Effects of Interferon-γ and Interleukin-4 on Proliferating Cell Nuclear Antigen Expression in Transplanted Bone Tumor Tissue

The rabbit VX2 bone tumor model is an ideal animal model for studying malignant bone tumors. Cytokines have been reported to play a role in tumor initiation and promotion, angiogenesis, and metastasis. However, few studies have investigated the relationship between cytokines and VX2 bone tumor development. This study investigated the effect of interferon-γ (IFN-γ) and interleukin-4 (IL-4) on proliferating cell nuclear antigen (PCNA) expression in tumor tissue. Thirty Japanese white rabbits were randomly divided into group A (n = 15) and group B (n = 15). The rabbit VX2 bone tumor model was constructed by implanting VX2 tumors on the medial side of the upper tibia. Group A was sacrificed in the first week of implantation, and group B in the second week of implantation. Peripheral venous blood, tumor tissue from the medullary cavity at the implantation site, and surrounding bone and soft tissue were harvested before implantation and execution in both experimental groups. IFN-γ and IL-4 expression levels in peripheral blood and PCNA levels in tumor tissues were measured by enzyme-linked immunosorbent assay (ELISA). The tumor tissue of the medullary cavity and surrounding bone and soft tissue was harvested for pathological examination. By the end of the experiment, 30 rabbits were included in the study. There was no significant difference in IFN-γ, IL-4 and PCNA expression levels in group A compared to group B before implantation (t = 1.187, p value = 0.255; t = 1.282, p value = 0.221; t = 0.499, p value = 0.626). IFN-γ and IL-4 expression levels before execution in group A were not significantly different from those before implantation (t = -1.280, p value = 0.213; t = 0.952, p value = 0.349), and PCNA expression levels were higher than those before implantation (t = 2.469, p value = 0.020). Group B had significantly lower IFN-γ expression levels before execution than before implantation (t = -3.741, p value = 0.001) and significantly higher IL-4 and PCNA expression levels before execution than before implantation (t = 6.279, p value < 0.01; t = 13.031, p value < 0.001). IFN-γ expression levels before execution in group B was significantly lower than those before execution in group A (t = 17.184, p value < 0.001), and IL-4 and PCNA expression before execution in group B was significantly higher than that before execution in group A (t = -26.235, p value < 0.001; t = -24.619, p value < 0.001). The correlation between IFN-γ and PCNA levels before execution in groups A and B was negative (r = -0.566, p value = 0.028; r = -0.604, p value = 0.017), and the correlation between IL-4 and PCNA levels was positive (r = 0.583, p value = 0.023; r = 0.884, p value < 0.001). In the rabbit VX2 bone tumor model, extending the period of time after tumor implantation resulted in a negative correlation between IFN-γ and PCNA levels and a positive correlation between IL-4 and PCNA levels.


