Interleukin 35 promotes progression of hepatocellular carcinoma by recruiting neutrophils

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.

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.

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.

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

PD1

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.

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], [6], [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], [9], [10], [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]. The current study shows that IL-35 is thought to have immunosuppressive effects, inhibiting the killing activity of multiple effector immune cells in TME. 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]. Blocking IL-35 might lift the inhibition of immune effector cells and enhance the immunotherapeutic effect, resolving the problem of ineffective PD1 monoclonal antibody. However, the effects of IL-35 on the HCC tumor microenvironment on tumor growth and immune responses are 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.

Materials and methods

Human samples, and collection of patient clinical data

A total of 360 patients with primary HCC who underwent radical resection at Zhongshan Hospital affiliated to Fudan 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.

Cell lines, mouse xenografts

LO2, HepG2, Huh7, PLC, Hep3B, MHCC97L, MHCC97H, HCCLM3 HCC and Hep1–6 cell lines were obtained from the Liver Cancer Institute, Zhongshan Hospital, Fudan University. All cell lines were cultured in a reasonable conditioned media at 37 °C in a humidified incubator containing 5 % CO₂, as previously described.

Male C57BL/6 and BALB/c-nu/nu mice (4–6 weeks old, from Shanghai Institute of Material Medicine, Chinese Academy of Science) were housed under pathogen-free conditions. Animals were cared for in accordance with the guidelines established by the Shanghai Medical Experimental Animal Care Commission. All experimental protocols were approved by the Zhongshan Hospital Research Ethics Committee. All experimental procedures involving animals were approved by the Animal Care and Use Committee of Zhongshan Hospital, Fudan University, China.

HCC-LM3 and Huh7 cells were subcutaneously injected into BALB/c-nu/nu mice to establish HCC transplanted tumor model. The mice were divided into four groups: HCC-LM3-Ctrl, HCC-LM3-KD, Huh7-Ctrl, and Huh7-OE with five mice in each group. On week 5, subcutaneous tumors for the xenograft model were removed and dissected. Hepa1–6 cells were subcutaneously injected into C57BL/6 mice to establish a tumor model. The mice were euthanized in the fifth week, and tumor samples were collected for subsequent experimental procedures. The activity, behavior, and diet of the mice were monitored daily before and throughout the experiment. Tumor dimensions (long and short diameters in mm) were measured every four days using vernier calipers. Tumor volume (V) was calculated using the formula V= ab²/2, and tumor growth curves were plotted.

IHC and immunofluorescence

The P35, EBI3, GP130, IL-12 Rβ2, P40, and P28 antibodies were used to detect the level of protein expression in tumor tissues using IHC, based on the protocol used in our previous study.

A number of pathology specialists were invited to evaluate the results, and H-scores were used to quantify P35, EBI3, GP130, IL12Rβ2, and other indexes. According to the calculation score of intensity and area, the intensity of positive staining was divided into 4 grades: 0, no staining; 1, weak staining; 2, moderate staining; and 3, strong staining. This was divided into 4 levels according to the area of colored cells: 0, <1 %; 1, 1–25 %; 2, 26–50 %; 3, >50 %. Dyeing was divided into the score of the intensity area, with the score of uneven dyeing requiring to be accumulated. The comprehensive score was divided into 4 grades: negative (-), 0 points; weak positive (+), 1–2 points; medium positive (+ +), 3–4 points; and strong positive (+ +), 5–9 points. Immune cells, such as CD4⁺, CD8⁺, CD34⁺, CD68⁺, neutrophils (CD66b⁺), and FOXP3⁺ cells were used to evaluate the level of infiltration. Five typical visual fields were randomly selected on the patient chip, and the average value was calculated as the immune cell count of the patient. The cutoff value of the statistical score was calculated using the X-tile software. The results of the chip were independently evaluated by 2 professionals; any contradictory results were discussed, and a joint decision was made.

Immunofluorescence was performed on the HCC cell line, where we explored the expression level of EBI3 and P35. Slides were prepared in the same manner as for IHC before incubation with antibodies. Next, incubation with anti-mouse and anti-rabbit antibodies was performed.

