Interleukin 35 promotes progression of hepatocellular carcinoma by recruiting neutrophils

Interleukin 35 promotes progression of hepatocellular carcinoma by recruiting neutrophils | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Interleukin 35 promotes progression of hepatocellular carcinoma by recruiting neutrophils WEI GAN, Guoqiang Sun, JinLong Huang, BaoYe Sun, ZhuTao Wang, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3856191/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background A growing number of therapeutic strategies against hepatocellular carcinoma (HCC) have emerged. However, their efficacy remains limited. This study investigated the mechanism of interleukin-35 (IL-35) in the progression of HCC and its potential application in HCC treatment. Methods The expression of IL-35,Gp130 ,IL12-Rβ2, CCL3,etc. in HCC tissues was detected by immunohistochemistry(IHC), and the expression of IL-35 in HCC cell lines was detected by fluorescence assay. Kaplan-Meier survival analysis of IL-35 and its receptor in relation to overall survival(OS) and recurrence free survival(RFS) in patients with HCC. The mouse subcutaneous tumor models to study the effects of IL-35 on HCC growth and immune cells. Western blot were used to detect the expression IL-35, CCL3, FGF2, and flow cytometric plot were performed to explore the immune cells infiltration in the tumor tissue. Results High expression of IL-35 in patients with HCC was associated with poor prognosis. Furthermore, IL-35 could facilitate tumor progression by affecting neutrophil infiltration, angiogenesis, and CD8 + T-cell infiltration. Additionally, CCL3 was a key factor mediating the recruitment of neutrophils by IL-35. FGF2 derived from neutrophils stimulated by IL-35 promoted intratumoral angiogenesis. IL-35 also facilitated the adhesion of tumors to endothelial cells, with neutrophils further enhancing this effect both. Anti-IL-35 antibody combined with anti-PD1 antibody significantly enhanced which therapeutic effect in HCC. Conclusion Our data show that the high expression of IL-35 in patients with HCC is an important tumor promoter. Combined treatment with anti-IL-35 and anti-PD1 antibodies have potential therapeutic effect against HCC. hepatocellular carcinoma interleukin 35 immunotherapy neutrophil anti-PD1 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Highlights High expression of IL-35 in patients with HCC is associated with poor prognosis IL-35 affects neutrophil infiltration, angiogenesis, and CD8 + T-cell infiltration CCL3 is a key factor mediating the recruitment of neutrophils by IL-35 IL-35 stimulates FGF2 secretion to promote intratumoral angiogenesis Anti-IL-35 antibody can be an effective treatment against HCC IL-35 facilitates the adhesion of tumors to endothelial cells, with neutrophils further enhancing this effect both in vitro and in vivo 1. Introduction Hepatocellular carcinoma (HCC), the most common type of primary liver cancer, is the fifth most common malignant tumor and second in mortality in China, based on 2020 data [ 1 , 2 ]. In recent years, treatments such as surgery, radiofrequency or microwave ablation of liver cancers have made great significant progress, leading to improved therapeutic outcomes. However, the rate of tumor recurrence and metastasis within 5 years post-operation remains high, at 60 to 70%[ 3 , 4 ]. The administration of Sorafenib, Lenvatinib, and Regorafenib has only modestly extended overall survival in patients with advanced HCC, by approximately 2.8 months [ 5 – 7 ]. Currently, immunotherapy, including programmed cell death protein 1 (PD1) and programmed cell death protein ligand 1 (PD-L1) antibodies, along with other immune checkpoint antibodies, has gained significant interest. Nevertheless, the efficacy of single-agent therapy in advanced HCC is limited, with success rates of only about 15 to 20%, highlighting the need for more effective treatment strategies[ 8 – 11 ]. Evidence suggests that combining immune checkpoint inhibitors(ICIs) with antiangiogenic antibodies can be effective in treating liver cancer, yielding potentially revolutionary results [ 12 ]. Consequently, our research focuses on exploring more effective combined treatments for HCC. Anti-angiogenic therapy, in combination with ICIs, exert anti-HCC effects by remodeling the tumor microenvironment (TME)[ 13 , 14 ]. By studying the TME of HCC, we can propose new therapeutic approaches for patients. Neutrophils, a significant component of the TME, exhibit both anti-tumor (TAN1) and pro-tumor (TAN2) phenotypes due to their diversity and plasticity[ 15 ]. In various cancers, neutrophils typically play a pro-tumorigenic role in processes such as tumorigenesis, tumor proliferation, metastasis, angiogenesis, neutrophils extracellular traps(NETs)and immunosuppression[ 16 ]. Neutrophil-secreted factors enhance tumor growth [ 17 , 18 ], while cytokines from tumor cells recruit neutrophils, creating a pro-tumor feedback loop [ 19 , 20 ]. In the TME, interleukins play a crucial role in recruiting neutrophils for tumor growth. For instance, interleukin 8(IL-8) recruits neutrophils in colorectal and thyroid cancers, altering the TME and promoting tumor growth[ 21 , 22 ]. Interleukin 6(IL-6) regulates neutrophil recruitment in breast cancer, and interleukin 1β(IL-1β) induces neutrophil extracellular traps (NETs) formation, facilitating lung metastasis in breast cancer [ 23 ]. However, the specific roles of interleukins and neutrophils in the TME of HCC remain to be fully elucidated. Our previous research has showed that interleukin 35(IL-35) expression correlates with HCC aggressiveness and is an independent prognostic factor for recurrence[ 24 ]. IL-35, a novel cytokine of the IL-12 family comprising P35 and EBI3 subunits, is primarily expressed by Treg and Breg cells [ 25 , 26 ]. In HCC, increased IL-35 expression in CCR4 + Tregs directly impairs CD8 + T-cell function[ 27 ], and also indirectly reduces antitumor IL-9 secretion by Th9 cells [ 28 ]. Other studies have indicated that in mouse of HCC and melanoma, IL-35 promotes the conversion of neutrophils to TAN2 phenotype and their infiltration into the TME, enhancing their pro-angiogenic and immunosuppressive functions[ 29 ]. However, the impact of IL-35 and neutrophils on tumor growth and immune response in human HCC is less understood. Our study reveals that IL-35, through autocrine secretion in HCC cells, affects the production of CCL3, which recruits neutrophils to the TME and promotes tumor angiogenesis and metastasis. Furthermore, IL-35 directly impedes CD8 + T cells infiltration and reshapes the immune environment. Thus, a combined treatment targeting IL-35 and PD1 antibodies holds promise as a potential therapeutic approach against HCC. 2. Materials and methods Detailed methods are presented in the Supplementary Material. 2.1. Human samples, and collection of patient clinical data A total of 360 patients with primary HCC who underwent radical resection at XX Hospital affiliated to XX University in 2012 were selected for this study. The obtained samples were confirmed as HCC based on pathological assessment. Clinicopathological data, such as liver function, alpha-fetoprotein (AFP), carbohydrate antigen 19 − 9 (CA19-9), carcinoembryonic antigen (CEA), tumor size, number and differentiation, lymph node metastasis, vascular invasion, cancer thrombus, and microvascular invasion (MVI) were collected from the medical history and pathological reports of enrolled patients. Our research has been registered at the web of research registry and the unique registration ID was researchregistryXXX. 2.2. Statistical analysis Statistical analyses were conducted using the IBM SPSS and GraphPad Prism. Each experiment was performed in triplicate, and values are presented as the mean ± SD, unless otherwise stated. The variance between the groups was statistically compared. The distributions of both the overall survival (OS) and recurrence-free survival (RFS) were depicted using the Kaplan–Meier method and analyzed by the log-rank test. Univariate and multivariate analyses for prognostic factors were based on the Cox proportional hazard model. P < 0.05 was considered significant. 3. Results 3.1. High expression of IL-35 in HCC is an independent risk factor for prognosis. Our study included 202 males (56.1%) and 158 females (43.9%), with an average age of 54.36 ± 11.038 (26–85) years. The average OS time was 51.87 ± 0.996 months, and the median OS was 49 ± 7.19 months. The average RFS time was 41.91 ± 1.27 months, and the median RFS was 22.0 ± 1.837 months (Table 1 ). Table 1 The correlation between clinicopathologic characters and IL35 characters Expression of IL-35 Low High P value Total patients 210 150 Gender, male/femeal 113/97 89/61 0.298 Age, < 60/≥60 133/77 107/43 0.112 HBsAg, negative/positive 24/186 22/128 0.364 AFP,<400/≥400 ng/mL 116/94 51/99 < 0.001 TBIL,<20/≥20 µmol/L 179/31 122/28 0.324 AST,<45/≥45 U/L 127/83 88/62 0.73 ALT,<50/≥50 U/L 171/39 116/34 0.341 ALB,<35/≥35g/L 15/195 5/145 0.12 cirrhosis,no/yes 55/155 29/121 0.129 tumor number,single/multiple 177/33 115/35 0.069 tumor thrombus,no/yes 204/6 110/40 < 0.001 tumor capsule,no/yes 59/151 49/101 0.351 tumor size,<5/≥5cm 138/72 80/70 0.018 LNM,no/yes 205/5 145/5 0.588 MVI, no/yes 189/21 17/133 < 0.001 differentiation, I-II/III-IV 140/70 75/75 0.001 Counts of CD34 + cells(low/high) 158/52 92/58 0.005 Counts of Treg cells(low/high) 134/76 90/60 0.462 Counts of CD8 + T cells(low/high) 120/90 112/38 0.001 Counts of CD4 + T cells(low/high) 181/29 130/20 0.897 Counts of CD66b + cells(low/high) 152/58 66/84 < 0.001 Counts of CD68 + cells(low/high) 138/72 89/61 0.216 BCLC stage(A/B/C) 132/72/6 64/46/40 < 0.001 APF, alpha fetal protein; LNM, lymph node metastasis; MVI, microvascular invasion;BCLC stage, Barcelona Clinic Liver Cancer staging system The P35 and EBI3 proteins widely expressed in tumor and stromal cells. Because of the lack of high-quality IL-35-specific antibodies, we examined the expression of P35 and EBI3 in serial tissue sections of HCC samples to determine the level of IL-35 expression, which has been reported as a reliable method in other studies [ 30 , 31 ]. As EBI3 forms IL-27 with P28 and P35 forms IL-12 with P40, we also examined the expression levels of P28 and P40 in consecutive tumor tissue. The expression of P35 and EBI3 was lower in adjacent normal tissue compared with tumor tissue of the same patient, we found that the expression level of both P28 and P40 in HCC was very low, and primarily located in stromal cells(Fig. 1 A-B). In addition, we found that P35 and EBI3 were mainly expressed in the cytoplasm rather than in the nucleus. The expression intensity of P35 and EBI3 was similar with overlapping spatial expression sites. Both proteins were expressed with higher intensity in HCCLM3 and MHCC97H and with lower intensity in SMMC-7721 and HUH7 (Fig. 1 D). To eliminate the effect of IL-12 and IL-27 co-subunits on IL-35 detection, we investigated the structural relationship of the four subunits in four cases of HCC using the CO-IP experiments. The P35 antibody immunoprecipitated EBI3 but rarely bound P40 and P28. Similarly, the same phenomenon was observed in CO-IP experiments in MHCC97H and HCCLM3 cell lines (Fig. S1 B). Our results identified IL-35 as the factor that is highly expressed in HCC, rather than IL-12 or IL-27. The high expression of both P35 and EBI3 was defined as the IL-35 high expression group (41.6%), whereas others were classified as the IL-35 low expression group (58.4%) (Fig. 1 C). The IL-35 high expression group had worse OS and RFS compared to the low expression group(Fig. 1 C). Significant correlations were found between the high expression of IL-35 and advanced BCLC stage. Moreover, we also found that the level of serum AFP was significantly increased in the IL-35 overexpression group (637.45 ± 32.8 vs 212.47 ± 18.9 ng/mL, P < 0.05). In addition, we noted that overexpression of IL-35 was closely related to an increased prevalence of portal vein tumor thrombus (PVTT), MVI, and large tumor size (P < 0.001). Multivariate analysis showed that overexpression of IL-35 was an independent risk factor for both OS (HR = 1.947, 95% CI, 1.046–3.624, P = 0.035) and RFS (HR = 2.442, 95% CI, 1.459–4.088) (Table 2 ). Therefore, the expression of IL-35 in HCC was considered an important reference index for prognosis. Table 2 The univariate and multivariate analysis for OS and RFS in the cohort OS RFS characters univariate P value multivariate Pvalue HR (95% CI) univariate P value multivariate P value HR (95% CI) Gender, male/femeal 0.559 NA NA 0.568 NA NA Age, < 60/≥60 0.266 NA NA 0.237 NA NA HBsAg, negative/positive 0.598 NA NA 0.394 NA NA TBIL,<20/≥20 µmol/L 0.528 NA NA 0.921 NA NA ALT,<50/≥50 U/L 0.716 NA NA 0.440 NA NA ALB,<35/≥35g/L 0.052 NA NA 0.075 NA NA AFP,<400/≥400 ng/mL 0.021 NS NS 0.001 NS NS AST,<45/≥45 U/L 0.094 NA NA 0.004 0.007 1.565(1.131–2.166) tumor size,<5/≥5cm <0.001 0.012 1.634(1.113–2.399) <0.001 NS NS tumor number, single/multiple <0.001 0.016 1.643(1.097–2.460) <0.001 NS NS tumor capsule,no/yes 0.158 NA NA 0.029 NS NS MVI, no/yes <0.001 NS NS <0.001 NS NS tumor throbus,no/yes <0.001 0004 2.073(1.262–3.403) <0.001 <0.001 2.507(1.610–3.906) differentiation, I-II/III-IV 0.015 NS NS 0.041 NS NS LNM,no/yes 0.023 NS NS 0.059 NA NA cirrhosis,no/yes 0.071 NA NA 0.010 NS NS Expression of IL-35 <0.001 0.035 1.947(1.046–3.624) <0.001 0.001 2.442(1.459–4.088) Counts of CD34 + cells (low/high) <0.001 <0.001 3.919(2.589–5.932) <0.001 <0.001 2.329(1.658–3.272) Counts of Treg cells (low/high) 0.972 NA NA 0.974 NA NA Counts of CD8 + T cells (low/high) 0.053 NA NA 0.027 NS NS Counts of CD4 + T cells (low/high) 0.770 NA NA 0.533 NA NA Counts of CD66b + cells (low/high) <0.001 0.022 1.625(1.074–2.460) <0.001 0.037 1.420(1.022–1.974) Counts of CD68 + cells (low/high) <0.001 NS NS <0.001 NS NS BCLC stage (A/B/C) <0.001 <0.001 2.205(1.709–2.844) <0.001 <0.001 1.911(1.539–2.374) Abbreviations: APF,alpha fetal protein;TBIL,total bilirubin;ALB,albumin; BCLC,Barcelona Clinic Liver Cancer staging system;NA, not applicable; NS, not significant It is of great significance to explore the expression of the GP130 and IL-12Rβ2 receptors of IL-35 in HCC [ 30 , 31 ]. We found that the expression of GP130 and IL-12Rβ2 in HCC was closely related (r = 0.39, P = 0.023, Fig. 2 B). Based on the expression of IL-35 and its receptors in HCC, we divided patients into 4 groups: IL-35R (+) IL-35 (high), IL-35R (+) IL-35 (low), IL-35R (-) IL-35 (high), and IL-35R (-) IL-35 (low). We found that patients in the IL-35R (+) IL-35 group (high) had the worst prognosis (P < 0.001), thus supporting the hypothesis that IL-35 facilitated the progression of HCC by directly acting on tumor cells in an autocrine or paracrine manner (Fig. 2 C). 