Co-expression analysis of P35 and EBI3 subunits in hepatocellular carcinoma could be used as a proxy for the level of IL-35.
At present, there is no high-quality specific antibody for IL-35. So in order to investigate the level of IL-35, we detected the expression of P35 and EBI3 in continuous tissue sections of HCC samples, which has been reported to be a reliable method in other research studies[22, 25]. As IL-12, IL-27, and IL-35 are known to share common subunits, we detected all 4 subunits, namely P35, EBI3, P40, and P28 in TMA of patients with HCC. The P35 and EBI3 proteins were observed to be mainly located in the cytoplasm, widely expressed in tumor cells, as well as in stromal cells. Whereas the expression of P35 and EBI3 was demonstrated to be lower in paracancerous compared with cancerous tissues of the same patient, we found that the expression level of both P28 and P40 in HCC was very low, and mainly located in stromal cells（FIG1.A,B）.
The proportional structure expression map of EBI3 and P35 was shown to be highly similar, but obviously different from that of P28 and P40. We further observed that the staining scores of EBI3 and P35 were strongly correlated (r = 0.698, P < 0.001). However, there was a poor association between EBI3 and P28 (r = 0.08, P < 0.05), as well as between P35 and P40 (r = 0.042, P < 0.001) (FIG1.D).
Furthermore, we used the HCCLM3, MHCC97H, SMMC-7721, and HUH7 cell lines to carry out cellular immunofluorescence experiments. We accordingly found that P35 and EBI3 were mainly expressed in the cytoplasm, and not in the nucleus. The expression intensity of P35 and EBI3 was shown to be basically the same, with the spatial expression sites basically overlapping. The expression intensity of these 2 proteins was found to be higher in HCCLM3 and MHCC97H, but low in SMMC-7721 and HUH7 (FIG1.C). In order to eliminate the effect of the cosubunits of IL-12 and IL-27 on the detection of IL-35, we used the CO-IP technique to study the structural relationship of the 4 subunits in 4 cases of HCC. The P35 antibody was demonstrated to successfully immunoprecipitate EBI3 but rarely bound to P40 and P28. Similarly, the same phenomenon was observed in CO-IP experiments in the MHCC97H and HCCLM3 cell lines (FIGS1.B).
Overexpression of IL-35 in hepatocellular carcinoma was an independent risk factor for prognosis.
We then analyzed the baseline characteristics of the 360 patients with HCC. Our study included 202 men (56.1 %) and 158 women (43.9 %), with an average age of 54.36 ± 11.038 (26–85) years and a median age of 54 years. The average overall survival (OS) time was 51.87 ± 0.996 months, and the median OS was 49 ± 7.19 months. The average recurrence-free survival (RFS) time was 41.91 ± 1.27 months, and the median RFS was 22.0 ± 1.837 months (Table1.).
The IHC staining score ++/+++ was stratified as high expression. 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 %) (FIG1.D). Significant correlations were found between the high expression of IL-35 and advanced Barcelona clinic liver cancer (BCLC) stage. Moreover, we also found that the level of serum alpha-fetoprotein (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), microvascular invasion (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) (Table2.). Therefore, the expression of IL-35 in HCC was considered an important reference index for judging prognosis.
Binding of cytokines to receptors is known to be an important link for the functional role cytokines play in cells. After binding to receptors, IL-35 is known to activate the intracellular signal transduction pathway. Therefore, it is of great significance to explore the expression of the GP130 and IL-12 Rβ2 receptors of IL-35 in HCC. At present, there has been no report on the expression of receptors of IL-35 in HCC. We found that the expression of GP130 and IL-12 Rβ2 in HCC was closely related (r = 0.39, P = 0.023)(FIG2B). Based on the expression of IL-35 and its receptors in HCC, we divided the 360 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(FIG2C).
Overexpression of IL-35 in patients with hepatocellular carcinoma was closely related to the infiltration of neutrophils and CD8+ in tumor microenvironment.
We further explored the relationship between the level of IL-35 and TME in HCC. Our results showed that the infiltration of neutrophils in HCC tissues with high levels of IL-35 was significantly higher than that in the low expression group (5.31 vs. 14.80 ± 1.34, P < 0.001). 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). Importantly, the number of microvessel density (MVD) labeling by CD34 in patients with overexpression of IL-35 was demonstrated to be significantly increased (86.63 ± 4.789 vs. 56.54 ± 2.308, P < 0.001). Additionally, the number of neutrophils infiltrating the tumor was shown to be positively correlated with MVD (r = 0.301, P < 0.001), suggesting that neutrophil infiltration might be an important factor in tumor angiogenesis(FIG2A-C).