ELISA, WB, coimmunoprecipitation (CO-IP), and quantitative reverse transcription PCR (qRT-PCR)

Western blotting (WB) was performed as previously described. Briefly, we generated total cell lysates. Proteins were separated on SDS-PAGE gels and then transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were washed and blocked. Next, membranes were incubated with primary and secondary antibodies, detected using enhanced chemiluminescent (ECL) substrate, and processed using the Image Lab software.

Coimmunoprecipitation (CO-IP) was performed as previously describe [50]. HCC-LM3 cells were seeded in 100 mm culture dishes. When the cell density reached approximately 70 %, a lysis buffer without SDS was added to lyse the cells or patient tissue. The lysate was thoroughly mixed and centrifuged to collect the supernatant, followed by the addition of 5× loading buffer. The mixture was heated at 95 °C for 5 min. The remaining supernatant was transferred to a new EP tube, and the primary antibody was added. The solution was gently mixed and incubated overnight at 4 °C. Agarose beads were then added and incubated for an additional 2 h at 4 °C.

The precipitate was collected by centrifugation at 14,000 rpm and 4 °C, and the supernatant was discarded. The precipitate was washed several times with pre-cooled PBS. Subsequently, 2× loading buffer was added to the precipitate, which was denatured by heating at 95 °C for 5 min. The prepared samples were analyzed using western blotting.

ELISA kits were used to detect the levels of secretory IL-35, CCL3, and FGF2 in the supernatants of cell cultures according to the manufacturer's instructions.

Quantitative reverse-transcription PCR was performed using a SYBR PrimeScript reverse-transcription PCR Kit (Takara Bio, Shiga, Japan) in accordance with the manufacturer’s instructions. The RT2 profiler array was probed using the Profiler PCR Array System and SYBR green/fluorescein quantitative PCR Master Mix (SABiosciences) in an ABI 7900 sequence analyzer (Applied Biosystems, Carlsbad, CA, USA) in accordance with the manufacturer’s protocol.

Wound-healing assay and transwell cell counting Kit-8

Analyses of cell proliferation were performed using the Cell Counting Kit-8 (CCK-8; Dojindo). Wound-healing assay and noncoated transwell chambers (BD Pharmingen) were used to assess the migratory ability of cells. The invasion ability of cells was assessed using Matrigel-coated transwell chambers (BD Pharmingen).

Cell adhesion assay and endothelial tube formation assay

Human umbilical vein endothelial cells (HUVECs) were cultured with ECM, in slices, and allowed to reach full confluence. Then, the monolayers were stained with a CytoPainter Cell Tracking Staining Kit (Abcam) and inserted into a flow chamber. HCC cells were labeled with Calcein AM (Donjindo) according to the manufacturer’ s instructions. Briefly, 1 × 10⁵ labeled HCC cells were added to HUVECs and cocultured for 2 h at 37 °C in a cell incubator. Next, the culture medium was removed, and the unattached tumor cells were washed twice with PBS before being replaced with complete culture medium. Adhered tumor cells on the monolayers were imaged using a fluorescence microscope and their average number was calculated in 5 random fields.

In vitro endothelial tube formation assays were performed using collagen gel. First, Matrigel was kept melt on ice, and then 250 mL of melted matrix glue was gently poured into each hole of a precooled 24-well plate to avoid bubbles. The plate was then placed into a 37 °C incubator for 35 min for solidification. HUVECs (8 × 10⁴ cells/well in a 24-well plate) were cultured on the surface of collagen gel using endothelial cell medium (ECM) and other conditioned media. The total tube length, the number of branch points, and the number of loops in the tube-like structures were quantified in 24 fields per group.

Flow cytometry

First, obtain tumor tissue samples from mice, cut the tissue into small pieces, and perform collagenase digestion to dissociate the cells into a single-cell suspension. Next, use a cell strainer to filter out large debris, resulting in a uniform single-cell suspension. Then, add pre-conjugated fluorescent antibodies to stain for various cell surface markers, such as Ly6G, CD11b, CD4, and CD8 antibody(BioLegend). After staining, the cell suspension is typically washed to remove unbound antibodies. Finally, analyze the stained cells using a flow cytometer, where the instrument’s laser excites the fluorescent signals. Cell types, counts, and states are assessed based on forward scatter (FSC), side scatter (SSC), and specific fluorescence signals. Data processing and analysis were performed using FlowJo 10.8.1 software.