3.2. High expression of IL-35 in patients with HCC is closely related to the infiltration of neutrophils and CD8 + T cells in tumor microenvironment. Our results showed that the expression of neutrophils marker CD66b in HCC tissues with high levels of IL-35 was significantly higher than that in the low expression group. In addition, we found that the number of infiltrated CD8 + T cells in tissues with high levels of IL-35 was significantly decreased (34.55 ± 2.758 vs. 56.61 ± 3.53, P < 0.001) by IHC experiment. Importantly, the number of microvessel density (MVD) labeling by CD34 in patients with overexpression of IL-35 was significantly increased (86.63 ± 4.789 vs. 56.54 ± 2.308, P < 0.001). The number of neutrophils infiltrating the tumor was positively correlated with MVD (r = 0.301, P < 0.001), suggesting that neutrophil infiltration might be an important factor in tumor angiogenesis (Fig. 2 A,C). 3.3. IL-35 affects HCC progression, angiogenesis, and adhesion via neutrophils and is negatively correlated with CD8 + T cell infiltration in vivo model. Based on the expression of IL-35 in HCC cell lines, we selected the HCCLM3 and MHCC97H cell lines, which exhibited a high expression level to construct IL-35 knocked-down cell lines. Whereas the HUH7 and SMMC-7721 cell lines, which showed a low expression level of IL-35 were selected to construct IL-35 overexpression cell lines. We also constructed IL-35 overexpression and knocked-down Hepa1-6 cells. (Fig. S1 A). First, we used the CCK8, transwell and wound healing to investigate the effect of IL-35 on HCC cells. Our results showed that IL-35 had no significant direct effects on the proliferation and migration of HCC cells in vitro . Surprisingly, we found that the formation rate of the subcutaneous tumor in the IL-35 overexpressing cell line in nude mice was significantly faster than that in the control group (volume mm 3 : 1186.81 ± 83. 53 vs. 612.82 ± 73. 49, P < 0. 001, tumor mass g: 1.17 ± 0.11 vs. 0.63 ± 0.09, P < 0.001), with the tumor growth rate being significantly slowed down in the knocked-down group (volume mm 3 : 495.48 ± 53.42 vs. 882.61 ± 73.25, P < 0.001, tumor mass g: 0.62 ± 0.07 vs. 1.11 ± 0.18, P < 0.001, Fig. 3 A). We also observed the same phenomenon in the immunocompetent C57BL/6 mouse model when exploring the effect of different expression levels of IL-35 on the growth of subcutaneous tumors (Fig. 4 A). Meanwhile, we found that the infiltration of neutrophils, as well as the number of MVD were both significantly increased in immunodeficient or immunocompetent mouse models injected with tumor cells overexpressing IL-35 (Fig. 3 B and 4 B,D). In the IL-35 knocked-down group, the number of neutrophils infiltrating the tumor, and the amount of MVD were shown to be significantly decreased (Fig. 3 B and 4 D). Thus, we assumed that IL-35 could promote intratumoral neovascularization by recruiting the infiltration of neutrophils in tumors. In addition, the number of infiltrating CD8 + T-cells was significantly decreased in the IL-35 overexpression group, whereas it was significantly increased in the knocked-down group (Fig. 4 C). We also found that IL-35 could enhance the adhesion of HCC cells to HUVECs in vitro. Following the overexpression of IL-35, the adhesion rate of SMMC-7721 or HUH7 cells to endothelial cells was significantly increased. In contrast, knocking down IL-35 strongly inhibited the number of HCCLM3 or MHCC97H cells adhered to endothelial cells. Meanwhile, we found that adding neutrophils to IL-35 overexpression group enhanced the amount of HUH7 or SMMC-7721 cells attached to endothelium cells by 46% and 54%, respectively (P 0.05) (Fig. S2 A). These results showed that IL-35 could increase the adhesion of HCC cells to endothelial cells, and neutrophils could further enhance this effect. Besides, we further verified this finding using the Hepa1-6 mouse HCC cell line (Fig. S3). 3.4. IL-35 promotes lung metastasis of HCC in the presence of neutrophils in vivo model. Next, we explored the role of IL-35 in tumor adhesion and lung metastasis in mouse model. Fluorescent dye-labeled HUH7 cells were injected into the body through the tail vein of nude mice, and fluorescent tracers were used to observe the remaining tumor cells in the lung tissue. We found no significant difference in the amount of tumor cells retained in the groups at 30 min. However, we observed a significant difference in the number of tumor cells retained in the lung tissue after 24h.The number of retained tumor cells in the NE + HUH7-OE group (279 ± 53) was significantly higher than that in the HUH7-OE group (103 ± 31) and the HUH7-OE-Ctrl group (62 ± 21) (P < 0.001), while the number of retained tumor cells in the HUH7-OE group was also significantly higher than that in the HUH7-OE-Ctrl group. We observed that in the NE + HUH7-OE group, many tumor cells adhered directly to neutrophils. We collected gross specimens of lung tissue and found that the number of metastatic nodules after HUH7-OE injection was significantly higher than that in the HUH7-OE-Ctrl group (Figure S2 B). The number of lung metastatic nodules further increased after simultaneous injection of a mixture of neutrophils and IL-35 overexpression cells. This number was significantly higher than that of the IL-35 overexpression lung metastasis model, which was consistent with the HE staining results of lung metastatic nodules. Meanwhile, we found that the lung metastasis rate was significantly reduced when IL-35 was knocked down (Figure S2 C). 3.5. IL-35 promotes neutrophil infiltration by increasing the expression of CCL3 in vitro We used transwell assay to verify the effect of the HCC-related expression of IL-35 on neutrophil chemotaxis. The chemotactic effect of the IL-35-KD conditioned medium (CM) on neutrophils was decreased by 64.5% and 56.3% (P < 0.05), whereas the IL-35 overexpression in CM increased by 3.97 and 4.67 times, respectively (P 0.05). These results showed that HCC-related IL-35 did not directly affect neutrophil infiltration (Fig. 5 A). We then investigated the pathway by which IL-35 affects the chemotaxis of neutrophils. After comparing the results of IL-35 positive related genes and sequencing in the TCGA database, we found that the expression of neutrophil-related chemokine genes was significantly increased after overexpression of IL-35. We further found that following overexpression of IL-35, the intracellular levels of the CCL3 protein were significantly increased. Whereas the intracellular levels of the CCL3 protein were significantly decreased after knocking down IL-35. CCL3 was significantly increased in IL-35 high expression patients group (P < 0.012, r = 0.431) (Fig. 5 B,D). To verify whether IL-35 chemotactically affected neutrophils through the expression of CCL3, we conducted CCL3 antibody block test. We accordingly discovered that CCL3 could significantly enhance the chemotactic effect on neutrophils, as the CCL3 antibody intervention experiment was demonstrated to significantly reduce the chemotactic effect of CM on neutrophils (Fig. 5 C). 3.6. IL-35 stimulates neutrophil secretion of FGF2 to promote angiogenesis To illustrate the roles and underlying mechanism of IL-35 in tumor angiogenesis, we conducted tube formation experiment in vitro . First, we stimulated HUVEC endothelial cells with rIL-35 or CM from IL-35 overexpression or knocked-down cells and found that the tube formation rate did not significantly change (Fig. 6 A). Considering that accumulation of neutrophils in HCC tissues has been reported to increase the production of angiogenesis factors and facilitate microvessel formation, we stimulated HUVECs with CM from the cocultivation of neutrophils and HCC cells and found that CM from neutrophils cocultured with IL-35 overexpression HCC cells could enhance tube formation (tube density: 212 ± 31 vs. 141 ± 19, P < 0.0024, tube branch: 365 ± 27 vs. 238 ± 24, P < 0.001). Conversely, the CM from IL-35 knocked-down HCC cells cocultured with neutrophils could significantly inhibit the tubule formation of endothelial cells (tube density: 119 ± 19 vs. 169 ± 23, P < 0.0056, tubule branch: 229 ± 24 vs. 315 ± 32, P < 0.0013) (Fig. 6 A). These results demonstrated that IL-35 stimulated neutrophils to produce angiogenesis factors. To further explore this, we isolated neutrophils from patients with HCC, stimulated them with human IL-35, and revealed that the expression of genes related to angiogenesis and adhesion factors in neutrophils was significantly increased. The KEGG pathway enrichment map showed that after neutrophils were stimulated by IL-35, the pathways of epidermal growth factor receptor (EGFR) and vascular endothelial growth factor (VEGF) were significantly activated. The FGF2 protein was demonstrated to be the most significantly elevated angiogenic factor, with the expression of the FGFR3 and FGFR4 receptors of FGF2 being also increased by 574 and 65 times, respectively. This was further confirmed using WB and ELISA analysis (Fig. 6 B). To determine whether FGF2 plays a significant role in mediating IL-35 to promote angiogenesis, we have conducted inhibition experiments. When anti-IL-35 and anti-FGF2 neutralizing antibodies were used, the tube formation rate was shown to be significantly abrogated. Furthermore, after IL-35 knocked-down HCC cells were cocultured with neutrophils in the presence of rIL-35, the tube formation rate was demonstrated to be significantly elevated. However, when anti-FGF2 neutralizing antibody was added to the above CM, tube formation was blocked (Fig. 6 C). 3.7. Anti-IL-35 antibody enhances the efficacy of PD1 antibody in the tumor model in mouse. Drug combination is an important way to explore better treatments of liver and other cancers. Therefore, we aimed to explore whether the IL-35 antibody could enhance the effect of the administration of the PD1 antibody in the treatment of HCC. We found that subcutaneous tumor models were established by Hepa1-6 cell in immunocompetent mice, and tumor growth was slightly inhibited after treatment with anti-IL-35 or PD1 neutralizing antibody. However, we observed more dramatic and durable responses, compared with the responses in the control treatment, when the anti-PD1 antibody was combined with the anti-IL-35 neutralizing antibody (Fig. 7 A). The infiltration of CD8 + T-cells was increased after treatment with the IL-35 and PD1 antibodies (P < 0.001). In contrast, neutrophil infiltration was decreased after treatment with the IL-35 antibody (P < 0.001), whereas no effect was observed in neutrophil infiltration after administration of the PD1 antibody. Nevertheless, we did not observe any significant difference in other cells, including macrophages and Treg cells. IHC analysis showed that single treatment with anti-PD1 antibody or anti-IL-35 antibody increased the infiltration of CD8 + T cells in the tumor. And the infiltration of CD8 + T cells was further increased in the combination treatment group compared with the single treatment group (P < 0.001). The neutrophil infiltration in the anti-IL-35 group and the combined treatment group was observed to be significantly lower than that in the control group (P < 0.001). However, there was no significant difference shown in neutrophil infiltration between the two groups. The PD1 antibody treatment group was also shown to have no effect on neutrophil infiltration (Fig. 7 B,C). 4. Discussion The tumor microenvironment(TME) is composed of cells such as cancer cells, tumor-associated macrophages (TAM), natural killer (NK) and dendritic cells (DC), T and B lymphocytes, etc. Apart from that, blood, lymphocytes, collagen fibers and glycoproteins constitute the extracellular matrix(ECM), while cancer-associated fibroblasts(CAFs) and mesenchymal stem cells lead to remodeling of the ECM. Cytokines, chemokines and other effectors exert either promotion or inhibition effects and are involved in the construction and multiple functions of the TME[ 32 ]. We found that IL-35 facilitates intratumoral neovascularization by neutrophils recruitment and reshapes the immune microenvironment by decreasing the infiltration of CD8 + T cells to promote the progression of HCC. In addition, the combination of anti-IL-35 and anti-PD1 antibodies showed a synergistic anticancer effect. Our results found that high expression of IL-35 in HCC is an independent risk factor for recurrence, while GP130 and IL-12Rβ2 receptors are expressed in HCC tissues, suggesting that IL-35 has a structural basis for autocrine functions in HCC. We further found that patients with high IL-35 (and its receptors) expression in tumor tissues have the worst prognosis, indicating that IL-35 might promote HCC progression. Despite the cancer-killing effect of neutrophils, in most cases the high level of neutrophil infiltration in solid tumors implies a poor clinical prognosis for patients[ 16 ]. In this study, the local infiltration of neutrophils and MVD are significantly increase, whereas that of CD8 + T cells is significantly decreased in tumors with high expression of IL-35. In vivo , IL-35 is closely associated with neutrophils infiltration and intratumoral angiogenesis, suggesting that the increased expression of IL-35 is related to local immune escape and tumor angiogenesis in HCC. Our study also showed a significant positive correlation between neutrophils and angiogenesis in HCC, along with a significant increase in angiogenesis and neutrophil infiltration in HCC tissues with high expression of IL-35. Tumor angiogenesis can also be indirectly affected by neutrophils, macrophages, Treg cells, stellate cells, and other interstitial cells known to affect TME [ 33 ]. It has been reported that IL-35 can also affect the secretion of CXCL1 and CXCL8 by monocytes and promote angiogenesis in pancreatic cancer [ 34 ]. Our results show that both direct stimulation of rIL-35 and IL-35 overexpression or knocked-down CM had no significant effect on microvessel formation, indicating that the tumor did not directly stimulate the formation of vascular endothelium by secreting IL-35. Therefore, we considered that tumor secretion of IL-35 promotes intratumoral angiogenesis through indirect mechanisms. It has reported that IL-35 can indirectly promote neutrophil polarization to TAN2 and tumor infiltration in mouse models of melanoma and HCC[ 29 ]. Our study showed that IL-35 is associated with neutrophil infiltration, indicating that IL-35 promotes intratumoral angiogenesis through neutrophils. Other studies reported that neutrophils (mainly TAN2) can participate in the reconstruction of the tumor extracellular matrix by secreting matrix metalloproteinases (MMPs), neutrophil