IL-35 facilitated tumor progression by affecting neutrophil infiltration, angiogenesis, and CD8+ T-cell infiltration in a mouse 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 overexpressing cell lines. We also constructed IL-35 overexpressing and knocked-down Hepa1-6 cells. These HCC cell lines were also used to detect the expression of the 2 subunits of the IL-35 receptor, GP130 and IL-12 Rβ2. These were observed to be structural foundations for liver cancer cells in affecting the function of tumor cells through autocrine IL-35(FIGS1A).
First, we used the CCK8 transwell and wound healing to test 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 mm3: 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 mm3: 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)(FIG3). 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(FIG4). Therefore, we speculated that IL-35 might promote the progression of liver cancer by affecting TME.
Meanwhile, we found that the infiltration of neutrophils (169 ± 47 vs. 62 ± 15, P < 0.001), as well as the number of MVD (149 ± 39 vs. 33 ± 21, P < 0.001) were both significantly increased in immunodeficient or immunocompetent mouse models injected with tumor cells overexpressing IL-35. In the IL-35 knocked-down group, the number of neutrophils infiltrating the tumor (31 ± 12 vs. 189 ± 43, P < 0.001), and the amount of MVD (43 ±21 vs. 125 ±34, P < 0.001) were shown to be significantly decreased(FIG3B). Thus, we assumed that IL-35 could promote intratumoral neovascularization by recruiting the infiltration of neutrophils in tumors. To test this hypothesis, we used a LY6G neutrophil antibody to deplete neutrophils in an IL-35-overexpressing subcutaneous tumor, and found that depleting neutrophils could significantly reverse the promoting effect of the overexpression of IL-35(FIG4D).
In addition, we also found that the number of infiltrating CD8+ T-cells was significantly decreased in the overexpression group, whereas it was significantly increased in the knocked-down group.
IL-35 promoted neutrophil infiltration by increasing the expression of CCL3 in vitro
We used a 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 shown to be decreased by 64.5 % and 56.3 % (P < 0.05), whereas the overexpression in CM increased by 3.97 and 4.67 times, respectively (P < 0.05). However, we noted that the recombinant IL-35 (rIL-35) had no significant effect on neutrophil chemotaxis (P > 0 05). These results showed that HCC-related IL-35 did not directly affect neutrophil infiltration(FIG5A).
We then aimed to further explore the pathway through 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(FIGS2A). We further found that following overexpression of IL-35, the intracellular levels of the CCL3 protein were significantly increased, whereas after knocking down IL-35, the intracellular levels of the CCL3 protein were shown to be significantly decreased. We further confirmed that CCL3 was significantly increased in IL-35-overexpressing patients (P < 0.012, r=0.431) (FIG5B). In order to verify whether IL-35 chemotactically affected neutrophils through the expression of CCL3, we carried out a CCL3 antibody block test. We accordingly discovered that CCL3 could significantly enhance the chemotactic effect on neutrophils, as the CCL3 antibody intervention test was demonstrated to significantly reduce the chemotactic effect of CM on neutrophils(FIG5A). In summary, we found that IL-35 could promote the chemotactic effect of neutrophils by promoting the expression of CCL3 in HCC.
IL-35 stimulated neutrophil secretion of FGF2 to promote angiogenesis
To illustrate the roles and underlying mechanism of IL-35 in tumor angiogenesis, we carried out a tube formation experiment in vitro. First, we stimulated HUVEC endothelial cells with rIL-35 or CM from IL-35- overexpressing or knocked-down cells, and found that the tube formation rate did not significantly change. This suggested that IL-35 did not directly stimulate the formation of vascular endothelium. Considering that accumulation of neutrophils in HCC tissues has been reported to increase the production of angiogenesis factors and facilitate microvessel formation, we speculated that IL-35 might indirectly affect tumor angiogenesis by stimulating neutrophils. To further assess this hypothesis, we stimulated HUVECs with CM from the coculturing of neutrophils and HCC cells. We found that CM from neutrophils cocultured with IL-35-overexpressing 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).(FIG5C) 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 then sequenced them. Our sequencing results were subjected to GO and KEGG analysis, where it was 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 (FIGS2B,FIG6A). This finding suggested that there might be a mechanism by which IL-35 stimulates the positive feedback secretion of FGF2 by neutrophils. This was further confirmed by WB analysis.