Introduction
The VX2 tumor model is established by the VX2 cell line, which originated from virus-induced human papillomaderived squamous cell carcinoma. As a result of its stable and rapid proliferation ability, VX2 has been widely used to generate rabbit tumor models. A number of studies have shown that VX2 tumors can lead to osteolytic changes, which is similar to the biological characteristics of human malignant bone tumors (Pezeshki et al. 2015;Chen et al. 2015;Melancon et al. 2018).
T helper (Th) cells play an important role in tumor immune regulation. After activation, Th cells differentiate into two subsets with distinct cytokine expression patterns: Th1 and Th2. Changes in the levels of Th1 and Th2 cells affect the antitumor immune response and tumor proliferation (Melssen and Slingluff 2017;Horiuchi et al. 2007). IFN-γ secreted by Th1 cells, which are an important cell subgroup in the body's antitumor immune response, kills cancer cells. IFN-γ induces the differentiation of Th0 cells into Th1 cells, enhances the recognition of transformed cells by the body's immune system, and provides tumor immune surveillance for immunoreactive hosts (Candeias and Gaipl 2016; Ikeda et al. 2002). Moreover, IL-4 secreted by Th2 cells has an inhibitory effect on the differentiation of Th1 cells, and IL-4 induces the differentiation of Th0 cells to Th2 cells, which play an important role in the proliferation of tumor-associated macrophages (May and Fung 2015;Ito et al. 2017).
Proliferating cell nuclear antigen (PCNA) is a circular homotrimer that is mainly involved in the coordination of DNA replication and repair (Wang et al. 2017;De March and De Biasio 2017). PCNA surrounds and slides along the DNA molecule in the nucleus. DNA polymerase, helicase, exonuclease, ligase, cell cycle regulator, acetyltransferase, chromatin reconstitution, and histone chaperone interact with DNA by binding with PCNA (Kilpatrick et al. 2006). PCNA is expressed in all cell types in normal tissues, and its expression level is relatively stable (Kilpatrick et al. 2001). In some rabbit experiments (Slade 2018), it was shown that PCNA expression was downregulated by trauma and significantly upregulated in tumor tissue; thus, PCNA can be a good marker of tumor proliferation without the interference of surgical trauma factors.
However, there are few studies on the relationship between cytokines and tumor proliferation in the rabbit VX2 bone tumor model. We hypothesized that as VX2 tumors grow, the expression level of IFN-γ decreases, the expression level of IL-4 increases, the antitumor immunomodulatory function of the body was inhibited, and the expression level of PCNA increases. To test this hypothesis and explore the effect of IFN-γ and IL-4 on PCNA expression, we conducted enzyme-linked immunosorbent assay (ELISA) to quantify the levels of IFN-γ and IL-4 in peripheral blood as well as PCNA expression levels in tumor tissue.

Laboratory Animals, Tumor Lines
According to the random number table method, thirty Japanese white rabbits (aged 3 months, male or female, weighing 2.0-2.5 kg) were divided into group A (n = 15) and group B (n = 15). Group A was sacrificed in the first week of implantation, and group B was sacrificed in the second week of implantation. They were purchased from the Beijing Changyang Xishan farm, where the animal license number is SCXK (Beijing) 2016-0007. According to the relevant regulations of Chengde Medical College, the rabbits were housed in standard rearing cages with an internal temperature of 25 °C and 50% humidity and subjected to 12/12-h light/dark cycle. Access to food and water was limited (2 times per day). The VX2 tumor cell line was purchased from Chongqing Mengbo Biotechnology Incorporated Company. The process of animal use in this experiment was reviewed and approved by the Affiliated Hospital of Chengde Medical College Committee on Animal Care and Use following the requirements of medical ethics (Approval batch number: LL047). The study was conducted in strict accordance with the recommendations of the National Institutes of Health's guidelines for the Care and Use of Experimental Animals.

Main Experimental Equipment and Reagents
Optical microscope (Model: Olympus-BX53, Japan), microplate reader of the multi-wavelength measurement system (Model: Multiskan FC, USA), Elisa kit was purchased from Shanghai Yubo Biotechnology incorporated company. We used them to detect IFN-γ, IL-4 and PCNA.The other instruments were derived from our laboratory. For example, scissors, leather knives, cotton swabs, cotton balls, etc.

Preparation of VX2 Tumor Cell Line
The cryopreservation tube of VX2 tumor tissue block was taken out from the sealed dry ice box and quickly resuscitated in 37 °C warm water. After being rinsed with phosphate buffered saline (PBS) for 3 times on the sterile operating table, 1640 cell culture medium containing 10% fetal bovine serum was added for temporary storage. A part of VX2 tumor cells was injected into the lateral femoral muscle of rabbits for continuous passage culture. Four weeks later, when a mass of 3 cm2 in size was palpable subcutaneously, and anesthesia was performed by intravenous injection of pentobarbital sodium (35 mg/kg). The disappearance of corneal reflex was regarded as the successful sign of anesthesia. The rabbit hair was removed from the tumor area and fixed on the animal experimental table in prone position. The operation area was disinfected with Iodophor, covered with sterile pore towel, skin was cut, and subcutaneous tissue and tumor were separated. After the tumor was completely removed, the tumor was moved to a sterile worktable and washed in PBS at 37 °C 3 times to remove the blood stain, necrotic tissue, and fibrous tissue in the tumor. The tumor was cut into 1 mm3 tissue block with ophthalmic scissors. After adding 1640 cell culture medium containing 10% fetal bovine serum, it was stored in the refrigerator at -80 °C. The VX2 tumor tissues used in this experiment were obtained after one passage in rabbits.