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. The p-value is calculated using a t-test. P < 0.05 was considered significant. *P < 0.005, **P < 0.001, ***P < 0.0001.

Results

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 (Table 1). 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.

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/≥35 g/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. 1A and 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. 1D). 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. S1B). Our results identified IL-35 as the factor that is highly expressed in HCC, rather than IL-12 or IL-27.

Fig. 1.

High expression of IL-35 is associated with poor prognosis in HCC patients. Serial 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. A. 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. B. 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 (absent -, low +, middle ++, and high +++) are shown. C. 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. D. 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.

We referred to the method of grouping based on IL-35 expression in previous studies [34]. 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. 1C). The IL-35 high expression group had worse OS and RFS compared to the low expression group(Fig. 1C). 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). Therefore, the expression of IL-35 in HCC was considered an important reference index for prognosis.

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. 2B). 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. 2C).

Fig. 2.

High IL-35 expression in HCC correlates with the infiltration of neutrophils, the formation of MVD, and the decrease of CD8⁺T cell. A. 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. B. 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. C. Quantification of MVD, CD66b⁺, CD8 counts per field. Pearson correlation analysis of CD34⁺ and CD66b⁺was 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, #PP > > 0.05, *PP << 0.005, **PP < < 0.001, ***PP < < 0.0001.

High expression of IL-35 in patients with HCC is 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. 2A,C).

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. S1A).

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 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, Fig. 3A). 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. 4A).

Fig. 3.

Overexpression of IL-35 significantly promoted HCC neovascularization, progression and recruited neutrophils in nude mouse model. A. 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. B. IL-35 increased intratumoral Ly6G⁺ neutrophil infiltration and MVD in nude mouse models. Scale bar, 50 μm , ***P < 0.001.

Fig. 4.

IL-35 overexpression significantly promoted HCC neovascularization, progression and recruited Ly6G⁺ neutrophils while influencing the infiltration of CD8⁺T cells. A. IL-35 knockdown in Hepa 1–6 cell significantly slowed down the progression of HCC, and increased IL-35 facilitated tumor growth in vivo experiments. B. IL-35 increased intratumoral Ly6G⁺ neutrophil infiltration. C. IL-35 influenced the infiltrated number of CD8⁺T cells. D. Overexpression of IL-35 increased neovascularization in the tumor, and MVD decreased when IL-35 was knocked down. Scale bar,50 μm, ***P < 0.0001.

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. 3B and 4B,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. 3B and 4D). Thus, we assumed that IL-35 might 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. 4C).

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 < 0.001). However, there was no significant change observed in the adhesion of tumor cells cocultured with neutrophils in the knocked-down group (P > 0.05) (Fig. S2A). 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).

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 24 h.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- 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-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-Ctrl group (Figure S2B). 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 S2C).

IL-35 may 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). However, 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 (Fig. 5A).

Fig. 5.

IL-35 might recruits neutrophils by increasing the expression of CCL3. A. The expression of IL-35 in HCC cell line, but not recombinant IL-35 (rIL-35), could influence neutrophils infiltration. B. Western blot and ELISA were used to detect the expression relationships between IL-35 and CCL3. C. The infiltration of neutrophils derived from IL-35 could be inhibited by anti-CCL3 antibody in vitro experiments. D. IL-35 expression was highly correlated with CCL3 in HCC patient tissues. OE, overexpression, Ctrl, control, KD: knockdown. Scale bar, 50 μm, #P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001.

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. 5B,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 enhance the chemotactic effect on neutrophils, as the CCL3 antibody intervention experiment was demonstrated to reduce the chemotactic effect of CM on neutrophils (Fig. 5C).

IL-35 may 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. 6A). 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. 6A). These results demonstrated that IL-35 stimulated neutrophils to produce angiogenesis factors.

Fig. 6.

Vascularization capability of neutrophils was activated in an FGF2-dependent manner while it was stimulated by IL-35. A. IL-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. B. IL-35 could significantly promote the secretions of FGF2 from neutrophils. Neutrophils proangiogenic capability was activated by increased FGF2. C. 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 > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001.

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. 6B).

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. 6C).

