u003eThe lungs are the main site of metastatic spread. Due to their extensive vascularization and large surface area, which is essential for normal lung function, tumors have many opportunities to stop, extravasate, and colonize this organ. The poor prognosis of patients with recurrent and metastatic cervical cancer has always been a daunting challenge for clinicians. Thus, we developed a tumor lung metastasis model. The untreated group was used as control group. On the 3 th day, GUPS-DC+HPV was given intradermal injection alone was the GUPS-DC+HPV group. mAbPD-1 (100 \u0026mu;g/animal) were intraperitoneally injected into the mAbPD-1 group, on the 6, 9, 12，15, 18, and 21th days respectively (Fig. 6A). It was found that after the tumor cells were injected through the tail vein, they reached the lungs of the mice through the blood vessels and formed lung metastases. Compared with the control group, HPV-DC, mAbPD-1 alone and HPV-DC combined with mAbPD-1 significantly reduced the number of lung metastases, and the HPV-DC combined with mAbPD-1 had the best effect (Fig. 6B). MDSCs and Tregs contribute to tumor growth and the inhibition of antitumor immune responses\u003csup\u003e[2\u003c/sup\u003e\u003csup\u003e9-\u003c/sup\u003e\u003csup\u003e32]\u003c/sup\u003e. On day 21, the spleens were collected to detect the immune cells，and found that the GUPS-DC+HPV, mAbPD-1 and GUPS-DC+HPV+mAbPD-1 groups significantly reduced the proportion of Tregs cells and significantly increased the proportion of activated CD4+ and CD8\u003csup\u003e+\u003c/sup\u003eT cells compared with the control group(Fig. 6C). The combination of HPV-DC to PD-1 blocking antibodies provides an inhibitory environment for the tumor microenvironment, which regulate immunosuppression and activates immune cells. Therefore, the reduction of inhibitory cells and induction of CD8\u003csup\u003e+\u003c/sup\u003eT cell response together inhibited tumor growth in GUPS-DC+HPV+mAbPD-1 group, and proposing a new therapeutic strategy for the treatment of cervical cancer. \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFig.6. GUPS-DC+HPV combined with mAbPD-1 or mAbPD-L1 to treat lung metastatic tumor mice.\u0026nbsp;\u003c/p\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eAlthough a large number of clinical trials have proved that DC vaccine has the advantages of high safety and induced antigen-specific immune response, its clinical efficacy needs to be improved, especially the insufficient maturity of DC in vitro, resulting in the limited degree of immune response\u003csup\u003e[33-35]\u003c/sup\u003e. Therefore, this study used GUPS as an adjuvant of HPV-DC vaccine to improve the immunogenicity of the vaccine. Following vaccination of normal C57BL/6 mice and stimulating HPV-DC prepared from mature DC with GUPS, the vaccine significantly enhanced CD8\u003csup\u003e+\u003c/sup\u003eT cell activation and antigen-specific CTL response compared with the HPV vaccine prepared from immature DC, which not only inhibited tumor growth but also increased survival of tumor mice. Transcriptome data revealed that compared to the control group, the GM-DC+HPV group exhibited upregulation of numerous anti-tumor immune-related pathways, particularly cytokine-cytokine receptor interactions, chemokine signaling, cell adhesion molecules, and natural killer (NK) cell-mediated cytotoxicity. Notably, genes associated with cytokine-cytokine receptor interactions\u0026mdash;including \u003cem\u003eIfng, Ccl10, Ccl14, Il15,\u003c/em\u003e and \u003cem\u003eIl17\u003c/em\u003e\u0026mdash;were significantly upregulated. Within the NK cell-mediated cytotoxicity pathway, cytotoxic-related genes such as\u003cem\u003e\u0026nbsp;Fas, GZMB, Prf1, and CD244\u003c/em\u003e also showed increased expression. These findings strongly suggest that GM-DC+HPV therapy induces a Th1-polarized anti-tumor immune response by activating key cytokines. However, further functional gene analysis revealed elevated expression of CD274 (PD-L1) in the GM-DC+HPV group, indicating persistent immunosuppressive signals within the tumor microenvironment (TME).\u003c/p\u003e\n\u003cp\u003eTraditionally, cancer was attributed to the accumulation of oncogenic and tumor suppressor gene mutations. However, recent paradigm shifts have established the TME as a critical driver of tumor progression. The TME comprises diverse cellular components\u0026mdash;lymphocytes, immune cells, cancer-associated fibroblasts, endothelial cells, pericytes, and tissue-resident cells\u0026mdash;These host cells were once considered bystanders in tumorigenesis, but they are now known to play a critical role in cancer progression\u003csup\u003e[36]\u003c/sup\u003e. Tumor cells evade immune surveillance through multiple mechanisms: loss of tumor antigen expression or downregulation of MHC-I molecules, secretion of immunosuppressive cytokines (\u003cem\u003eIL-10, TGF-\u003c/em\u003e\u003cem\u003e\u0026beta;\u003c/em\u003e), expansion of regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs), activation of immune-inhibitory pathways, and overexpression of immune checkpoint molecules such as \u003cem\u003ePD-1, BTLA\u003c/em\u003e, and \u003cem\u003eCTLA-4\u003c/em\u003e\u003csup\u003e[37]\u003c/sup\u003e. Overcoming TME-induced immune tolerance/suppression thus demands novel therapeutic strategies. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTumor cells frequently overexpress PD-L1 to suppress T-cell cytotoxicity and evade CTL-mediated killing\u003csup\u003e[38-40]\u003c/sup\u003e, making immune checkpoint inhibitors a focal point in cancer immunotherapy. FDA-approved CTLA-4 and PD-1/PD-L1 inhibitors have demonstrated clinical efficacy, while next-generation checkpoint inhibitors (IDO inhibitors)\u003csup\u003e[41]\u003c/sup\u003e are under clinical investigation. Rigo et al\u003csup\u003e[42]\u003c/sup\u003e. reported that combined anti-PD-L1/PD-1 and anti-CD4 immunotherapy achieved cure in syngeneic disseminated neuroblastoma. In non-small cell lung cancer, radiotherapy synergized with anti-PD-L1 antibodies to enhance antitumor responses\u003csup\u003e[43]\u003c/sup\u003e. Ren et al\u003csup\u003e[44]\u003c/sup\u003e. further demonstrated that imiquimod potentiates the therapeutic effect of exogenous BM-DC vaccines in murine melanoma.Massarelli et al\u003csup\u003e[45]\u003c/sup\u003e. reported that ISA101, an HPV-16 synthetic long peptide vaccine combined with nivolumab, a PD-1 immune checkpoint inhibitor, exhibited promising efficacy outcomes in patients with recurrent HPV-16\u0026ndash;positive cancer. the statistical power was reduced to77.6%. with anORR of 33% and responses were durable with63%. Liuet al\u003csup\u003e[46]\u003c/sup\u003e. reported that Combinatorial therapy with ADEC205-SC-E7 vaccine and anti-PD-L1 blockade showed significantly enhanced therapeutic effects. Penget al\u003csup\u003e[47]\u003c/sup\u003e. reported that intratumoral injection of TA-CIN vaccine can induce a strong E7-specific CD8+T cell response both locally and systemically. When combining the intratumoral TA-CIN vaccine with PD-1 blockade, combination therapy has much stronger antitumor effects compared to either TA-CIN vaccination or anti-PD-1 antibody administration alone.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe designed a combination therapy integrating GM-DC+HPV with anti-PD-L1 or anti-PD-1 antibodies for TC-1 tumor treatment. As we expected, GUPS-DC+HPV, mAbPD-1 and mAbPD-L1 significantly inhibited tumor growth and had no significant effect on the weight of mice, with no significant side effects on the mice. The survival rate of the GUPS-DC+HPV+mAbPD-1 group was 87.5% and produced a strong memory immune response. Consistently, we also observed that Compared with the control group, HPV-DC, mAbPD-1 alone and HPV-DC combined with mAbPD-1 significantly reduced the number of lung metastases.\u003c/p\u003e\n\u003cp\u003eGUPS demonstrate significant potential in immunomodulation, antitumor activity, and chronic disease management. However, current clinical applications of GUPS are predominantly limited to adjuvant therapy, such as alleviating cancer-related fatigue and chemotherapy-induced side effects. Their potential as standalone therapeutic agents still requires validation through large-scale clinical trials. Future efforts must focus on advancing clinical translational research, overcoming technical bottlenecks, and facilitating its transition from laboratory research to clinical practice. \u0026nbsp;As an adjuvant for human papillomavirus (HPV)-dendritic cell (DC) vaccines, GUPS enhances antigen-specific immune responses, promotes CD8\u003csup\u003e+\u003c/sup\u003eT cell infiltration into tumor tissues, suppresses tumor growth, and improves survival rates in tumor-bearing mice. The combination of mAbPD-1 and GUPS-DC+HPV further enhanced anti-tumor efficacy and increased the survival rate of mice . These findings provide a critical research foundation for developing therapeutic vaccines and combination strategies, potentially benefiting patients with advanced, recurrent, or metastatic cervical cancer and offering robust data support for clinical investigations. \u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eBM, bone marrow; CTL\u003cem\u003e,\u0026nbsp;\u003c/em\u003ecytotoxic T lymphocytes; DC, dendritic cell;\u0026nbsp;GUPS，\u003cem\u003eGlycyrrhiza uralensis polysaccharides;\u0026nbsp;\u003c/em\u003eHPV, human papillomavirus; LN, lymph node; Th cell, T helper cell.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003e\u0026nbsp;Conflict of Interest\u003c/h2\u003e\n\u003cp\u003eThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\u003c/p\u003e\n\u003ch2\u003eEthics Statement\u003c/h2\u003e\n\u003cp\u003eApproval of the research protocol by an Institutional Review Board: Ethics Committee-approved protocols (XJUAE-2023-030).