Interleukin-34 is associated with hepatocellular carcinoma in chronic hepatitis B patients

Backgrounds: IL ‐ 34 is involved in a number of auto-immunities and cancers. We explored the relationship between serum/hepatic IL ‐ 34 and hepatitis B virus (HBV) related hepatocellular carcinoma (HBV ‐ HCC) patients. Methods: Serum was obtained from the HBV patients or healthy control with written consents. Liver tissue was obtained from liver biopsy in CHB, HBV related cirrhosis patients or curative resection in HBV ‐ HCC patients. Serum IL ‐ 34 and MCSF were measured, using ELISA. HepaticIL ‐ 34, MCSF and CD68 were determined, using immunohistochemistry. Results: Serum IL ‐ 34 was 1.7, 1.6 or 1.7 fold higher in HBV ‐ HCC than that of the other three groups (CHB, HBV related cirrhosis, and healthy control). Serum IL ‐ 34 was signicantly reduced after trans ‐ hepatic arterial chemoembolization (TACE) in HBV ‐ HCC patients. There was signicant correlation between the incidence of HBV ‐ HCC and IL ‐ 34 (r s =0.160, p<0.05) as well as AFP (r s =0.442, p<0.01). Furthermore, intrahepatic IL ‐ 34 was higher in HBV ‐ HCC than that of the other three groups. Intra-hepatic IL ‐ 34 was associated with high HBV ‐ DNA, HBeAg - , low tumor differentiation and small tumor size of HBV ‐ HCC patients. Intra-hepaticCD68 + TAMs were increased 1.6 or 1.3 fold in HBV ‐ HCC compared to that from CHB or HBV-cirrhosis. Intra-hepatic CD68 + TAMs were associated with high HBV ‐ DNA, high tumor differentiation, big tumor size, abnormal AFP and more tumor number. Conclusions: IL ‐ 34, correlated with HBV ‐ HCC and IL ‐ 34, may be used as a therapeutic target in precise medicine for management of HBV ‐ HCC.


Introduction
Hepatitis B virus (HBV) infection, a major health problem worldwide (1), is one of the major causes of hepatocellular carcinoma (HCC) (2). HBV related HCC (HBVHCC) is a common primary liver cancer with high mortality, high recurrence and low post-operative survival rate, mainly is due to later diagnosis. It is well known that host immunity plays a critical role in the carcinogenesis, which is elegantly demonstrated by the Nobel laureates for Medicine in 2018 (3).
Tumor microenvironment, including tumor cells, activate macrophages, cytokines and blood vessels, has an important role on the occurrence and development of tumor (4,5). Tumor associated macrophage (TAMs) regulate the microenvironment (6), but the role of TAMs is controversial (7,8). TAMs promotes tumor invasion, formation of blood vessels and lymphatic vessel, migration of tumor cells (9,10), perhaps via enhancing immunosuppression environment (11). In contrast, TAM may be involved in inhibiting cancer growing and metastasis (12). The explanation of such discrepancy might be due to differential polarization of macrophages during their maturation, namely macrophage 1 (M1) and M2 (12)(13)(14)(15). The differential polarization of macrophages perhaps due to different microenvironments in different regions and/or in different individuals, mediated by different regulators (14,16).
Higher circulating IL34 and MCSF are detected in hepatitis C virus (HCV) patients with high brosis scores than that from the low scores or healthy subjects (19). Furthermore, serum IL34 may be associated with in ammatory activity and liver brosis in chronic hepatitis B (CHB) (28). There is a close correlation between the severity of hepatic brosis and the incidence of HCC from both chronic hepatitis B and C patients (29). It is unclear the relationship between IL34 and the clinical presentation of HBVHCC, as well as the association between IL34 and TAM/MCSF. Understand the precise underlying mechanism of IL34 in regulating macrophage mediated tumorogenesis in HBVHCC, it could contribute to development of therapeutic agents, particularly in precision medicine.
Interestingly, intrahepatic MCSF and CD68 + TAMs were signi cantly lower than HCs (p < 0.05).  All datum were *10 3 image unites. The reference range of AFP is 0-9 µg/L.The histopathological classi cation is well described in the published Literature.