The anti-IL-35 antibody boosts the effectiveness of the PD1 antibody in mouse HCC model

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 PD1 antibody was combined with the anti-IL-35 neutralizing antibody (Fig. 7A).

Fig. 7.

Anti-IL-35 significantly improved the therapeutic effect of PD1 antibody in a mouse subcutaneous HCC model. A. Either anti-IL-35 antibody or anti-PD1 antibody could apparently slow down HCC progression in vivo experiments. Combination therapy with both antibodies has significant and more durable responses for the treatment of HCC. B. 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⁺ 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. C. CD8⁺T cells were significantly increased in both PD1 antibody group and combined treatment group. Scale bar, 50 μm, #P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001.

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. 7B and C).

Discussion

Cytokines, chemokines, and other effectors in the tumor microenvironment (TME) play either promoting or inhibitory roles, contributing to the construction and diverse functions of the TME [32]. We found that IL-35 promotes tumor angiogenesis by recruiting neutrophils and reshapes the TME by reducing CD8+ T cell infiltration, thereby facilitating the progression of HCC. Furthermore, the combination of anti-IL-35 and anti-PD1 antibodies exhibits synergistic anti-cancer effects.

Our study found that high expression of IL-35 in HCC is an independent risk factor for recurrence. Additionally, the expression of GP130 and IL-12Rβ2 receptors in HCC tissues suggests that IL-35 has a structural basis for autocrine functions in HCC. Further analysis revealed that patients with high expression of IL-35 and its receptors in tumor tissues had the worst prognosis, indicating that IL-35 may promote HCC progression. While prior research has focused on immune cell-derived IL-35, our study highlights the role of tumor cell-derived IL-35 in the tumor microenvironment (TME).

Despite the cancer-killing capabilities of neutrophils, high levels of neutrophil infiltration in solid tumors are often associated with poor clinical outcomes [16]. In our study, tumors with high IL-35 expression exhibited significantly increased local infiltration of neutrophils and microvascular density (MVD), alongside significantly reduced CD8+ T cell infiltration. In vivo, IL-35 was associated with neutrophil infiltration and intratumoral angiogenesis, suggesting that high IL-35 expression contributes to local immune evasion and angiogenesis in HCC. This finding helps explain the poor prognosis of patients with high neutrophil infiltration. Tumor angiogenesis can also be indirectly influenced by stromal cells such as neutrophils, macrophages, Treg cells, and stellate cells, which participate in TME remodeling [33]. Studies have reported that IL-35 can influence monocyte secretion of CXCL1 and CXCL8, promoting angiogenesis in pancreatic cancer [34]. Our findings showed no significant impact of IL-35 directly stimulating microvessel formation or through conditioned medium (CM) from overexpression or knockdown models, suggesting that IL-35 promotes angiogenesis through indirect mechanisms.

IL-35 has been reported to indirectly polarize neutrophils into TAN2 and induce tumor infiltration in mouse models of melanoma and HCC [29]. Our study complements these findings with human data, showing a correlation between IL-35 and neutrophil infiltration, suggesting that IL-35 promotes angiogenesis in HCC through neutrophils. Other studies have shown that neutrophils, particularly TAN2, can promote tumor metastasis by remodeling the extracellular matrix through the secretion of matrix metalloproteinases (MMPs), neutrophil elastase (NE), and cathepsin G [23,35]. In breast cancer, neutrophils promote metastasis by binding to circulating tumor cells (CTCs) through vascular cell adhesion molecule 1 (VCAM1) [36]. Our in vitro experiments demonstrated that IL-35 and neutrophils enhanced the adhesion of HCC cells to endothelial cells. This finding aligns with in vivo observations where IL-35 promotes neutrophil-induced lung metastasis in HCC. Factors such as CCL3 and FGF2 may mediate neutrophil tumor infiltration and angiogenesis. CCL3-recruited neutrophils promote lung metastasis in breast cancer, and neutrophil-secreted FGF2 drives liver metastasis in colorectal cancer [23,37]. Our results showed that IL-35 affects CCL3 expression in HCC cells, promoting neutrophil infiltration in tumors. Additionally, IL-35 enhances tumor vascular growth and metastatic colonization by promoting neutrophil secretion of the angiogenic factor FGF2. Furthermore, IL-35 stimulates the expression of FGF2 receptors FGFR3 and FGFR4 in neutrophils, indicating a possible positive feedback loop within the TME.