Establishment of Rabbit VX2 Bone Tumor Model
Thirty Japanese white rabbits were anesthetized by intravenous injection of 3% pentobarbital sodium (35 mg/kg), and successful anesthesia was indicated by the disappearance of the corneal reflex. The rabbits were then fixed on the operating table in the supine position to remove the hair of the hind limbs. After routine disinfection, we covered the animal with a sterile hole sheet. A 2.0 cm incision was made at the medial level of the tibia. The skin, subcutaneous tissue, fascia and periosteum were dissected and separated layer by layer to reveal the tibia. We used a 1.6 mm Kirschner wire to drill perpendicular to the medial tibia. The depth of the hole was approximately 1.5 cm. We placed 2-3 prepared tissue blocks with a volume of 1 mm3 into the bone marrow cavity. We used bone wax to seal the tumor graft hole, distilled water to clean the surrounding tissue of the implant hole and silk thread to suture the subcutaneous tissue and skin layer by layer. The incision was disinfected with iodophor and bandaged with sterile gauze. The rabbits were placed back in the cage after complete recovery. Rabbits were intramuscularly injected with 800 000 U penicillin once a day for 3 consecutive days. The rabbits were also given ibuprofen (10 mg/kg) for 3 days to relieve any pain.

Pathological and ELISA Examination
Group A (n = 15) and B (n = 15) rabbits were anesthetized by intravenous injection of 3% pentobarbital sodium (35 mg/kg) at the ear margin before implantation and before execution, and successful anesthesia was marked by the disappearance of the corneal reflex.
Before implantation and before execution, 3 ml of venous blood from the ear margin was extracted by a vacuum vessel with anticoagulant. The blood was left at room temperature for 1 h and then centrifuged at 2000 r/min for 10 min to collect plasma. The levels of IFN-γ and IL-4 in the plasma were determined by ELISA. The experimental process was carried out in strict accordance with the instructions of the kit. Tumor tissue in the medullary cavity and surrounding bone and soft tissue was removed before implantation and prior to execution. Hematoxylin eosin staining (HE staining) and microscopic examination were performed. One milligram of tumor tissue was transferred into a glass grinder on a sterile operating table. PBS (1 mg: 9 ml) was added to homogenize the sample fully, and the sample was centrifuged at 2000 R/min for 10 min. The supernatant was collected. The results of microscopic examination were performed by the same senior pathologist in a blinded manner. The level of PCNA in the tumor tissue was determined by ELISA. The experimental process was carried out in strict accordance with the instructions of the kit. After the experiment, intraperitoneal injection anesthesia (pentobarbital sodium, 40 mg/kg) and ear vein air embolism were used to sacrifice the rabbits, with the disappearance of palpable chest heartbeat as the indicator of mortality. The carcasses were disposed of by the pathology staff in accordance with the relevant regulations on the use of experimental animals.

Statistical Data Processing
SPSS 25.0 (SPSS Inc., Chicago, IL, USA) was used to analyze the data. The expression levels of IFN-γ and IL-4 in peripheral blood between group A and group B were analyzed by independent samples t test. A paired samples t test was used to analyze the expression levels of IFN-γ and IL-4 in the peripheral blood within group A and group B. The Pearson correlation coefficient was used to analyze the correlation between peripheral blood IFN-γ and IL-4 levels and PCNA levels in tumor tissue before execution in group A and group B. A p value < 0.05 was considered statistically significant.

Construction of Rabbit Bone Tumor Model
Thirty experimental rabbits were successfully transplanted with VX2 tumor tissue as determined by histopathological examination. There were no infections or deaths in the experimental animals during the study. All rabbits were included in the final experiment.