To explore whether FGF2 plays a decisive role in mediating IL-35 to promote angiogenesis, we performed blocking experiments. When anti-IL-35 and 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, tubule formation was blocked.(FIG6B)
IL-35 facilitated the adhesion of tumor to endothelial cells, with neutrophils further enhancing this effect in vitro.
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 demonstrated to be significantly increased. In contrast, knocking down IL-35 strongly inhibited the number of HCCLM3 or MHCC97H cells adhered to endothelial cells. At the same time, we also found that when neutrophils were added to overexpressing IL-35 trials, the number of SMMC-7721 or HUH7 cells adhered to endothelial cells was increased by 46 % and 54 %, respectively (P < 0.001). However, there was no significant change observed in the adhesion of tumor cells cocultured with neutrophils in the knocked-down group (P > 0.05). These results showed that IL-35 could increase the adhesion of hepatocellular carcinoma cells to endothelial cells, and neutrophils could further enhance this effect. In addition, we further verified this finding using the Hepa1-6 mouse HCC cell line (FIG7A).
The lung metastasis model of the tail vein in nude mice further showed the promoting ability of IL-35 and neutrophils.
In the in vivo experiment, we first labeled tumor cells with a fluorescent dye. Following the intravenous injection of HCC cells into the tail vein of nude mice, the remaining tumor cells in the lung tissue were observed using a fluorescence tracer. We found that there was no significant difference in the retention of tumor cells in each group at 30 min; however we observed a significant difference in the number of tumor cells stranded in lung tissue 24 h later. The number of retained tumor cells in the coinjection group (279 ± 53)was significantly higher than that in the overexpression (103 ± 31) and control (62 ±21) groups (P < 0.001), whereas the number of tumor cells retained in the overexpression group was also significantly higher than that in the control group. We observed that in the coinjection group, a large number of tumor cells adhered directly to neutrophils. We collected gross specimens of lung tissue, and found that the number of metastatic nodules after injecting HUH7-OE was significantly higher than that in the control group (FIG7A). After simultaneous injection of mixed cells of neutrophils and OE strains, the number of pulmonary metastatic nodules was demonstrated to be further increased. This number was significantly higher than that of the OE stable lung metastasis model, consistent with the results of HE staining of lung metastasis nodules. Meanwhile, we found that the rate of lung metastasis was significantly decreased when IL-35 was knocked down (FIG7B).
Anti-IL-35 neutralizing antibody enhanced the efficacy of PD-1 antibody
The combined use of drugs is an important way to explore better treatments of liver and other cancers. Therefore, we aimed to explore whether an IL-35 antibody could enhance the effect of the administration of the PD-1 antibody in the treatment of HCC.
We found that in the subcutaneous tumor model of immunocompetent mice recipients bearing Hepa1-6 cells, tumor growth was slightly suppressed after treatment with either an anti-IL-35 or a PD-1 neutralizing antibody. However, we observed more dramatic and durable responses, compared with the responses in the control treatment, when the anti-PD-1 antibody was combined with the anti-IL-35 neutralizing antibody(FIG8A). We also constructed an in vivo model using the IL-35-OE Hepa1-6 cell line, followed by the administration of both the anti-PD-1 and anti-IL-35 antibodies. Our results showed that the anti-PD-1 antibody combined with treatment with the anti-IL-35 antibody could reverse the increased tumor growth induced by IL-35OE Hepa1-6 cells in C57BL/6 mice (FIGS3).
As shown in Figure8B-C, the infiltration of CD8+ T-cells was increased after treatment with the IL-35 and PD-1 antibodies (P < 0.001). In contrast, we found that 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 PD-1 antibody. Nevertheless, we did not observe any significant difference in other cells, including macrophages and Treg cells. Immunohistochemical analysis showed that treatment with the PD-1 and IL-35 antibodies could increase the infiltration of CD8+ T-cells in the tumor, with the combined treatment group being shown to further increase the infiltration of CD8+ T-cells compared with the single treatment group (P < 0.001). The neutrophil infiltration in the IL-35 antibody 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 2 groups. The PD-1 antibody treatment group was also shown to have no effect on neutrophil infiltration.