elastase(NE), and cathepsin G, thus promoting tumor metastasis [ 35 , 23 ]. Neutrophils in breast cancer have been demonstrated to bind to circulating tumor cells (CTCs) entering the circulatory system through vascular cell adhesion protein 1 (VCAM1) to promote metastasis [ 36 ]. The results of in vitro experiments illustrated that IL-35 with neutrophils enhances the adhesion of HCC cells to endothelial cells. Since IL-35 with neutrophils enhances the adhesion of tumor cells to epithelial cells, this explains the in vivo experiments in which IL-35 promotes neutrophil-induced lung metastasis of HCC. Factors involved in neutrophil tumor infiltration and angiogenesis might be CCL3 and FGF2. CCL3-recruited neutrophils promote breast cancer lung metastasis, and neutrophil-secreted FGF2 in colorectal cancer lead to the development of liver metastasis[ 23 , 37 ].Our results showed that IL-35 can affect the expression of CCL3 in HCC cells and promote the infiltration of neutrophils in tumors. Meanwhile, IL-35 can lead to tumor vascular growth and metastatic tumor colonization by promoting the secretion of FGF2 angiogenic factor of neutrophils. Moreover, IL-35 was demonstrated to stimulate the expression of the FGFR3 and FGFR4 neutrophil FGF2 receptors, indicating that there might be a positive feedback loop in TME regarding the IL-35-stimulated secretion of neutrophil FGF2. Tumor immunotherapy represented by immune checkpoint antibody therapy is a hot spot of tumor immunotherapy in recent years. So far, it has been reported to achieve exciting results in a variety of tumors, including HCC, and has received extensive attention [ 38 – 44 ]. However, immunotherapy only benefits no more than 20% of liver cancer patients, and approximately 30% of liver cancer patients develop intrinsic resistance[ 45 ].Combination drugs against multiple key targets might be a good solution. IL-35 could significantly reduce the infiltration of CD8 + T cells in tumor tissues of immunocompetent mouse, suggesting that IL-35 have a role in suppressing tumor immunity. We found that the IL-35 antibody could enhance the efficacy of PD1. IL-35 is mainly expressed by Treg and Breg and exerts anti-tumor effects in the tumor microenvironment by suppressing immunosuppression, leading to PD1 antibody resistance. Treg derived IL-35 synergistically promotes BLIMP-dependent depletion of CD8 + T cells with IL-10, limiting effective immune antitumor effects[ 46 ]. IL-35 produced by Breg in pancreatic ductal adenocarcinoma inhibits the infiltration and effector function of CD8 + T cells within the tumor by activating the transcription factor STAT3 to inhibit the expression of CXCR3, CCR5, and IFNγ[ 47 ]. STING agonist therapy in pancreatic cancer results in elevated IL-35 production by Breg, and the TING-IL-35 axis reduces NK cell proliferation and attenuates NK-driven antitumor responses[ 48 ].It appears that IL-35 is the culprit for the poor results of immune anti-tumor therapy. Our results further complement the role of IL-35 in HCC TME. HCC cell-derived IL-35 can play a role in angiogenesis, metastasis, and immune evasion by autocrine-induced self-production of CCL3, which recruits neutrophils for infiltration in tumors. Antibody therapy with IL-35 enhances the infiltration of anti-tumor immune cells, reduces the infiltration of tumor-associated neutrophils, and decreases the inhibitory effect of IL-35 on anti-tumor immune cells[ 49 ].In addition, inhibition of IL-35 blocked neutrophil-mediated intra-tumor angiogenesis, reducing nutrient supply to tumor cells as well as metastasis[ 16 ].IL-35 antibody combined with PD1 antibody in HCC has a synergistic effect, inhibiting intra-tumor angiogenesis, reversing the immunosuppressive microenvironment, and enhancing the efficacy of PD1 antibody therapy. We conclude that IL-35 secreted by HCC cells recruits neutrophils to accumulate in the TME to cause lung metastasis through autocrine induction of CCL3 production by their own cells, and that neutrophils induce tumor angiogenesis through secretion of FGF2. Meanwhile, IL-35 and neutrophil cells exert simultaneous immunesuppressive anti-tumor effects. IL-35 antibody combined with PD1 antibody can inhibit the growth of HCC by increasing the infiltration of CD8 + T cells with decreasing neutrophil recruitment in the TME and reducing tumor angiogenesis(Fig. 8 ). This study also has some limitations. At first, the IL-35 antibody is an anti-single subunit antibody, and it is difficult to fully assess the inhibition effect of IL-35. Hence we could not rule out the possibility of blocking the effect of IL-12 or IL-27. Second, IL-35 is a widely secreted cytokine. The use of an IL-35 antibody could not only affect IL-35 secreted by HCC but also IL-35 secreted by other cells, including Tregs. Finally, the clinical samples were obtained from a retrospective single-center study, and as such verification of these clinical data require an extended study. 5. Conclusions Our data show that the high expression of IL-35 in patients with HCC is an important tumor promoter. Combined treatment with anti-IL-35 and anti-PD1 antibodies have potential therapeutic effect against HCC (Fig. 8 ). Abbreviations IL-35 interleukin-35 AFP alpha fetal protein CA19-9 carbohydrate antigen 19 − 9 CEA carcinoembryonic antigen TBIL total bilirubin ALB albumin BCLC Barcelona Clinic Liver Cancer staging system HCC Hepatocellular carcinoma TMA tissue microarray PD 1 programmed cell death protein 1 PD-L1 programmed cell death protein ligand 1 DCs dendritic cells TME tumor microenvironment CTL cytotoxic T-lymphocytes IHC immunohistochemistry CO-IP Coimmunoprecipitation HUVECs Human umbilical vein endothelial cells OS overall survival RFS recurrence-free survival PVTT portal vein tumor thrombus MVI microvascular invasion MVD microvessel density CCL3 C-C motif chemokine ligand 3 EGFR epidermal growth factor receptor VEGF vascular endothelial growth factor FGF2 fibroblast growth factor 2 IFN interferon CTCs circulating tumor cells VCAM1 vascular cell adhesion protein 1 NETs neutrophils extracellular traps TANs tumor-associated neutrophils ECM extracellular matrix CAFs cancer-associated fibroblasts ICIs immune checkpoint inhibitors. Declarations Ethics approval and consent to participate Animal experiment protocols were approved by the ethics committee of the XX Hospital of XX University. And the study protocol was approved by the ethics committee of the XX Hospital of XX University Consent for publication Not applicable. Availability of data and materials Reasonable requests for data will be made available for review. Competing interests The authors declare that they have no competing interests. Funding This work was supported by the National Natural Science, Foundation of China (grant number XX,XX,XX)，the Clinical Research Project of XX Hospital (NO.XX and XX). Authors' contributions XX performed the in vivo and cellular experiments and wrote the manuscript. XX, XX and XX discussed and participated in the data collection and analysis. XX collected data and performed some statistical analysis. XX, XX, XX helped to design the experiments. 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B cell-Derived IL35 Drives STAT3-Dependent CD8(+) T-cell Exclusion in Pancreatic Cancer. Cancer Immunol Res. 2020;8(3):292-308. Li S, Mirlekar B, Johnson BM, Brickey WJ, Wrobel JA, Yang N, et al. STING-induced regulatory B cells compromise NK function in cancer immunity. Nature. 2022;610(7931):373-80. Xue R, Zhang Q, Cao Q, Kong R, Xiang X, Liu H, et al. Liver tumour immune microenvironment subtypes and neutrophil heterogeneity. Nature. 2022;612(7938):141-7. Supplementary Files supplementarymaterial.doc figS1.png Fig.S1. Construction of stable IL-35 cell line. A. The expression of IL-35 in HCC cell line was detected by ELISA assay. We established HCC-LM3 and MHCC97H IL-35 knockdown stable cell line and IL-35 overexpression of SMCC-7721 and HUH7 stable cell line. Hepa1-6 cell line of IL-35 knockdown and overexpression was established. B. 4 cases of HCC samples and 2 cell lines were used to investigate the structural relationship of the 4 subunits by CO-IP experiments. The P35 antibody was demonstrated to successfully immunoprecipitate EBI3 but rarely bound to P40 and P28. figS22.png Figure S2. IL-35 promoted the adhesion between tumor cell and HUVEC and facilitated the pulmonary metastasis in vivo . A.IL-35 overexpression could significantly promote the adhesion of HCC to HUVEC in vitro adhesion experiment, and co-cultivation with neutrophils could further enhance this effect. More HCC adhered to the surface of HUVEC (Red: HUVEC, Green: HCC). B. During in vivo experiment, the remaining tumor cells in lung tissue were observed by fluorescence tracer. No significant difference in the retention of tumor cells in each group at 30 minutes. A significant difference in the number of tumor cells stranded in lung tissue 24 hours later. (Red: HCC, Green: Neutrophils) C. The number of HE staining pulmonary metastatic nodules in the lung metastasis model. Significantly increased metastatic nodules in the IL-35 overexpression co-inject with neutrophils group. The lung metastasis rate decreased significantly when IL-35 was knocking down (arrows: metastatic tumor). NE, neutrophils; Fig A, NE+, co-culture with neutrophils; Fig B and C, NE+, co-inject with neutrophils; Fig A CO, co-culture; Fig B and C, CO, co-inject. Scale bar, 50 μm, #P > 0.05, *P < 0.005, **P < 0.001, ***P < 0.0001. FIGS32.png Fig.S3. Overexpression of IL-35 could significantly promote the adhesion of Hepa1-6 to HUVEC in vitro adhesion experiment. Overexpression of IL-35 could significantly promote the adhesion of Hepa1-6 to HUVEC in vitro adhesion experiment. Cocultivation with neutrophils could further enhance this effect. Moreover, Hepa1-6 cells adhered to the surface of HUVEC (Red: HUVEC, Green: Hepa1-6). Cite Share Download PDF Status: Posted Version 1 posted 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. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3856191","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":269660137,"identity":"1c44c487-3322-40dd-ae71-5c14378967fa","order_by":0,"name":"WEI GAN","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABE0lEQVRIie2RsWrDMBCGLwjk5RKvVyj1K8gESodCx76Gu7sh0CVDMQKDp4BXQ5+ib6AiaBaVrh0yuEunDNlrSs+lWYKM10L1DYcQ+vSfTgCBwN+FMOYq+qUEmBgAM6qcnuiDIn/OjypwqcyvAmNKcv1iBK4ucP66acVyVSxmSdka6LYQP2ivkrpFJtARnhunROPsnZRSmUn1AbT1J6U6V+K2YuVpHX1OK3NTSWBFW1CU+ZV6x8oX4byMWjGtClaiPTc2rCTUp2hCxZezIlhBHoUcVhTtlO2eCcmh4kf1b8mX3J5FehtIqfP0vbkvruKaJ4Y8saTcPLb7zp7FzUCKOfqC7FDRe75P0Ucb/psDgUDgX/MNHXpYNyDDmVwAAAAASUVORK5CYII=","orcid":"","institution":"Zhongshan Hospital Fudan University","correspondingAuthor":true,"prefix":"","firstName":"WEI","middleName":"","lastName":"GAN","suffix":""},{"id":269660138,"identity":"b5580281-5a17-4ef4-b1d1-8dc81c9ee06f","order_by":1,"name":"Guoqiang Sun","email":"","orcid":"","institution":"Zhongshan Hospital Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Guoqiang","middleName":"","lastName":"Sun","suffix":""},{"id":269660139,"identity":"882ca01c-c482-4ade-a755-ee899ab4a775","order_by":2,"name":"JinLong Huang","email":"","orcid":"","institution":"Zhongshan Hospital Fudan University","correspondingAuthor":false,"prefix":"","firstName":"JinLong","middleName":"","lastName":"Huang","suffix":""},{"id":269660140,"identity":"62d66c91-5453-49fa-b585-6280d6fd218d","order_by":3,"name":"BaoYe Sun","email":"","orcid":"","institution":"Zhongshan Hospital Fudan University","correspondingAuthor":false,"prefix":"","firstName":"BaoYe","middleName":"","lastName":"Sun","suffix":""},{"id":269660141,"identity":"d8a7b81f-a091-441a-a2fa-ebc2687c6fef","order_by":4,"name":"ZhuTao Wang","email":"","orcid":"","institution":"Zhongshan Hospital Fudan University","correspondingAuthor":false,"prefix":"","firstName":"ZhuTao","middleName":"","lastName":"Wang","suffix":""},{"id":269660142,"identity":"a689356f-0f24-4286-9ea7-99ad2904f350","order_by":5,"name":"ZhangFu Yang","email":"","orcid":"","institution":"Zhongshan Hospital Fudan University","correspondingAuthor":false,"prefix":"","firstName":"ZhangFu","middleName":"","lastName":"Yang","suffix":""},{"id":269660143,"identity":"46d00037-46fc-48e7-b523-0d56cc79b974","order_by":6,"name":"Cheng Zhou","email":"","orcid":"","institution":"Zhongshan Hospital Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Cheng","middleName":"","lastName":"Zhou","suffix":""},{"id":269660144,"identity":"3aa9217a-866e-4ef9-af0e-0e30da2fdaf0","order_by":7,"name":"Yong Yi","email":"","orcid":"","institution":"Zhongshan Hospital Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Yong","middleName":"","lastName":"Yi","suffix":""},{"id":269660145,"identity":"866d66e6-9090-45ec-8d57-43d020164b3d","order_by":8,"name":"ShuangJian Qiu","email":"","orcid":"https://orcid.org/0000-0003-2056-6127","institution":"Zhongshan Hospital Fudan University","correspondingAuthor":false,"prefix":"","firstName":"ShuangJian","middleName":"","lastName":"Qiu","suffix":""}],"badges":[],"createdAt":"2024-01-12 08:10:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3856191/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3856191/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":50391095,"identity":"7004046d-4416-48ca-96ad-35c5ea131b58","added_by":"auto","created_at":"2024-01-30 18:45:11","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":5225806,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHigh expression of IL-35 is associated with poor prognosis in HCC patients.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSerial sections of HCC tissue microarray (TMA) were used to explore the expression levels of the two subunits of the IL-35 ligand: EBI3 and P35. \u003cstrong\u003eA.\u003c/strong\u003e A significantly higher expression of P35 and EBI3 in HCC tumor was found in comparison with adjacent normal tissue. P35 expression levels were higher compared to P40 and P28 in consecutive sections of HCC tissue. \u003cstrong\u003eB.\u003c/strong\u003e The expression levels of P35 and EBI3 were highly consistent. Representative images of P35 and EBI3 from low to high depending on the expression level (-,+,++,+++) are shown. \u003cstrong\u003eC\u003c/strong\u003e. The distribution histogram of P35, EBI3, P28, P40 in 360 HCC patients and staining extent correlation among them. We have also illustrated the Kaplan–Meier survival analysis of OS and RFS according to different IL-35 levels. \u003cstrong\u003eD\u003c/strong\u003e. Cell fluorescent microscopy images demonstrating that the expression of P35 (red), EBI3 (green) was higher in HCC tumor cells HCC-LM3 and MHCC-97H than SMMC-7721 and HUH7. Scale bar，50 μm.