Results of IFN-γ, IL-4 and PCNA Expression Analysis
The expression levels of IFN-γ, IL-4 and PCNA in group A before tumor transplantation were not significantly different from those in group B before tumor transplantation (t = 1.187, p value = 0.255; t = 1.282, p value = 0.221; t = 0.499, p value = 0.626). In group A, the expression levels of IFN-γ and IL-4 before execution were not significantly different from those before implantation (t = -1.280, p value = 0.213; t = 0.952, p value = 0.349), and the expression levels of PCNA before execution were significantly higher than those before implantation (t = 2.469, p value = 0.020). In group B, the expression levels of IFN-γ before execution were significantly lower than those before implantation (t = -3.741, p value = 0.001), and the expression levels of IL-4 and PCNA before execution were significantly higher than those before implantation (t = 6.279, p value < 0.001; t = 13.031, p value < 0.001). The level of IFN-γ expression before execution in group B was significantly lower than that before execution in group A (t = 17.184, p value < 0.001). The levels of IL-4 and PCNA expression before execution in group B were significantly higher than those before execution in group A (t = -26.235, p value < 0.001; t = -24.619, p value < 0.001). (Table 1).

Results of Correlation Analysis Between IFN-γ and PCNA levels and IL-4 and PCNA Levels Before Execution
The correlation between IFN-γ and PCNA levels before execution in groups A and B was negative (r = -0.566, p value = 0.028; r = -0.604, p value = 0.017), and the correlation between IL-4 and PCNA levels was positive (r = 0.583, p value = 0.023; r = 0.884, p value < 0.001).

Results of Histopathological Examinations
In Group A, pathological examinations revealed no tumor cells in the surrounding soft tissues. Seven rabbits had tumor cells located in the bone cortex and 8 rabbits had tumor cells found in the bone marrow cavity. In group B, pathological examinations revealed that tumor cells were found in the peripheral soft tissues in 9 rabbits and in the bone cortex or bone marrow cavity in 6 rabbits. (Fig. 1).