\u003c/p\u003e\n\u003ch2\u003eAuthor Contributions\u003c/h2\u003e\n\u003cp\u003ePA: Writing\u0026ndash;original draft, Conceptualization, Data curation, Formal Analysis, Methodology, Validation. SC: Writing\u0026ndash;original draft, Data curation，Methodology, Validation. BC: Writing\u0026ndash;review \u0026amp; editing, Conceptualization. NA: Writing\u0026ndash;original draft, Conceptualization, Visualization. LH: Validation, Supervision, Funding acquisition, Conceptualization. AA: Writing\u0026ndash;review \u0026amp; editing, Validation, Supervision, Funding acquisition, Conceptualization.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThis work was supported by the\u0026nbsp;Key research and development program in Xinjiang Uygur Autonomous Region (2022B03018-5) and the Natural Science Foundation of Xinjiang Uygur Autonomous Region (2022D01C97), Fudan University Ministry of Education / National Health Commission / Academy of Medical Sciences Key Laboratory of Molecular Virology Open Projects (FDMV-2024003) and the Jiayin Hospital Horizontal Project (No. 202306140004) .\u003c/p\u003e\n\u003ch2\u003eData Availability Statement\u003c/h2\u003e\n\u003cp\u003eThe original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding authors.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eIgarashi Y, Sasada T. 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Cancer Immunol Immunother. 2021 Apr;70(4):1049-1062.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"npj-vaccines","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjvaccines","sideBox":"Learn more about [npj Vaccines](http://www.nature.com/npjvaccines/)","snPcode":"41541","submissionUrl":"https://submission.springernature.com/new-submission/41541/3?","title":"npj Vaccines","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-8390778/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8390778/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe activation of naive CD8\u003csup\u003e+\u003c/sup\u003eT cells by mature dendritic cells (DCs) through antigen presentation is essential for initiating effective anti-tumor immunity. In our previous study, Glycyrrhiza uralensis polysaccharides (GUPS) were prepared and found that they significantly promoted the maturation and cytokine secretion of human Mo-DC and murine BM-DC through TLR4 signaling pathways. In this study, HPV-DC vaccine prepared with GUPS (GUPS-DC\u0026thinsp;+\u0026thinsp;HPV) significantly suppressed the tumor growth and improved the survival of tumor mice, which was correlated with the induction of HPV-16-specific cellular response and the increased infiltration of CD8\u003csup\u003e+\u003c/sup\u003eT cells in tumors. Although HPV-DC vaccine can induce CTL response and inhibit tumor growth, the late treatment effect needs to be further improved. Therefore, the combination treatment of HPV-DC vaccine and anti-PD-1 Ab could further improve the therapeutic efficacy, improve the survival rate of tumor mice, generated memory immune response and suppressed lung metastasis of tumors. These findings suggest GUPS as a potent adjuvant that promotes DC maturation, and offering a combination strategy for the treatment of advanced tumors.\u003c/p\u003e","manuscriptTitle":"Glycyrrhiza uralensis polysaccharides as a DC-based Vaccine Adjuvant: Enhanced Immunotherapy Combined with PD-1 Blockade","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-23 08:03:11","doi":"10.21203/rs.3.rs-8390778/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-02-12T16:30:11+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-10T15:37:29+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-28T08:39:53+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-21T09:32:13+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"223347962275508233452266738924098435500","date":"2026-01-21T01:15:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"31506542802278681953854273105239034384","date":"2026-01-20T21:23:28+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"298289062507283277272554825021180416396","date":"2026-01-20T11:34:00+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-20T11:26:14+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-14T15:17:40+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-25T05:45:28+00:00","index":"","fulltext":""},{"type":"submitted","content":"npj Vaccines","date":"2025-12-18T03:58:51+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"npj-vaccines","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjvaccines","sideBox":"Learn more about [npj Vaccines](http://www.nature.com/npjvaccines/)","snPcode":"41541","submissionUrl":"https://submission.springernature.com/new-submission/41541/3?","title":"npj Vaccines","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"97b0d803-68ae-4272-b2be-c4e7c43c1549","owner":[],"postedDate":"January 23rd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":61495390,"name":"Biological sciences/Cancer"},{"id":61495391,"name":"Biological sciences/Immunology"}],"tags":[],"updatedAt":"2026-05-13T05:08:14+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-23 08:03:11","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8390778","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8390778","identity":"rs-8390778","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}