\u003c/p\u003e","description":"","filename":"fig1a.png","url":"https://assets-eu.researchsquare.com/files/rs-3856191/v1/d8c621bbdf5d122715af9ffb.png"},{"id":50391097,"identity":"622f41cf-0f84-49cd-a18e-b43a73140235","added_by":"auto","created_at":"2024-01-30 18:45:11","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":6007450,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHigh IL-35 expression in HCC correlates with the infiltration of neutrophils, the formation of MVD, and the decrease of CD8\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eT cell.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e Representative images of immunohistochemistry (IHC) staining for P35, EBI3, CD66b, CD8, CD34. According to the expression of P35 and EBI3, patients were distributed into high IL-35 group and low IL-35 group. \u003cstrong\u003eB.\u003c/strong\u003e The expression levels of Gp130 and IL12-Rβ2 were also consistent with IL-35. Representative images of Gp130 and IL12-Rβ2 from low to high depending on the expression level (absent -, low +, middle ++, high +++) are shown. \u003cstrong\u003eC.\u003c/strong\u003e Quantification of MVD, CD66b\u003csup\u003e+\u003c/sup\u003e, CD8 counts per field. Pearson correlation analysis of CD34\u003csup\u003e+\u003c/sup\u003e and CD66b\u003csup\u003e+ \u003c/sup\u003ewas carried out. Kaplan–Meier survival analysis of OS and RFS based on the corporation of IL-35 expression and IL-35 receptors were performed. Scale bar， 50 μm, *P \u0026lt; 0.005, **P \u0026lt; 0.001, ***P \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"fig2a.png","url":"https://assets-eu.researchsquare.com/files/rs-3856191/v1/1935f0748be7c4179ac234e3.png"},{"id":50391285,"identity":"19688ae1-f231-40e4-927e-d5787560579f","added_by":"auto","created_at":"2024-01-30 18:53:10","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1998171,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOverexpression of IL-35 significantly promoted HCC neovascularization, progression and recruited neutrophils in nude mouse model.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e IL-35 knockdown in HCC-LM3 cell line significantly slowed down the progression of HCC. IL-35 overexpression facilitated tumor growth in nude mouse model. \u003cstrong\u003eB.\u003c/strong\u003e IL-35 increased intratumoral Ly6G\u003csup\u003e+\u003c/sup\u003e neutrophil infiltration and MVD in nude mouse models. Scale bar， 50 μm , ***P \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"FIG3.png","url":"https://assets-eu.researchsquare.com/files/rs-3856191/v1/31a9dbeb2c0516895cdbee6f.png"},{"id":50391287,"identity":"f0a90d35-df9e-4925-8053-17cdacbf9684","added_by":"auto","created_at":"2024-01-30 18:53:11","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2631448,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIL-35 overexpression significantly promoted HCC neovascularization, progression and recruited Ly6G\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e neutrophils while influencing the infiltration of CD8\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eT cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIL-35 knockdown in Hepa 1-6 cell significantly slowed down the progression of HCC, and increased IL-35 facilitated tumor growth in\u003cem\u003e vivo\u003c/em\u003e experiments. \u003cstrong\u003eB.\u003c/strong\u003e IL-35 increased intratumoral Ly6G\u003csup\u003e+\u003c/sup\u003e neutrophil infiltration. \u003cstrong\u003eC\u003c/strong\u003e. IL-35 influenced the infiltrated number of CD8\u003csup\u003e+\u003c/sup\u003eT cells. \u003cstrong\u003eD\u003c/strong\u003e. Overexpression of IL-35 increased neovascularization in the tumor, and MVD decreased when IL-35 was knocked down. Scale bar,50 μm, ***P \u0026lt; 0.0001\u003c/p\u003e","description":"","filename":"fig4a.png","url":"https://assets-eu.researchsquare.com/files/rs-3856191/v1/fc6abe271622dff9b8d2da6c.png"},{"id":50391098,"identity":"d72cbf73-ea31-4ae8-a494-7fbfe19f4a01","added_by":"auto","created_at":"2024-01-30 18:45:11","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":5871074,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIL-35 might recruits neutrophils by increasing the expression of CCL3.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA. \u003c/strong\u003eThe expression of IL-35 in HCC cell line, but not recombinant IL-35 (rIL-35), could influence neutrophils infiltration. \u003cstrong\u003eB.\u003c/strong\u003e Western blot and ELISA were used to detect the expression relationships between IL-35 and CCL3. \u003cstrong\u003eC.\u003c/strong\u003eThe infiltration of neutrophils derived from IL-35 could be inhibited by anti-CCL3 antibody in \u003cem\u003evitro\u003c/em\u003e experiments. \u003cstrong\u003eD.\u003c/strong\u003e IL-35 expression was highly correlated with CCL3 in HCC patient tissues. OE, overexpression, Ctrl, control, KD: knockdown. Scale bar, 50 μm, #P \u0026gt; 0.05, *P \u0026lt; 0.005, **P \u0026lt; 0.001, ***P \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-3856191/v1/01fdcd3edcdda7c1252038bf.png"},{"id":50391090,"identity":"3c5d4fd2-a43d-448c-96e3-4d08cbb14df1","added_by":"auto","created_at":"2024-01-30 18:45:10","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2142870,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVascularization capability of neutrophils was activated in an FGF2-dependent manner while it was stimulated by IL-35.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA. \u003c/strong\u003eIL-35 could not independently facilitate the angiogenesis of HUVEC. Nonetheless, the conditional medium (CM) from the cocultivation of HUH7-OE and neutrophils could significantly promote the generation of HUVEC vessel density and branch points. Simultaneously, the formations decrease when cultured with the co-cultural conditional medium from HCC-LM3-KD and neutrophils. \u003cstrong\u003eB\u003c/strong\u003e. IL-35 could significantly promote the secretions of FGF2 from neutrophils. Neutrophils proangiogenic capability was activated by increased FGF2. \u003cstrong\u003eC\u003c/strong\u003e. Anti-FGF2 antibody was used to block IL-35 mediated neutrophils’ proangiogenic capability. Blockade of IL-35 with IL-35 antibody could also decrease the vessel density and branch points in the co-culture system of HCC and neutrophils. CM, conditional medium; OE-CM, conditional medium from IL-35 overexpression HCC cell line; KD-CM, conditional medium from IL-35 knock down HCC cell line; NE, neutrophils, NE+; col-culture with neutrophils, #P \u0026gt; 0.05, *P \u0026lt; 0.005, **P \u0026lt; 0.001, ***P \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"fig6a.png","url":"https://assets-eu.researchsquare.com/files/rs-3856191/v1/8c6e606364e2b43ab5c70bef.png"},{"id":50391099,"identity":"1a60e689-1692-4f32-843b-b3ee074386b1","added_by":"auto","created_at":"2024-01-30 18:45:11","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2952460,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnti-IL-35 significantly improved the therapeutic effect of PD1 antibody in a mouse subcutaneous tumor model.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. Either anti-IL-35 antibody or anti-PD1 antibody could apparently slow down HCC progression in\u003cem\u003e vivo\u003c/em\u003e experiments. Combination therapy with both antibodies has significant and more durable responses for the treatment of HCC. \u003cstrong\u003eB.\u003c/strong\u003e IHC and flow cytometric plot were performed to explore the immune cells infiltration in the tumor tissue. Anti-PD1 antibody has no effect on the infiltration of Ly6G\u003csup\u003e+\u003c/sup\u003e neutrophils, but significant differences were found in the combined treatment group and anti-IL-35 group. There was less infiltration of neutrophils among these two groups. \u003cstrong\u003eC.\u003c/strong\u003e CD8\u003csup\u003e+\u003c/sup\u003eT cells were significantly increased in both PD1 antibody group and combined treatment group. Scale bar, 50 μm, #P \u0026gt; 0.05, *P \u0026lt; 0.005, **P \u0026lt; 0.001, ***P \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-3856191/v1/5013c7805cb3049678aa4ed7.png"},{"id":50391096,"identity":"8d2343c7-ef70-4f66-ac99-6697a9834fc1","added_by":"auto","created_at":"2024-01-30 18:45:11","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":828975,"visible":true,"origin":"","legend":"\u003cp\u003eThe expression of IL-35 in HCC can facilitate tumor progression by affecting neutrophil infiltration, angiogenesis, and CD8\u003csup\u003e+\u003c/sup\u003e T-cell infiltration. IL-35 can significantly promote the secretion of CCL3 by an autocrine pathway in HCC, attracting neutrophils into the tumor tissues. Neutrophils facilitate the FGF2 secretion, contributing to an environment of intratumoral angiogenesis. Meanwhile, CD8\u003csup\u003e+\u003c/sup\u003eT cells tend to decrease and exhaustion when stimulated by IL-35. HCC-derived IL-35 can facilitate pulmonary metastasis by interacting with neutrophils. The mouse xenograft tumor model suggests that the application of anti-IL-35 antibody and treatment combined anti-IL-35 antibody with anti-PD1 antibody can decrease HCC growth by increasing the infiltration of CD8\u003csup\u003e+\u003c/sup\u003eT and decreasing the infiltration of neutrophils in the tumor microenvironment.\u003c/p\u003e","description":"","filename":"fig8.png","url":"https://assets-eu.researchsquare.com/files/rs-3856191/v1/582fc41539a9c3b478c33eaa.png"},{"id":50946472,"identity":"f0e02f41-61d6-4e8b-aec8-d9af57e164fd","added_by":"auto","created_at":"2024-02-10 06:55:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":10560473,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3856191/v1/16020fd9-ce5b-4c64-b388-02cceac90118.pdf"},{"id":50391092,"identity":"8b6812dd-3fa0-4761-a9f0-6000aeac5fe5","added_by":"auto","created_at":"2024-01-30 18:45:10","extension":"doc","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":30208,"visible":true,"origin":"","legend":"","description":"","filename":"supplementarymaterial.doc","url":"https://assets-eu.researchsquare.com/files/rs-3856191/v1/c24cb20abf953d9da5a1def8.doc"},{"id":50391093,"identity":"db7f3dee-bab2-4f8c-83b5-60a57a2984a0","added_by":"auto","created_at":"2024-01-30 18:45:11","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1499377,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig.S1. Construction of stable IL-35 cell line.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e The expression of IL-35 in HCC cell line was detected by ELISA assay. We established HCC-LM3 and MHCC97H IL-35 knockdown stable cell line and IL-35 overexpression of SMCC-7721 and HUH7 stable cell line. Hepa1-6 cell line of IL-35 knockdown and overexpression was established.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB.\u003c/strong\u003e 4 cases of HCC samples and 2 cell lines were used to investigate the structural relationship of the 4 subunits by CO-IP experiments. The P35 antibody was demonstrated to successfully immunoprecipitate EBI3 but rarely bound to P40 and P28.\u003c/p\u003e","description":"","filename":"figS1.png","url":"https://assets-eu.researchsquare.com/files/rs-3856191/v1/96a7ecdf6184b87d01c8478a.png"},{"id":50391101,"identity":"ac2d2782-31e9-4e51-b93d-133839676803","added_by":"auto","created_at":"2024-01-30 18:45:11","extension":"png","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":3962952,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure S2. IL-35 promoted the adhesion between tumor cell and HUVEC and facilitated the pulmonary metastasis in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003evivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003eIL-35 overexpression could significantly promote the adhesion of HCC to HUVEC in\u003cem\u003e vitro\u003c/em\u003e adhesion experiment, and co-cultivation with neutrophils could further enhance this effect. More HCC adhered to the surface of HUVEC (Red: HUVEC, Green: HCC). \u003cstrong\u003eB\u003c/strong\u003e. During \u003cem\u003ein vivo\u003c/em\u003e experiment, the remaining tumor cells in lung tissue were observed by fluorescence tracer. No significant difference in the retention of tumor cells in each group at 30 minutes. A significant difference in the number of tumor cells stranded in lung tissue 24 hours later. (Red: HCC, Green: Neutrophils) \u003cstrong\u003eC.\u003c/strong\u003e The number of HE staining pulmonary metastatic nodules in the lung metastasis model. Significantly increased metastatic nodules in the IL-35 overexpression co-inject with neutrophils group. The lung metastasis rate decreased significantly when IL-35 was knocking down (arrows: metastatic tumor). NE, neutrophils; Fig A, NE+, co-culture with neutrophils; Fig B and C, NE+, co-inject with neutrophils; Fig A CO, co-culture; Fig B and C, CO, co-inject. Scale bar, 50 μm, #P \u0026gt; 0.05, *P \u0026lt; 0.005, **P \u0026lt; 0.001, ***P \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"figS22.png","url":"https://assets-eu.researchsquare.com/files/rs-3856191/v1/7624a8981ba493a4355057c3.png"},{"id":50391286,"identity":"cd93d705-a3ff-4b41-ad02-b27b96eade76","added_by":"auto","created_at":"2024-01-30 18:53:11","extension":"png","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":844541,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig.S3. \u003c/strong\u003eOverexpression of IL-35 could significantly promote the adhesion of Hepa1-6 to HUVEC \u003cem\u003ein vitro\u003c/em\u003e adhesion experiment.\u003c/p\u003e\n\u003cp\u003eOverexpression of IL-35 could significantly promote the adhesion of Hepa1-6 to HUVEC in vitro adhesion experiment. Cocultivation with neutrophils could further enhance this effect. Moreover, Hepa1-6 cells adhered to the surface of HUVEC (Red: HUVEC, Green: Hepa1-6).