IL-2 Modulates TAMs Derived Exosomal MiRNAs to Ameliorate Hepatocellular Carcinoma Development and Progression

Background. Interleukin-2 (IL-2) is proved to play an irreplaceable role in antitumor regulation in numerous experimental and clinical trials. Tumor-associated macrophages (TAMs) are able to release exosomes to promote the development and progression of hepatocellular carcinoma (HCC) as essential component of microenvironment. In this study, our intention is to explore the effects of the exosomes from TAMs with IL-2 treatment on HCC development. TAMs were collected and cultured from HCC tissues. The exosomes from the TAMs treated with IL-2 (ExoIL2-TAM) or not (ExoTAM) were identified and used to treat HCC cells in vivo and in vitro. The proliferation, apoptosis, and metastasis of HCC cells were measured. The changes of miRNAs in exosomes were explored to clarify the possible mechanisms. Both decrease of cell proliferation and metastasis and increase of apoptosis were observed with ExoIL2-TAM treatment compared with ExoTAMin vivo and in vitro. miR-375 was obviously augmented in ExoIL2-TAM and HCC cells treated with ExoIL2-TAM. Taken together, IL-2 may modulate exosomal miRNAs from TAMs to ameliorate hepatocellular carcinoma development. This study provides a new perspective to explain the mechanism by which IL-2 inhibits hepatocellular carcinoma and implies the potential clinical value of exosomal miRNAs released by TAMs.


Introduction
Primary hepatocellular carcinoma (HCC) is the third most common malignant tumor in cancer mortality [1]. e treatment of HCC is mainly surgical resection and liver transplantation [2]. Because of its high degree of malignancy, the five-year survival rate is still less than 50% and the recurrence rate is high. Recurrence and metastasis have become the biggest obstacle to improve the therapeutic effect and survival rate of HCC [3]. e occurrence, development, invasion, and metastasis of tumors are not only determined by the malignant tumor cells themselves but also closely related to the tumor microenvironment [4][5][6].
It has been proved that there are a large number of tumor-associated macrophages (TAMs) in tumor microenvironment. Recent studies have confirmed that TAMs are mainly M2 type macrophages, which can promote tumor growth by affecting angiogenesis, immunosuppression, invasion, and metastasis [7][8][9]. Exosomes are membrane vesicles with a diameter of 30-150 nm [10]. ey can be secreted by the cells and widely exist in various body fluids. e exosomes carry RNA and protein components and are involved in signal transduction and immune escape of tumor as well as the diagnosis and treatment of a large number of diseases [11,12]. Compared with adjacent normal tissues, the expression levels of miRNAs changed in many malignant tumors [1]. It has been reported that the exosomes derived from TAMs promoted HCC cell proliferation and metastasis [13][14][15][16].
IL-2 is a multifunctional cytokine in the immune system which effectively activates and enhances the phagocytosis and killing ability of macrophage [17,18]. IL-2 has been used in lots of clinical trials for the treatment of HCC [2]. However, it remains unknown whether IL-2 regulates the exosomes released from TAMs. Given the emerging role of IL-2 in curing HCC and regulating the function of macrophages, this study is aimed to explore the effects of exosomes from IL-2-treated TAMs on HCC development and the possible mechanisms.

Patients and Primary Human TAMs Isolation and
Culture from Tissue Specimens. Primary human HCC specimens were collected from the patients who suffered from hepatectomy at Youjiang Medical College Affiliated Hospital. Patient's consent was obtained and the procedures were approved by the Ethics Committee of the Youjiang Medical College Affiliated Hospital. Human fresh tumor samples were minced with scissors. e macrophages were isolated and cultured by Percoll (GE Healthcare) density gradient centrifugation. TAMs were treated with IL-2 for 24 hours before the supernatants were collected. e treated concentration was 20 ng/ml.

Isolation and Labelling of Exosomes.
Exosomes were isolated from the cell culture media with Total Exosome Isolation Reagent ( ermo Scientific) according to the manufacturer's instructions. e purified exosomes were then labelled with PKH26 (Umibio) according to the manufacturer's directions.

Exosomes and TAMs Cells Observed by Transmission Electron Microscopy.
e samples were fixed with 2% glutaraldehyde and 2% paraformaldehyde in 0.1 mol/L sodium cacodylate buffer at pH 7.3 for 3 hours at room temperature. After air drying, samples were mounted on specimen stubs and visualized using transmission electron microscope.

RNA Isolation and qPCR.
Total RNA was isolated from cells or mouse tissues using TRIzol reagent (Invitrogen), following the manufacturer's instructions. e RNA was then analyzed using real-time qPCR with SYBR Green PCR Master Mix (Roche Applied Science, Mannheim, Germany).

Quantification of Apoptosis by Flow Cytometry.
Apoptosis was determined using an Annexin V-FITC/PI apoptosis detection kit (eBioscience, USA). e cells were assessed via flow cytometry.