Discussion
The rabbit VX2 bone tumor model is a nonosteogenic allogeneic transplantation model that possesses features that are similar to those found in human metastatic bone tumors, including rapid tumor growth, high tumor formation rate, osteolytic changes, and periosteal reaction. The possession of all of these features makes the rabbit VX2 model an ideal experimental animal model for the study of malignant bone tumors (Yu et al. 2019;Chen et al. 2019). Combined with previous literature reports and our group's preliminary results, we found that the growth of rabbit VX2 bone tumors was stable within 2 weeks, and distant metastasis could occur in the third week. Therefore, we chose the first and second weeks after tumor implantation to avoid the influence of possible metastasis on the accuracy of experimental data (Ibrahim et al. 2013).
The abnormally high expression of PCNA in most tumor tissues during the progression and invasion of tumors demonstrates that PCNA is an indicator of active tumor cell proliferation and objectively reflects the biological characteristics and growth status of tumors (Cai et al. 2017;Guo et al. 2017). Changes in PCNA expression in cells are periodic. Although PCNA was not discernible in the G0-G1 phase, it began to be expressed in the late G1 phase and peaked in the S phase. Finally, PCNA levels significantly decreased in G2/M phase. Moreover, as an auxiliary protein of DNA polymerase δ and a necessary component of DNA replication, PCNA regulates DNA proliferation and participates in the synthesis of the leading and lagging strand in the early stage of cell proliferation (Chen et al. 2018;Gu et al. 2018). In this study, histopathological examination revealed that the tumor had grown extensively in the medullary cavity before execution in group A and had invaded the bone cortex before execution in group B. The ELISA results indicated that the expression level of PCNA in the tumor tissue was higher than that before implantation. This finding indicates that PCNA can reflect the growth and infiltration of rabbit VX2 bone tumor tissue and cells, respectively. With the extension of time after VX2 tumor implantation in rabbits, the expression level of PCNA increased, and the abnormally high expression of PCNA enhanced tumor proliferation ability.
By regulating the expression level of the p21 receptor and RB phosphorylation, IFN-γ can induce G1 phase arrest and inhibit tumor cell proliferation. It can also induce tumor cell apoptosis through the death receptormediated exogenous pathway and mitochondrial-mediated endogenous pathway. In addition, it plays a synergistic role with the calcineurin B subunit to promote the transformation of tumor-associated macrophages into the M1 phenotype, which enhances the antitumor activity of macrophages and modifies the tumor microenvironment (Su et al. 2015;Bahrambeigi et al. 2014;Liu et al. 2012). Dighe et al.(Dighe et al. 1994). showed that the speed and type of tumorigenesis changed when IFN-γ receptordeficient mice were subjected to different chemicals. Our results demonstrated that the expression level of IFN-γ in group A was not significantly different before execution compared with that before tumor transplantation, and the expression level of PCNA was higher than that before tumor transplantation. The level of IFN-γ expression before execution was significantly lower in group B than in group A, and the level of PCNA expression was significantly higher in group B than in group A. Pearson analysis in both groups suggested a negative correlation between IFN-γ and PCNA levels. This result indicated that IFN-γ could inhibit the proliferation of tumor cells in the rabbit VX2 bone tumor model. However, with the prolongation of tumor implantation time, the expression level of IFN-γ decreased, which decreased apoptosis of tumor cells that drive tumor cell proliferation. In addition, VX2 tumors have a strong proliferation and replication ability, which means that when the expression level of PCNA gradually increases, the tumor will continue to proliferate, grow and invade the surrounding tissue (Gkouveris et al. 2018;Li et al. 2019).
IL-4 can inhibit the secretion of IFN-γ by Th1 cells. It decreases the activation of natural killer cells (NK) and the ability of cytotoxic lymphocytes (CTL) to recognize tumor antigens. It allows tumor cells to escape the killing effect of specific CTLs and act directly on CD8 + T cells, which can reduce their toxicity to tumor cells and increase the number of regulatory CD4 + T cells and myeloid-derived suppressor cells. It also plays an important role in the proliferation of tumor-associated macrophages, which can promote the production of M2 macrophages and other suppressive immune cells (Liu et al. 2016;Kim et al. 2016;May and Fung 2015). In previous studies, it was also found that with the increase in IL-4 levels in the body, the host's antitumor immunity decreased, resulting in the continuous growth of tumors and possible metastasis. When an anti-IL-4 neutralizing antibody was injected into transplanted tumor model mice, it was found that reducing the level of IL-4 enhanced the antitumor immune ability, inhibited the development of tumors, and prolonged the survival time of mice Ito et al. 2017). The results of this study showed that both IL-4 and PCNA levels were significantly elevated and positively correlated in group B prior to execution, and the histopathological examination indicated that the tumor had invaded the surrounding soft tissues. This finding indicates that as VX2 tumor cells proliferate and differentiate, they may produce large amounts of IL-4 in an autocrine manner, which inhibits the secretion of IFN-γ and reduces antitumor effects of IFN-γ. Moreover, IL-4 promotes the proliferation of tumor-associated macrophages, protects tumor cells from apoptosis, and mediates the proliferation of tumor cells through IL-4 receptors on tumor cells, which increases the proliferative and invasive capacity of tumor cells. Moreover, the expression levels of both IL-4 and PCNA increased in tumor tissues. Therefore, IL-4 plays a promoting role in the high expression of PCNA (Traub et al. 2017;Bojarska-Junak et al. 2018;Gaggianesi et al. 2017).
This study has some limitations. The rabbit VX2 bone tumor model is a nonosteogenic allogeneic transplantation model, but its biological characteristics still have certain shortcomings in mimicking human malignant bone tumors. Although the experimental animals were all homogenized, the results of the experimental data may be biased due to the small number of experimental animals. Only quantitative ELISA analysis of the collected specimens in this study, and no other relevant molecular biology experiments were conducted. The research group will continue to improve the design of the study and carry out further research to provide more support for clinical applications.

Conclusions
In summary, with the extension of tumor implantation time, the expression levels of IFN-γ and PCNA were significantly negatively correlated, and the expression levels of IL-4 and PCNA were significantly positively correlated. The summation of these data led us to conclude that IFN-γ may inhibit the proliferation of tumor cells and that IL-4 may promote the proliferation of tumor cells. The analysis of the relationship between the changes in the expression levels of IFN-γ, IL-4 and PCNA is conducive to the analysis of the relationship between immune regulation and tumor proliferation and growth, and the rabbit VX2 bone tumor model can assist in the discovery and development of new treatments for bone cancer patients.