\u003c/p\u003e","description":"","filename":"FIGS32.png","url":"https://assets-eu.researchsquare.com/files/rs-3856191/v1/f675b8a509b76858393357f1.png"}],"financialInterests":"","formattedTitle":"Interleukin 35 promotes progression of hepatocellular carcinoma by recruiting neutrophils","fulltext":[{"header":"Highlights","content":"\u003cul type=\"disc\"\u003e\n \u003cli\u003eHigh expression of IL-35 in patients with HCC is associated with poor prognosis\u003c/li\u003e\n \u003cli\u003eIL-35 affects neutrophil infiltration, angiogenesis, and CD8\u003csup\u003e+\u003c/sup\u003e T-cell infiltration\u003c/li\u003e\n \u003cli\u003eCCL3\u0026nbsp;is a key factor mediating the recruitment of neutrophils by IL-35\u003c/li\u003e\n \u003cli\u003eIL-35 stimulates FGF2 secretion to promote intratumoral angiogenesis\u003c/li\u003e\n \u003cli\u003eAnti-IL-35 antibody can be an effective treatment against HCC\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eIL-35 facilitates the adhesion of tumors to endothelial cells, with neutrophils further enhancing this effect both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"1. Introduction","content":"\u003cp\u003eHepatocellular carcinoma (HCC), the most common type of primary liver cancer, is the fifth most common malignant tumor and second in mortality in China, based on 2020 data [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. In recent years, treatments such as surgery, radiofrequency or microwave ablation of liver cancers have made great significant progress, leading to improved therapeutic outcomes. However, the rate of tumor recurrence and metastasis within 5 years post-operation remains high, at 60 to 70%[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The administration of Sorafenib, Lenvatinib, and Regorafenib has only modestly extended overall survival in patients with advanced HCC, by approximately 2.8 months [\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Currently, immunotherapy, including programmed cell death protein 1 (PD1) and programmed cell death protein ligand 1 (PD-L1) antibodies, along with other immune checkpoint antibodies, has gained significant interest. Nevertheless, the efficacy of single-agent therapy in advanced HCC is limited, with success rates of only about 15 to 20%, highlighting the need for more effective treatment strategies[\u003cspan additionalcitationids=\"CR9 CR10\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Evidence suggests that combining immune checkpoint inhibitors(ICIs) with antiangiogenic antibodies can be effective in treating liver cancer, yielding potentially revolutionary results [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Consequently, our research focuses on exploring more effective combined treatments for HCC.\u003c/p\u003e \u003cp\u003eAnti-angiogenic therapy, in combination with ICIs, exert anti-HCC effects by remodeling the tumor microenvironment (TME)[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. By studying the TME of HCC, we can propose new therapeutic approaches for patients. Neutrophils, a significant component of the TME, exhibit both anti-tumor (TAN1) and pro-tumor (TAN2) phenotypes due to their diversity and plasticity[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. In various cancers, neutrophils typically play a pro-tumorigenic role in processes such as tumorigenesis, tumor proliferation, metastasis, angiogenesis, neutrophils extracellular traps(NETs)and immunosuppression[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Neutrophil-secreted factors enhance tumor growth [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], while cytokines from tumor cells recruit neutrophils, creating a pro-tumor feedback loop [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In the TME, interleukins play a crucial role in recruiting neutrophils for tumor growth. For instance, interleukin 8(IL-8) recruits neutrophils in colorectal and thyroid cancers, altering the TME and promoting tumor growth[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Interleukin 6(IL-6) regulates neutrophil recruitment in breast cancer, and interleukin 1β(IL-1β) induces neutrophil extracellular traps (NETs) formation, facilitating lung metastasis in breast cancer [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. However, the specific roles of interleukins and neutrophils in the TME of HCC remain to be fully elucidated.\u003c/p\u003e \u003cp\u003eOur previous research has showed that interleukin 35(IL-35) expression correlates with HCC aggressiveness and is an independent prognostic factor for recurrence[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. IL-35, a novel cytokine of the IL-12 family comprising P35 and EBI3 subunits, is primarily expressed by Treg and Breg cells [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In HCC, increased IL-35 expression in CCR4\u0026thinsp;+\u0026thinsp;Tregs directly impairs CD8\u0026thinsp;+\u0026thinsp;T-cell function[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], and also indirectly reduces antitumor IL-9 secretion by Th9 cells [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Other studies have indicated that in mouse of HCC and melanoma, IL-35 promotes the conversion of neutrophils to TAN2 phenotype and their infiltration into the TME, enhancing their pro-angiogenic and immunosuppressive functions[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. However, the impact of IL-35 and neutrophils on tumor growth and immune response in human HCC is less understood. Our study reveals that IL-35, through autocrine secretion in HCC cells, affects the production of CCL3, which recruits neutrophils to the TME and promotes tumor angiogenesis and metastasis. Furthermore, IL-35 directly impedes CD8\u003csup\u003e+\u003c/sup\u003e T cells infiltration and reshapes the immune environment. Thus, a combined treatment targeting IL-35 and PD1 antibodies holds promise as a potential therapeutic approach against HCC.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cp\u003eDetailed methods are presented in the Supplementary Material.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Human samples, and collection of patient clinical data\u003c/h2\u003e \u003cp\u003eA total of 360 patients with primary HCC who underwent radical resection at XX Hospital affiliated to XX University in 2012 were selected for this study. The obtained samples were confirmed as HCC based on pathological assessment. Clinicopathological data, such as liver function, alpha-fetoprotein (AFP), carbohydrate antigen 19\u0026thinsp;\u0026minus;\u0026thinsp;9 (CA19-9), carcinoembryonic antigen (CEA), tumor size, number and differentiation, lymph node metastasis, vascular invasion, cancer thrombus, and microvascular invasion (MVI) were collected from the medical history and pathological reports of enrolled patients. Our research has been registered at the web of research registry and the unique registration ID was researchregistryXXX.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Statistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were conducted using the IBM SPSS and GraphPad Prism. Each experiment was performed in triplicate, and values are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, unless otherwise stated. The variance between the groups was statistically compared. The distributions of both the overall survival (OS) and recurrence-free survival (RFS) were depicted using the Kaplan\u0026ndash;Meier method and analyzed by the log-rank test. Univariate and multivariate analyses for prognostic factors were based on the Cox proportional hazard model. P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e3.1. High expression of IL-35 in HCC is an independent risk factor for prognosis.\u003c/h2\u003e \u003cp\u003eOur study included 202 males (56.1%) and 158 females (43.9%), with an average age of 54.36\u0026thinsp;\u0026plusmn;\u0026thinsp;11.038 (26\u0026ndash;85) years. The average OS time was 51.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.996 months, and the median OS was 49\u0026thinsp;\u0026plusmn;\u0026thinsp;7.19 months. The average RFS time was 41.91\u0026thinsp;\u0026plusmn;\u0026thinsp;1.27 months, and the median RFS was 22.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.837 months (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe correlation between clinicopathologic characters and IL35\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003echaracters\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eExpression of IL-35\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLow\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHigh\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eP value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal patients\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e210\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e150\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGender, male/femeal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e113/97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e89/61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.298\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge, \u0026lt;\u0026thinsp;60/\u0026ge;60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e133/77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e107/43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.112\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHBsAg, negative/positive\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e24/186\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e22/128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.364\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAFP,\u0026lt;400/\u0026ge;400 ng/mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e116/94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e51/99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTBIL,\u0026lt;20/\u0026ge;20 \u0026micro;mol/L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e179/31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e122/28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.324\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAST,\u0026lt;45/\u0026ge;45 U/L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e127/83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e88/62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.73\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eALT,\u0026lt;50/\u0026ge;50 U/L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e171/39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e116/34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.341\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eALB,\u0026lt;35/\u0026ge;35g/L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e15/195\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5/145\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ecirrhosis,no/yes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e55/155\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e29/121\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.129\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003etumor number,single/multiple\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e177/33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e115/35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.069\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003etumor thrombus,no/yes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e204/6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e110/40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003etumor capsule,no/yes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e59/151\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e49/101\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.351\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003etumor size,\u0026lt;5/\u0026ge;5cm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e138/72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e80/70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.018\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLNM,no/yes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e205/5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e145/5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.588\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMVI, no/yes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e189/21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e17/133\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003edifferentiation, I-II/III-IV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e140/70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e75/75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCounts of CD34\u003csup\u003e+\u003c/sup\u003e\u0026thinsp;cells(low/high)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e158/52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e92/58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.005\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCounts of Treg cells(low/high)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e134/76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e90/60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.462\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCounts of CD8\u003csup\u003e+\u003c/sup\u003eT cells(low/high)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e120/90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e112/38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCounts of CD4\u003csup\u003e+\u003c/sup\u003eT cells(low/high)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e181/29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e130/20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.897\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCounts of CD66b\u003csup\u003e+\u003c/sup\u003e\u0026thinsp;cells(low/high)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e152/58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e66/84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCounts of CD68\u003csup\u003e+\u003c/sup\u003e\u0026thinsp;cells(low/high)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e138/72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e89/61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.216\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBCLC stage(A/B/C)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e132/72/6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e64/46/40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e \u003cp\u003eAPF, alpha fetal protein; LNM, lymph node metastasis; MVI, microvascular invasion;BCLC stage, Barcelona Clinic Liver Cancer staging system\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe P35 and EBI3 proteins widely expressed in tumor and stromal cells. Because of the lack of high-quality IL-35-specific antibodies, we examined the expression of P35 and EBI3 in serial tissue sections of HCC samples to determine the level of IL-35 expression, which has been reported as a reliable method in other studies [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. As EBI3 forms IL-27 with P28 and P35 forms IL-12 with P40, we also examined the expression levels of P28 and P40 in consecutive tumor tissue. The expression of P35 and EBI3 was lower in adjacent normal tissue compared with tumor tissue of the same patient, we found that the expression level of both P28 and P40 in HCC was very low, and primarily located in stromal cells(Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-B). In addition, we found that P35 and EBI3 were mainly expressed in the cytoplasm rather than in the nucleus. The expression intensity of P35 and EBI3 was similar with overlapping spatial expression sites. Both proteins were expressed with higher intensity in HCCLM3 and MHCC97H and with lower intensity in SMMC-7721 and HUH7 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). To eliminate the effect of IL-12 and IL-27 co-subunits on IL-35 detection, we investigated the structural relationship of the four subunits in four cases of HCC using the CO-IP experiments. The P35 antibody immunoprecipitated EBI3 but rarely bound P40 and P28. Similarly, the same phenomenon was observed in CO-IP experiments in MHCC97H and HCCLM3 cell lines (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB). Our results identified IL-35 as the factor that is highly expressed in HCC, rather than IL-12 or IL-27.