Immune checkpoint antibody therapy, a prominent approach in tumor immunotherapy, has achieved remarkable outcomes in various cancers, including HCC, and has garnered significant attention [[38], [39], [40], [41], [42], [43], [44]]. However, immunotherapy benefits fewer than 20 % of liver cancer patients, with approximately 30 % exhibiting intrinsic resistance [45]. Combination therapies targeting multiple key pathways may provide an effective solution. IL-35 significantly reduces CD8+ T cell infiltration in tumors of immunocompetent mice, suggesting its role in suppressing antitumor immunity. We found that IL-35 antibody could enhance the efficacy of PD1 antibody therapy. In the TME, IL-35 functions as an immunosuppressive factor by promoting BLIMP-dependent CD8+ T cell exhaustion and suppressing their infiltration and effector function via STAT3 activation, reducing CXCR3, CCR5, and IFNγ expression. It also diminishes NK cell activity, facilitating immune escape [[46], [47], [48]]. Thus, IL-35 appears to be a critical factor undermining the efficacy of immunotherapy. Our findings further elucidate the role of IL-35 in the HCC TME. Tumor cell-derived IL-35 promotes angiogenesis, metastasis, and immune evasion by inducing autocrine production of CCL3, recruiting neutrophils to the tumor. IL-35 antibody therapy enhances the infiltration of antitumor immune cells, reduces tumor-associated neutrophil infiltration, and alleviates the immunosuppressive effects of IL-35 [49]. Additionally, IL-35 inhibition blocks neutrophil-mediated intratumoral angiogenesis, reducing nutrient supply and metastasis [16]. IL-35 antibody combined with PD1 antibody exerts a synergistic effect in HCC by inhibiting intratumoral angiogenesis, reversing the immunosuppressive TME, and enhancing the efficacy of PD1 antibody therapy.

This study has several limitations. First, the IL-35 antibody used targets a single subunit, making it challenging to fully assess the inhibition of IL-35, and potential effects on IL-12 or IL-27 cannot be excluded. Second, IL-35 is a broadly secreted cytokine, so the use of an IL-35 antibody may affect IL-35 from other sources, including Tregs, in addition to HCC. Third, the clinical samples were derived from a retrospective single-center study, necessitating further validation through broader investigations. Lastly, the precise mechanisms by which IL-35 upregulates CCL3 in tumor cells and how CCL3 influences neutrophil-derived FGF2 require further exploration.

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).

Fig. 8.

The expression of IL-35 in HCC can facilitate tumor progression by affecting neutrophil infiltration, angiogenesis, and CD8⁺ 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⁺T cells tend to decrease and exhaustion when stimulated by IL-35. HCC-derived IL-35 can facilitate pulmonary metastasis by interacting with neutrophils. The C57BL/6 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⁺T and decreasing the infiltration of neutrophils in the tumor microenvironment.

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.

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 min. A significant difference in the number of tumor cells stranded in lung tissue 24 h 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.

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).

Ethics statement

Animal experiment protocols were approved by the ethics committee of the zhongshan Hospital of Fudan University. And the study protocol was approved by the ethics committee of the Zhongshan Hospital of Fudan University. The ethics approval no.:B2021–159R.

Funding

This work was supported by the China Postdoctora Science Foundation 2020M671002.

CRediT authorship contribution statement

Wei Gan: Investigation, Writing – original draft. Guo-Qiang Sun: Investigation. Jin-Long Huang: Investigation. Bao-Ye Sun: Investigation. Zhu-Tao Wang: Investigation. Zhang-Fu Yang: Investigation. Cheng Zhou: Investigation. Yong Yi: Writing – review & editing. Shuang-Jian Qiu: Writing – review & editing.

Declaration of competing interest

This paper has not been submitted elsewhere for consideration of publication. The authors certify that they have participated sufficiently in the work to take public responsibility for the appropriateness of the experimental design and method, and the collection, analysis, and interpretation of the data. All authors have reviewed the final version of the manuscript and approved to submit it to your journal. There is no conflict of interest of any authors in relation to the submission.

Data availability

Reasonable requests for data will be made available for review.