Discussion
In the present study, we evaluated circulating and intra-hepatic IL34 in HBV related liver diseases. Serum IL34 from HBVHCC patients was signi cantly higher than that of CHB, HBVcirrhosis and HCs. IL34 and AFP were correlated with the incidence of HBVHCC. Furthermore, serum IL34 was decreased following antitumor TACE treatment in HBVHCC. Intrahepatic IL34 from these groups was consistent with serum IL34. Intrahepatic IL34 was associated with high HBVDNA, HBeAg − , low tumor differentiation and small tumor size of HBV-HCC patients. Intrahepatic CD68 + TAMs were increased in HBVHCC compared to that from CHB and HBVcirrhosis. Intrahepatic CD68 + TAMs were associated with high HBVDNA, high tumor differentiation, big tumor size, abnormal AFP and more tumor number.
Chronic HBV infection is a major risk factor for HCC globally, which is supported by the ndings, showing a close correlation among chronic viral hepatitis, liver cirrhosis after hepatitis, primary liver cancer (29). It is well documented there is close association between HBV infection, as well as, the level of HBsAg, and the incidence of HCC (30,31). Based on the APSAL guideline, it is considered that CHB, liver cirrhosis, HCC are re ecting the different stages of the liver diseases. Up to date, two major therapeutic strategies are applied for CHB, i.e. direct acting antiviral drugs and immune modulatory agents (32). However, there is no effective treatment to cure HBV infection, resulting in certain CHB patients progress to cirrhosis and HCC (1). Therefore, early detection, diagnosis and treatment are the key points in reducing the incidence of HBVHCC.
In our current study, serum IL34 from HBVHCC patients was signi cantly higher than that of CHB, HBVcirrhosis, and HCs, suggesting that IL34 may contribute to tumorigenesis of HCC, enhancing progression from CHB patients to cirrhosis and nally HCC. Furthermore, intra-hepatic IL34 was consistent with the result of serum. Our results are in line with previous studies documenting high IL34 in pathological conditions (22,23,33). IL34 is overexpressed in the in amed synovium of patients with rheumatoid arthritis, where it perhaps acting synergistic with TNF and IL1β, induces osteoclastogenesis and contributes to tissue in ammation and bone erosion (34).
The development of tumor is closely related to the microenvironment, including tumor cells, monocytes/macrophages, cytokines and neovasculization. TAMs are mixed phenotype, expressing M1 or M2 markers (13), and may be in uenced by different microenvironments in different regions and/or in different individuals. CD68, regardless the status of their polarization in many studies, has been widely used as a marker in marking TAMs which is in ltrating in solid tumors (35). In our current study, intrahepatic CD68 + TAMs were increased gradually with the order from CHB, HBVcirrhosis to HBVHCC patients. Our current observation invites speculation that the increased in ltrating CD68 + TAMs may be M1 dominant, contributing to antitumor activity, which will be determined in our future experiments.
It was reported that IL34 was upregulated in hepatitis C virus infection and inhibited the production of IFNγ (19), and IL34 may also be associated with in ammatory activity and liver brosis in CHB (28). Moreover, baseline serum IL34 levels seem to serve as a prognostic factor for progression in such patients. In our current study, there was only IL34 signi cantly decreased in postantitumor treatment compared to that of pre-treatment of HBVHCC in serum. Serum IL34 was signi cantly correlated with the incidence of HBVHCC. This further suggests that IL34 could be used as a predictor for HBVHCC, which will be further determined in vitro and in vivo.
IL34 induces differentiation of leukemia cells into monocytelike, macrophagelike cells and mature macrophages through the JAK/STAT and PI3K/Akt signaling pathways (36), suggesting IL34 enhances differentiation of other cancers (15,21). This is consistent with our current nding that IL34 was correlated with the differentiation and tumor size of HBVHCC. In addition, intrahepatic IL34 was associated with HBVDNA, HBeAg, tumor differentiation and tumor size of HBVHCC patients. Our data may provide an explanation for the possible role of IL34 in the development of HBVHCC, i.e. IL34 also regulates HBVHCC differentiation, which would have potential clinical relevance regarding IL34 as a therapeutic target for malignancy. IL-34 is able to inhibit HBV replication in vivo and in vitro (37). Such nding is in line with our current clinical study, showing that IL-34 is bene cially to the HCC patients for potential therapeutic target.
We acknowledge that there are limitations in the current study. There is no kinetics of intrahepatic or circulating IL34 during the development and management of HCC, and no correlation between IL34 and prognosis is detected. These two interesting points will be determined in our future study. We also acknowledge that the sample size in the current study is relatively small, which may not perfectly re ect the real cases. However the current experiment is just a proof of concept. We will explore the underlying mechanism with large sample size and multiple center studies in future.
In summary, the current study improves our understanding of the role of IL34 in HBV related liver disease.
Increased IL34 may contribute to the transformation of the tumor, which is a potential predictor of HBVHCC. The underlying mechanism of IL34 in HBVHCC is being currently investigated.