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe high expression of both P35 and EBI3 was defined as the IL-35 high expression group (41.6%), whereas others were classified as the IL-35 low expression group (58.4%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). The IL-35 high expression group had worse OS and RFS compared to the low expression group(Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Significant correlations were found between the high expression of IL-35 and advanced BCLC stage. Moreover, we also found that the level of serum AFP was significantly increased in the IL-35 overexpression group (637.45\u0026thinsp;\u0026plusmn;\u0026thinsp;32.8 vs 212.47\u0026thinsp;\u0026plusmn;\u0026thinsp;18.9 ng/mL, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In addition, we noted that overexpression of IL-35 was closely related to an increased prevalence of portal vein tumor thrombus (PVTT), MVI, and large tumor size (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Multivariate analysis showed that overexpression of IL-35 was an independent risk factor for both OS (HR\u0026thinsp;=\u0026thinsp;1.947, 95% CI, 1.046\u0026ndash;3.624, P\u0026thinsp;=\u0026thinsp;0.035) and RFS (HR\u0026thinsp;=\u0026thinsp;2.442, 95% CI, 1.459\u0026ndash;4.088) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Therefore, the expression of IL-35 in HCC was considered an important reference index for prognosis.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe univariate and multivariate analysis for OS and RFS in the cohort\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003eOS\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c9\" namest=\"c6\"\u003e \u003cp\u003eRFS\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003echaracters\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eunivariate\u003c/p\u003e \u003cp\u003eP value\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003emultivariate\u003c/p\u003e \u003cp\u003ePvalue\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHR\u003c/p\u003e \u003cp\u003e(95% CI)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eunivariate\u003c/p\u003e \u003cp\u003eP value\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003emultivariate\u003c/p\u003e \u003cp\u003eP value\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eHR\u003c/p\u003e \u003cp\u003e(95% CI)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGender, male/femeal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.559\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.568\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge, \u0026lt;\u0026thinsp;60/\u0026ge;60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.266\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.237\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHBsAg, negative/positive\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.598\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.394\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTBIL,\u0026lt;20/\u0026ge;20 \u0026micro;mol/L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.528\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.921\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eALT,\u0026lt;50/\u0026ge;50 U/L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.716\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.440\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eALB,\u0026lt;35/\u0026ge;35g/L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.052\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.075\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAFP,\u0026lt;400/\u0026ge;400 ng/mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e0.021\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAST,\u0026lt;45/\u0026ge;45 U/L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.094\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e0.004\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e0.007\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003e\u003cb\u003e1.565(1.131\u0026ndash;2.166)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003etumor size,\u0026lt;5/\u0026ge;5cm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e0.012\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.634(1.113\u0026ndash;2.399)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003eNS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003etumor number,\u003c/p\u003e \u003cp\u003esingle/multiple\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.016\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.643(1.097\u0026ndash;2.460)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003etumor capsule,no/yes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.158\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e0.029\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMVI, no/yes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003etumor throbus,no/yes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e0004\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e2.073(1.262\u0026ndash;3.403)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003e\u003cb\u003e2.507(1.610\u0026ndash;3.906)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003edifferentiation, I-II/III-IV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e0.015\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e0.041\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLNM,no/yes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e0.023\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.059\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ecirrhosis,no/yes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.071\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.010\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eExpression of IL-35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e0.035\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.947(1.046\u0026ndash;3.624)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003e2.442(1.459\u0026ndash;4.088)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCounts of CD34\u003csup\u003e+\u003c/sup\u003e\u0026thinsp;cells\u003c/p\u003e \u003cp\u003e(low/high)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.919(2.589\u0026ndash;5.932)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003e2.329(1.658\u0026ndash;3.272)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCounts of Treg cells\u003c/p\u003e \u003cp\u003e(low/high)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.972\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.974\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCounts of CD8\u003csup\u003e+\u003c/sup\u003eT cells\u003c/p\u003e \u003cp\u003e(low/high)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.053\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e0.027\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCounts of CD4\u003csup\u003e+\u003c/sup\u003eT cells\u003c/p\u003e \u003cp\u003e(low/high)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.770\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.533\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCounts of CD66b\u003csup\u003e+\u003c/sup\u003e\u0026thinsp;cells\u003c/p\u003e \u003cp\u003e(low/high)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e0.022\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e1.625(1.074\u0026ndash;2.460)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e0.037\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003e1.420(1.022\u0026ndash;1.974)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCounts of CD68\u003csup\u003e+\u003c/sup\u003e\u0026thinsp;cells\u003c/p\u003e \u003cp\u003e(low/high)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBCLC stage (A/B/C)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e2.205(1.709\u0026ndash;2.844)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003e\u003cb\u003e1.911(1.539\u0026ndash;2.374)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"9\" nameend=\"c9\" namest=\"c1\"\u003e \u003cp\u003eAbbreviations: APF,alpha fetal protein;TBIL,total bilirubin;ALB,albumin; BCLC,Barcelona Clinic Liver Cancer staging system;NA, not applicable; NS, not significant\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eIt is of great significance to explore the expression of the GP130 and IL-12Rβ2 receptors of IL-35 in HCC [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. We found that the expression of GP130 and IL-12Rβ2 in HCC was closely related (r\u0026thinsp;=\u0026thinsp;0.39, P\u0026thinsp;=\u0026thinsp;0.023, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Based on the expression of IL-35 and its receptors in HCC, we divided patients into 4 groups: IL-35R (+) IL-35 (high), IL-35R (+) IL-35 (low), IL-35R (-) IL-35 (high), and IL-35R (-) IL-35 (low). We found that patients in the IL-35R (+) IL-35 group (high) had the worst prognosis (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), thus supporting the hypothesis that IL-35 facilitated the progression of HCC by directly acting on tumor cells in an autocrine or paracrine manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e3.2. High expression of IL-35 in patients with HCC is closely related to the infiltration of neutrophils and CD8\u003c/b\u003e \u003csup\u003e \u003cb\u003e+\u003c/b\u003e \u003c/sup\u003e \u003cb\u003eT cells in tumor microenvironment.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eOur results showed that the expression of neutrophils marker CD66b in HCC tissues with high levels of IL-35 was significantly higher than that in the low expression group. In addition, we found that the number of infiltrated CD8\u003csup\u003e\u003cb\u003e+\u003c/b\u003e\u003c/sup\u003e T cells in tissues with high levels of IL-35 was significantly decreased (34.55\u0026thinsp;\u0026plusmn;\u0026thinsp;2.758 vs. 56.61\u0026thinsp;\u0026plusmn;\u0026thinsp;3.53, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) by IHC experiment. Importantly, the number of microvessel density (MVD) labeling by CD34 in patients with overexpression of IL-35 was significantly increased (86.63\u0026thinsp;\u0026plusmn;\u0026thinsp;4.789 vs. 56.54\u0026thinsp;\u0026plusmn;\u0026thinsp;2.308, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). The number of neutrophils infiltrating the tumor was positively correlated with MVD (r\u0026thinsp;=\u0026thinsp;0.301, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), suggesting that neutrophil infiltration might be an important factor in tumor angiogenesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eA,C).\u003c/p\u003e \u003cp\u003e \u003cb\u003e3.3. IL-35 affects HCC progression, angiogenesis, and adhesion via neutrophils and is negatively correlated with CD8\u003c/b\u003e \u003csup\u003e \u003cb\u003e+\u003c/b\u003e \u003c/sup\u003e \u003cb\u003eT cell infiltration in\u003c/b\u003e \u003cb\u003evivo\u003c/b\u003e \u003cb\u003emodel.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eBased on the expression of IL-35 in HCC cell lines, we selected the HCCLM3 and MHCC97H cell lines, which exhibited a high expression level to construct IL-35 knocked-down cell lines. Whereas the HUH7 and SMMC-7721 cell lines, which showed a low expression level of IL-35 were selected to construct IL-35 overexpression cell lines. We also constructed IL-35 overexpression and knocked-down Hepa1-6 cells. (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eFirst, we used the CCK8, transwell and wound healing to investigate the effect of IL-35 on HCC cells. Our results showed that IL-35 had no significant direct effects on the proliferation and migration of HCC cells \u003cem\u003ein vitro\u003c/em\u003e. Surprisingly, we found that the formation rate of the subcutaneous tumor in the IL-35 overexpressing cell line in nude mice was significantly faster than that in the control group (volume mm\u003csup\u003e3\u003c/sup\u003e: 1186.81\u0026thinsp;\u0026plusmn;\u0026thinsp;83. 53 vs. 612.82\u0026thinsp;\u0026plusmn;\u0026thinsp;73. 49, P\u0026thinsp;\u0026lt;\u0026thinsp;0. 001, tumor mass g: 1.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11 vs. 0.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), with the tumor growth rate being significantly slowed down in the knocked-down group (volume mm\u003csup\u003e3\u003c/sup\u003e: 495.48\u0026thinsp;\u0026plusmn;\u0026thinsp;53.42 vs. 882.61\u0026thinsp;\u0026plusmn;\u0026thinsp;73.25, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001, tumor mass g: 0.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 vs. 1.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). We also observed the same phenomenon in the immunocompetent C57BL/6 mouse model when exploring the effect of different expression levels of IL-35 on the growth of subcutaneous tumors (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMeanwhile, we found that the infiltration of neutrophils, as well as the number of MVD were both significantly increased in immunodeficient or immunocompetent mouse models injected with tumor cells overexpressing IL-35 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eB and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eB,D). In the IL-35 knocked-down group, the number of neutrophils infiltrating the tumor, and the amount of MVD were shown to be significantly decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eB and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Thus, we assumed that IL-35 could promote intratumoral neovascularization by recruiting the infiltration of neutrophils in tumors. In addition, the number of infiltrating CD8\u003csup\u003e\u003cb\u003e+\u003c/b\u003e\u003c/sup\u003e T-cells was significantly decreased in the IL-35 overexpression group, whereas it was significantly increased in the knocked-down group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eWe also found that IL-35 could enhance the adhesion of HCC cells to HUVECs in vitro. Following the overexpression of IL-35, the adhesion rate of SMMC-7721 or HUH7 cells to endothelial cells was significantly increased. In contrast, knocking down IL-35 strongly inhibited the number of HCCLM3 or MHCC97H cells adhered to endothelial cells. Meanwhile, we found that adding neutrophils to IL-35 overexpression group enhanced the amount of HUH7 or SMMC-7721 cells attached to endothelium cells by 46% and 54%, respectively (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). However, there was no significant change observed in the adhesion of tumor cells cocultured with neutrophils in the knocked-down group (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA). These results showed that IL-35 could increase the adhesion of HCC cells to endothelial cells, and neutrophils could further enhance this effect. Besides, we further verified this finding using the Hepa1-6 mouse HCC cell line (Fig. S3).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e3.4. IL-35 promotes lung metastasis of HCC in the presence of neutrophils in \u003cem\u003evivo\u003c/em\u003e model.\u003c/h2\u003e \u003cp\u003eNext, we explored the role of IL-35 in tumor adhesion and lung metastasis in mouse model. Fluorescent dye-labeled HUH7 cells were injected into the body through the tail vein of nude mice, and fluorescent tracers were used to observe the remaining tumor cells in the lung tissue. We found no significant difference in the amount of tumor cells retained in the groups at 30 min. However, we observed a significant difference in the number of tumor cells retained in the lung tissue after 24h.The number of retained tumor cells in the NE\u0026thinsp;+\u0026thinsp;HUH7-OE group (279\u0026thinsp;\u0026plusmn;\u0026thinsp;53) was significantly higher than that in the HUH7-OE group (103\u0026thinsp;\u0026plusmn;\u0026thinsp;31) and the HUH7-OE-Ctrl group (62\u0026thinsp;\u0026plusmn;\u0026thinsp;21) (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), while the number of retained tumor cells in the HUH7-OE group was also significantly higher than that in the HUH7-OE-Ctrl group. We observed that in the NE\u0026thinsp;+\u0026thinsp;HUH7-OE group, many tumor cells adhered directly to neutrophils. We collected gross specimens of lung tissue and found that the number of metastatic nodules after HUH7-OE injection was significantly higher than that in the HUH7-OE-Ctrl group (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eB). The number of lung metastatic nodules further increased after simultaneous injection of a mixture of neutrophils and IL-35 overexpression cells. This number was significantly higher than that of the IL-35 overexpression lung metastasis model, which was consistent with the HE staining results of lung metastatic nodules. Meanwhile, we found that the lung metastasis rate was significantly reduced when IL-35 was knocked down (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eC).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e3.5. IL-35 promotes neutrophil infiltration by increasing the expression of CCL3 \u003cem\u003ein vitro\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eWe used transwell assay to verify the effect of the HCC-related expression of IL-35 on neutrophil chemotaxis. The chemotactic effect of the IL-35-KD conditioned medium (CM) on neutrophils was decreased by 64.5% and 56.3% (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), whereas the IL-35 overexpression in CM increased by 3.97 and 4.67 times, respectively (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). However, the recombinant IL-35 (rIL-35) had no significant effect on neutrophil chemotaxis (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05). These results showed that HCC-related IL-35 did not directly affect neutrophil infiltration (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe then investigated the pathway by which IL-35 affects the chemotaxis of neutrophils. After comparing the results of IL-35 positive related genes and sequencing in the TCGA database, we found that the expression of neutrophil-related chemokine genes was significantly increased after overexpression of IL-35. We further found that following overexpression of IL-35, the intracellular levels of the CCL3 protein were significantly increased. Whereas the intracellular levels of the CCL3 protein were significantly decreased after knocking down IL-35. CCL3 was significantly increased in IL-35 high expression patients group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.012, r\u0026thinsp;=\u0026thinsp;0.431) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003eB,D). To verify whether IL-35 chemotactically affected neutrophils through the expression of CCL3, we conducted CCL3 antibody block test. We accordingly discovered that CCL3 could significantly enhance the chemotactic effect on neutrophils, as the CCL3 antibody intervention experiment was demonstrated to significantly reduce the chemotactic effect of CM on neutrophils (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003eC).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.6. IL-35 stimulates neutrophil secretion of FGF2 to promote angiogenesis\u003c/h2\u003e \u003cp\u003eTo illustrate the roles and underlying mechanism of IL-35 in tumor angiogenesis, we conducted tube formation experiment \u003cem\u003ein vitro\u003c/em\u003e. First, we stimulated HUVEC endothelial cells with rIL-35 or CM from IL-35 overexpression or knocked-down cells and found that the tube formation rate did not significantly change (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Considering that accumulation of neutrophils in HCC tissues has been reported to increase the production of angiogenesis factors and facilitate microvessel formation, we stimulated HUVECs with CM from the cocultivation of neutrophils and HCC cells and found that CM from neutrophils cocultured with IL-35 overexpression HCC cells could enhance tube formation (tube density: 212\u0026thinsp;\u0026plusmn;\u0026thinsp;31 vs. 141\u0026thinsp;\u0026plusmn;\u0026thinsp;19, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0024, tube branch: 365\u0026thinsp;\u0026plusmn;\u0026thinsp;27 vs. 238\u0026thinsp;\u0026plusmn;\u0026thinsp;24, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Conversely, the CM from IL-35 knocked-down HCC cells cocultured with neutrophils could significantly inhibit the tubule formation of endothelial cells (tube density: 119\u0026thinsp;\u0026plusmn;\u0026thinsp;19 vs. 169\u0026thinsp;\u0026plusmn;\u0026thinsp;23, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0056, tubule branch: 229\u0026thinsp;\u0026plusmn;\u0026thinsp;24 vs. 315\u0026thinsp;\u0026plusmn;\u0026thinsp;32, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0013) (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). These results demonstrated that IL-35 stimulated neutrophils to produce angiogenesis factors.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further explore this, we isolated neutrophils from patients with HCC, stimulated them with human IL-35, and revealed that the expression of genes related to angiogenesis and adhesion factors in neutrophils was significantly increased. The KEGG pathway enrichment map showed that after neutrophils were stimulated by IL-35, the pathways of epidermal growth factor receptor (EGFR) and vascular endothelial growth factor (VEGF) were significantly activated. The FGF2 protein was demonstrated to be the most significantly elevated angiogenic factor, with the expression of the FGFR3 and FGFR4 receptors of FGF2 being also increased by 574 and 65 times, respectively. This was further confirmed using WB and ELISA analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e6\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eTo determine whether FGF2 plays a significant role in mediating IL-35 to promote angiogenesis, we have conducted inhibition experiments. When anti-IL-35 and anti-FGF2 neutralizing antibodies were used, the tube formation rate was shown to be significantly abrogated. Furthermore, after IL-35 knocked-down HCC cells were cocultured with neutrophils in the presence of rIL-35, the tube formation rate was demonstrated to be significantly elevated. However, when anti-FGF2 neutralizing antibody was added to the above CM, tube formation was blocked (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e6\u003c/span\u003eC).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.7. Anti-IL-35 antibody enhances the efficacy of PD1 antibody in the tumor model in mouse.\u003c/h2\u003e \u003cp\u003eDrug combination is an important way to explore better treatments of liver and other cancers. Therefore, we aimed to explore whether the IL-35 antibody could enhance the effect of the administration of the PD1 antibody in the treatment of HCC.\u003c/p\u003e \u003cp\u003eWe found that subcutaneous tumor models were established by Hepa1-6 cell in immunocompetent mice, and tumor growth was slightly inhibited after treatment with anti-IL-35 or PD1 neutralizing antibody. However, we observed more dramatic and durable responses, compared with the responses in the control treatment, when the anti-PD1 antibody was combined with the anti-IL-35 neutralizing antibody (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e7\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe infiltration of CD8\u003csup\u003e\u003cb\u003e+\u003c/b\u003e\u003c/sup\u003e T-cells was increased after treatment with the IL-35 and PD1 antibodies (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). In contrast, neutrophil infiltration was decreased after treatment with the IL-35 antibody (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), whereas no effect was observed in neutrophil infiltration after administration of the PD1 antibody. Nevertheless, we did not observe any significant difference in other cells, including macrophages and Treg cells. IHC analysis showed that single treatment with anti-PD1 antibody or anti-IL-35 antibody increased the infiltration of CD8\u003csup\u003e+\u003c/sup\u003e T cells in the tumor. And the infiltration of CD8\u003csup\u003e+\u003c/sup\u003e T cells was further increased in the combination treatment group compared with the single treatment group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). The neutrophil infiltration in the anti-IL-35 group and the combined treatment group was observed to be significantly lower than that in the control group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). However, there was no significant difference shown in neutrophil infiltration between the two groups. The PD1 antibody treatment group was also shown to have no effect on neutrophil infiltration (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e7\u003c/span\u003eB,C).\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe tumor microenvironment(TME) is composed of cells such as cancer cells, tumor-associated macrophages (TAM), natural killer (NK) and dendritic cells (DC), T and B lymphocytes, etc. Apart from that, blood, lymphocytes, collagen fibers and glycoproteins constitute the extracellular matrix(ECM), while cancer-associated fibroblasts(CAFs) and mesenchymal stem cells lead to remodeling of the ECM. Cytokines, chemokines and other effectors exert either promotion or inhibition effects and are involved in the construction and multiple functions of the TME[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. We found that IL-35 facilitates intratumoral neovascularization by neutrophils recruitment and reshapes the immune microenvironment by decreasing the infiltration of CD8\u003csup\u003e+\u003c/sup\u003eT cells to promote the progression of HCC. In addition, the combination of anti-IL-35 and anti-PD1 antibodies showed a synergistic anticancer effect.\u003c/p\u003e \u003cp\u003eOur results found that high expression of IL-35 in HCC is an independent risk factor for recurrence, while GP130 and IL-12Rβ2 receptors are expressed in HCC tissues, suggesting that IL-35 has a structural basis for autocrine functions in HCC. We further found that patients with high IL-35 (and its receptors) expression in tumor tissues have the worst prognosis, indicating that IL-35 might promote HCC progression.\u003c/p\u003e \u003cp\u003eDespite the cancer-killing effect of neutrophils, in most cases the high level of neutrophil infiltration in solid tumors implies a poor clinical prognosis for patients[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In this study, the local infiltration of neutrophils and MVD are significantly increase, whereas that of CD8\u003csup\u003e\u003cb\u003e+\u003c/b\u003e\u003c/sup\u003e T cells is significantly decreased in tumors with high expression of IL-35. \u003cem\u003eIn vivo\u003c/em\u003e, IL-35 is closely associated with neutrophils infiltration and intratumoral angiogenesis, suggesting that the increased expression of IL-35 is related to local immune escape and tumor angiogenesis in HCC. Our study also showed a significant positive correlation between neutrophils and angiogenesis in HCC, along with a significant increase in angiogenesis and neutrophil infiltration in HCC tissues with high expression of IL-35. Tumor angiogenesis can also be indirectly affected by neutrophils, macrophages, Treg cells, stellate cells, and other interstitial cells known to affect TME [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. It has been reported that IL-35 can also affect the secretion of CXCL1 and CXCL8 by monocytes and promote angiogenesis in pancreatic cancer [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Our results show that both direct stimulation of rIL-35 and IL-35 overexpression or knocked-down CM had no significant effect on microvessel formation, indicating that the tumor did not directly stimulate the formation of vascular endothelium by secreting IL-35. Therefore, we considered that tumor secretion of IL-35 promotes intratumoral angiogenesis through indirect mechanisms.