Study population
All of the patients were identi ed between April 2015 and July 2017 in Department of Infectious Diseases, Shanghai Ruijin Hospital. Serum and liver tissues were obtained from the patients with informed consents. It was selected as healthy controls (HCs) that age and sex matched healthy people for routine health check in our hospital without liver disease/HBsAg negative/negative image in CT or MRI. The selection of treatment for HBV-HCC patients were based on the guideline for treatment of primary liver cancer in China, and was conducted with a multidisciplinary diagnosis and treatment team of Ruijin Hospital, as described previously (38). HBV-HCC patients selected for the current study were received eithertranshepatic arterial chemoembolization (TACE) or curative resection treatment. TACE, an interventional treatment, is one of the most common nonsurgical treatments for liver cancer (38).Curative resection is a surgical procedure that hepatocellular cancerous tissue and a certain amount of normal tissue to be removed to obtain adequate margins. The purpose is to minimize the risk of any cancer cells being left behind (39).
The inclusion criteria of CHB patients were: rst, adult with consecutive HBsAg + for at least six months, nucleos(t)ide (NA)naïve without cirrhosis or carcinoma.
Second,1) Adult with consecutive HBsAg + for at least six months; 2) Diagnosed as primary HCC con rmed with pathology; 3) alpha-fetoprotein (AFP) > 400 µg/L, no other active liver disease, pregnancy, embryonic source sex reproductive system tumor and metastatic liver cancer, and could touch a swelling or hard of the liver with tumor, or imaging examination, such as computerized tomography (CT)(40), magnetic resonance imaging (MRI) scans (41), and ultrasound examinations, revealed liver occupying lesions characteristic; 4) AFP ≤ 400 µg/L, more than two imaging examinations revealed liver occupying lesions which has characteristic of HCC.
Third, 1) adult with consecutive HBsAg + for at least six months; 2) diagnosed cirrhosis by biopsy of the liver; 3) or imaging examination, such as CT, MRI, FibroScan or ultrasound examinations, detected enlarged livers, abnormally nodular livers, enlarged spleens, and uid in the abdomen, suggesting cirrhosis; 4) the hospitalized patients under any event can also be diagnosed as decompensation liver cirrhosis: abdominal cavity effusion, esophageal gastric varices burst out of the blood, hepatic encephalopathy, infection.
The exclusion criteria of healthy people were: 1) Undergone liver disease before the study; 2) Had abnormal liver function recently; 3) alcoholism (amount of alcohol: female ≥ 20 g/d, male ≥ 30 g/d).
This study complies with the declaration of Helsinki, and the study protocol was approved by the Human Ethics Committee, Ruijin Hospital. Written informed consent was obtained from all of the patients according to standards of the local ethics committees. . HBeAg + is de ned as positive hepatitis B envelop antigen, and HBeAg − is de ned as negative hepatitis B envelop antigen. The Scheuer's scoring system was applied for pathology diagnosis of in ammation and brosis grading of liver tissue (42). Liver cirrhosis is de ned as ≥ S4 of Scheuer's scoring system. Serum ALT, AST, AKP, rGT, TBil, and Alb (re ecting liver function) were quanti ed using Beckman coulter AU5800 automatic biochemical analyzer. HBsAg, anti-HBs, HBeAg and anti-HBe were determined using commercial ELISA kits (Abbott Diagnostics, IL). Serum HBV DNA levels were measured using qPCR, Roche Amplicor (Roche Diagnostic Systems, Branchburg, NJ, USA). Serum IL34 and MCSF were quanti ed using ELISA (R&D Systems, Lille, France). Results were expressed as a concentration of cytokine production.

Immunohistochemistry (IHC)
The liver tissue blocks were obtained from surgery for HBVHCC (n = 30) or from liver biopsy for diagnosis of CHB liver (n = 5) and HBV-cirrhosis liver (n = 5) or the off cuts from liver transport donors for HCs (n = 5). HCs did not present liver disease/ HBsAg negative/negative image in CT or MRI. Hepatic IL-34, MCSF and CD68 were determined using immunohistochemistry, using 3, 3'diaminobenzidine (DAB) color development, as described previously (43). The primary antibodies were polyclonal rabbit antihuman IL34 (bs-18170R, Beijing Biosynthesis Biotechnology, China), polyclonal rabbit anti-human MCSF (Abcam, Cambridge, UK) and monoclonal mouse antihuman CD68 (Dako, Copenhagen, Denmark). The secondary antibody (Beijing Sequoia Jinqiao Biological Technology) was used. The speci c target(s) was visualized with DAB detection kit and counterstained with hematoxylin. The IHC was repeated for twice. Negative control was applied in each labeling for every primary rabbit negative control. Intra-hepaticIL-34 or MCSF is localized in the cytoplasma of hepatocytes, which has been well documented in our previous publications (44). IHC was quanti ed using a computer-assisted genuine color image analysis system (ImageProplus 9.0) for hepatic IL34, MCSF or CD68, as described previously (44, 45).

Statistical analysis
Continuous variables were expressed as means ± standard deviation or median (inter-quartile range) where appropriate. Differences between two groups were determined by unpaired t test or the MannWhitney U test. Among three groups were used by analysis of variance (ANOVA) or the KruskalWallis H nonparametric test. Chisquare or Fisher's exact test was employed to compare nominal variables. Figure 1