\u003c/p\u003e \u003cp\u003eIt has reported that IL-35 can indirectly promote neutrophil polarization to TAN2 and tumor infiltration in mouse models of melanoma and HCC[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Our study showed that IL-35 is associated with neutrophil infiltration, indicating that IL-35 promotes intratumoral angiogenesis through neutrophils. Other studies reported that neutrophils (mainly TAN2) can participate in the reconstruction of the tumor extracellular matrix by secreting matrix metalloproteinases (MMPs), neutrophil elastase(NE), and cathepsin G, thus promoting tumor metastasis [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Neutrophils in breast cancer have been demonstrated to bind to circulating tumor cells (CTCs) entering the circulatory system through vascular cell adhesion protein 1 (VCAM1) to promote metastasis [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The results of in \u003cem\u003evitro\u003c/em\u003e experiments illustrated that IL-35 with neutrophils enhances the adhesion of HCC cells to endothelial cells. Since IL-35 with neutrophils enhances the adhesion of tumor cells to epithelial cells, this explains the in \u003cem\u003evivo\u003c/em\u003e experiments in which IL-35 promotes neutrophil-induced lung metastasis of HCC. Factors involved in neutrophil tumor infiltration and angiogenesis might be CCL3 and FGF2. CCL3-recruited neutrophils promote breast cancer lung metastasis, and neutrophil-secreted FGF2 in colorectal cancer lead to the development of liver metastasis[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].Our results showed that IL-35 can affect the expression of CCL3 in HCC cells and promote the infiltration of neutrophils in tumors. Meanwhile, IL-35 can lead to tumor vascular growth and metastatic tumor colonization by promoting the secretion of FGF2 angiogenic factor of neutrophils. Moreover, IL-35 was demonstrated to stimulate the expression of the FGFR3 and FGFR4 neutrophil FGF2 receptors, indicating that there might be a positive feedback loop in TME regarding the IL-35-stimulated secretion of neutrophil FGF2.\u003c/p\u003e \u003cp\u003eTumor immunotherapy represented by immune checkpoint antibody therapy is a hot spot of tumor immunotherapy in recent years. So far, it has been reported to achieve exciting results in a variety of tumors, including HCC, and has received extensive attention [\u003cspan additionalcitationids=\"CR39 CR40 CR41 CR42 CR43\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. However, immunotherapy only benefits no more than 20% of liver cancer patients, and approximately 30% of liver cancer patients develop intrinsic resistance[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e].Combination drugs against multiple key targets might be a good solution. IL-35 could significantly reduce the infiltration of CD8\u0026thinsp;+\u0026thinsp;T cells in tumor tissues of immunocompetent mouse, suggesting that IL-35 have a role in suppressing tumor immunity. We found that the IL-35 antibody could enhance the efficacy of PD1. IL-35 is mainly expressed by Treg and Breg and exerts anti-tumor effects in the tumor microenvironment by suppressing immunosuppression, leading to PD1 antibody resistance. Treg derived IL-35 synergistically promotes BLIMP-dependent depletion of CD8\u003csup\u003e+\u003c/sup\u003eT cells with IL-10, limiting effective immune antitumor effects[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. IL-35 produced by Breg in pancreatic ductal adenocarcinoma inhibits the infiltration and effector function of CD8\u003csup\u003e+\u003c/sup\u003e T cells within the tumor by activating the transcription factor STAT3 to inhibit the expression of CXCR3, CCR5, and IFNγ[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. STING agonist therapy in pancreatic cancer results in elevated IL-35 production by Breg, and the TING-IL-35 axis reduces NK cell proliferation and attenuates NK-driven antitumor responses[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e].It appears that IL-35 is the culprit for the poor results of immune anti-tumor therapy. Our results further complement the role of IL-35 in HCC TME. HCC cell-derived IL-35 can play a role in angiogenesis, metastasis, and immune evasion by autocrine-induced self-production of CCL3, which recruits neutrophils for infiltration in tumors. Antibody therapy with IL-35 enhances the infiltration of anti-tumor immune cells, reduces the infiltration of tumor-associated neutrophils, and decreases the inhibitory effect of IL-35 on anti-tumor immune cells[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e].In addition, inhibition of IL-35 blocked neutrophil-mediated intra-tumor angiogenesis, reducing nutrient supply to tumor cells as well as metastasis[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].IL-35 antibody combined with PD1 antibody in HCC has a synergistic effect, inhibiting intra-tumor angiogenesis, reversing the immunosuppressive microenvironment, and enhancing the efficacy of PD1 antibody therapy.\u003c/p\u003e \u003cp\u003eWe conclude that IL-35 secreted by HCC cells recruits neutrophils to accumulate in the TME to cause lung metastasis through autocrine induction of CCL3 production by their own cells, and that neutrophils induce tumor angiogenesis through secretion of FGF2. Meanwhile, IL-35 and neutrophil cells exert simultaneous immunesuppressive anti-tumor effects. IL-35 antibody combined with PD1 antibody can inhibit the growth of HCC by increasing the infiltration of CD8\u0026thinsp;+\u0026thinsp;T cells with decreasing neutrophil recruitment in the TME and reducing tumor angiogenesis(Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThis study also has some limitations. At first, the IL-35 antibody is an anti-single subunit antibody, and it is difficult to fully assess the inhibition effect of IL-35. Hence we could not rule out the possibility of blocking the effect of IL-12 or IL-27. Second, IL-35 is a widely secreted cytokine. The use of an IL-35 antibody could not only affect IL-35 secreted by HCC but also IL-35 secreted by other cells, including Tregs. Finally, the clinical samples were obtained from a retrospective single-center study, and as such verification of these clinical data require an extended study.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eOur data show that the high expression of IL-35 in patients with HCC is an important tumor promoter. Combined treatment with anti-IL-35 and anti-PD1 antibodies have potential therapeutic effect against HCC (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIL-35\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003einterleukin-35\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAFP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ealpha fetal protein\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCA19-9\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecarbohydrate antigen 19\u0026thinsp;\u0026minus;\u0026thinsp;9\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCEA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecarcinoembryonic antigen\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTBIL\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003etotal bilirubin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eALB\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ealbumin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBCLC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eBarcelona Clinic Liver Cancer staging system\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHCC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHepatocellular carcinoma\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTMA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003etissue microarray\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePD 1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eprogrammed cell death protein 1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePD-L1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eprogrammed cell death protein ligand 1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDCs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003edendritic cells\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTME\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003etumor microenvironment\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCTL\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecytotoxic T-lymphocytes\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIHC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eimmunohistochemistry\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCO-IP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCoimmunoprecipitation\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHUVECs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHuman umbilical vein endothelial cells\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eOS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eoverall survival\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRFS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003erecurrence-free survival\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePVTT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eportal vein tumor thrombus\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMVI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emicrovascular invasion\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMVD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emicrovessel density\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCCL3\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eC-C motif chemokine ligand 3\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eEGFR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eepidermal growth factor receptor\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eVEGF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003evascular endothelial growth factor\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFGF2\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003efibroblast growth factor 2\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIFN\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003einterferon\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCTCs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecirculating tumor cells\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eVCAM1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003evascular cell adhesion protein 1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNETs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eneutrophils extracellular traps\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTANs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003etumor-associated neutrophils\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eECM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eextracellular matrix\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCAFs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecancer-associated fibroblasts\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eICIs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eimmune checkpoint inhibitors.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnimal experiment protocols were approved by the ethics committee of the\u0026nbsp;XX\u0026nbsp;Hospital of\u0026nbsp;XX\u0026nbsp;University. And the study protocol was approved by the ethics committee of the\u0026nbsp;XX\u0026nbsp;Hospital of\u0026nbsp;XX\u0026nbsp;University\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eReasonable requests for data will be made available for review.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science, Foundation of China (grant number\u0026nbsp;XX,XX,XX)，the Clinical Research Project of XX Hospital (NO.XX and XX).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eXX\u0026nbsp;performed the in vivo and cellular experiments and wrote the manuscript.\u0026nbsp;XX,\u0026nbsp;XX\u0026nbsp;and\u0026nbsp;XX\u0026nbsp;discussed and participated in the data collection\u0026nbsp;and\u0026nbsp;analysis.\u0026nbsp;XX\u0026nbsp;collected data and performed some statistical analysis.\u0026nbsp;XX,\u0026nbsp;XX,\u0026nbsp;XX\u0026nbsp;helped to design the experiments.\u0026nbsp;XX,\u0026nbsp;XX,\u0026nbsp;XX\u0026nbsp;and\u0026nbsp;XX\u0026nbsp;designed the manuscript, evaluated the results, and revised the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eVogel A, Meyer T, Sapisochin G, Salem R, Saborowski A. 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Nature. 2022;612(7938):141-7.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"hepatocellular carcinoma, interleukin 35, immunotherapy, neutrophil, anti-PD1","lastPublishedDoi":"10.21203/rs.3.rs-3856191/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3856191/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eA growing number of therapeutic strategies against hepatocellular carcinoma (HCC) have emerged. However, their efficacy remains limited. This study investigated the mechanism of interleukin-35 (IL-35) in the progression of HCC and its potential application in HCC treatment.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThe expression of IL-35,Gp130 ,IL12-Rβ2, CCL3,etc. in HCC tissues was detected by immunohistochemistry(IHC), and the expression of IL-35 in HCC cell lines was detected by fluorescence assay. Kaplan-Meier survival analysis of IL-35 and its receptor in relation to overall survival(OS) and recurrence free survival(RFS) in patients with HCC. The mouse subcutaneous tumor models to study the effects of IL-35 on HCC growth and immune cells. Western blot were used to detect the expression IL-35, CCL3, FGF2, and flow cytometric plot were performed to explore the immune cells infiltration in the tumor tissue.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eHigh expression of IL-35 in patients with HCC was associated with poor prognosis. Furthermore, IL-35 could facilitate tumor progression by affecting neutrophil infiltration, angiogenesis, and CD8\u0026thinsp;+\u0026thinsp;T-cell infiltration. Additionally, CCL3 was a key factor mediating the recruitment of neutrophils by IL-35. FGF2 derived from neutrophils stimulated by IL-35 promoted intratumoral angiogenesis. IL-35 also facilitated the adhesion of tumors to endothelial cells, with neutrophils further enhancing this effect both. Anti-IL-35 antibody combined with anti-PD1 antibody significantly enhanced which therapeutic effect in HCC.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eOur data show that the high expression of IL-35 in patients with HCC is an important tumor promoter. Combined treatment with anti-IL-35 and anti-PD1 antibodies have potential therapeutic effect against HCC.\u003c/p\u003e","manuscriptTitle":"Interleukin 35 promotes progression of hepatocellular carcinoma by recruiting neutrophils","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-30 18:45:06","doi":"10.21203/rs.3.rs-3856191/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9526caca-3ce5-40ad-896b-be10ad8b3f9b","owner":[],"postedDate":"January 30th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-02-10T06:47:34+00:00","versionOfRecord":[],"versionCreatedAt":"2024-01-30 18:45:06","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3856191","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3856191","identity":"rs-3856191","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}