EdU
Assays. For EdU assays, HepG2 cells were added to 24-well plates, and, after 24 h of incubation with exosomes, EdU (Sigma-Aldrich) staining was conducted based on the protocols.

Migration Assay.
Cell migration assays were conducted on 24-well Transwell cell culture chambers with 8 μm sized pores (Corning, USA). HepG2 cells were suspended in 500 μL of medium and added to the upper inserts. After 24 hours of incubation, the cells remaining in the upper chamber were removed, and the cells on the lower surface of the chamber were fixed with 4% paraformaldehyde and stained with 0.5% crystal violet.

Scratch Test.
Scratch assays were performed to assess cell migration in vitro. First, HepG2 cells were seeded in 6well plates until a confluent monolayer was formed. en, upon confluence, cells were scratched with a 10 μL sterile pipette tip. Pictures of the scratch were then taken at different time points under the microscope. e cell migration rate was calculated as (width at 0 hours -width at 24 hours)/ width at 0 hours.

Xenograft Mouse Models.
All animal experiments were performed after obtaining the approval of the Animal Ethics Committee of Youjiang Medical University for Nationalities. QJY-7703 cells in logarithmic growth phase were prepared into cell suspension. Axillary subcutaneous injection of cells (5 × 10 5 ) of BALB/c nude mice (4-6 weeks old, n = 6 per group) was performed. After the mean tumor volume had grown to be palpable, PBS or the indicated exosomes were injected every 2 days for 7 times. Tumor volumes were 2 Journal of Oncology measured after 10 days every 5 days until mice were sacrificed 30 days later.

e Liver and Lung Metastasis
Experiment. e 6-8week-old nude mice were divided into three randomized groups (n = 12 per group), and QJY-7703 (5 × 10 5 ) was injected into the mice via tail vein with or with out the indicated exosomes. 30 days after cell injection, the mice were euthanized and were necropsied to assess the metastatic ability of HCC cells. e liver and lung tissues of mice were further examined by H&E staining.

TUNEL Staining.
Apoptotic cells in tumor tissues were detected by TUNEL assay according to the standard procedure. After being fixed in 4% paraformaldehyde, the tissues were stained by 50 μL TUNEL reaction mixture (Roche) for 60 min at 37°C. e cell nucleus was stained with DAPI and observed using the Olympus microscope.

Difference in Expression.
e data came from the miRNAseq data of level 3 BCGSC miRNA Profiling in the LIHC (hepatocellular carcinoma) project of TCGA (https:// portal.gdc.cancer.gov/). e software for analyzing unpaired sample data was R (version 3.6.3) (statistical analysis and visualization), the R package was ggplot2 [version 3.3.3] (for visualization), and the molecule was hsa-miR-375 (MIMAT0000728). ere were a total of 425 cases in the unpaired sample. When analyzing the paired samples, the paired samples in the data (424 cases) were retained. At the same time, on the UALCAN website, we also showed the expression of hsa-miR-375 in different cancer stages. In order to show the expression level of miR-375 in different stages, we entered the target molecule on the UALCAN website and basically selected hepatocellular carcinoma to get the results.

Clinical Significance.
e R packages for drawing survival curves were survminer package (version 0.4.9) (for visualization) and survival package (version 3.2-10) (for statistical analysis of survival data); the prognosis type was overall survival. e R packages for ROC curve drawing were pROC package (version 1.17.0.1) (for analysis) and ggplot2 package (version 3.3.3) (for visualization), and the clinical variables were Normal versus Tumor. Interpretation of ROC curve results were as follows: the abscissa was the false positive rate (FPR), and the ordinate was the true positive rate (TPR). e value of the area under the ROC curve was between 0.5 and 1. e closer the AUC was to 1, the better the diagnostic effect is. AUC is less accurate when it is from 0.5 to 0.7, and it has a certain accuracy when it was between 0.7 and 0.9. ere is higher accuracy when AUC is above 0.9.

Correlation Scatter Plot.
For correlation analysis, we also downloaded the level 3 HTSeq-FPKM format RNAseq data in the TCGA (https://portal.gdc.cancer.gov/) LIHC (hepatocellular carcinoma) project, which is the protein expression data. RNAseq data in FPKM (Fragments Per Kilobase per Million) format were converted to TPM (transcripts per million reads) format and log2 conversion was performed.

Statistical
Analysis. Data were presented as mean ± SD. Comparisons between groups were performed using one-way ANOVA, and Tukey's procedure for multiple range tests was performed. All experiments for cell cultures were performed independently at least three times and in triplicate each time. P < 0.05 was considered to be significant. All analyses were performed with Graphpad Prism 7.0 and SPSS18.0.

e Internalization of Exosomes by HCC Cells.
To obtain exosomes, we collected HCC tissues to isolate and culture TAMs.
e exosomes were isolated from macrophage supernatants with ultracentrifugation and were observed by transmission electron microscope (TEM) which were hemisphere with one side depression with typical characteristics (Figure 1(a)). e diameter of exosomes ranged from 30 to 150 nm, as shown in Figure 1(b). Western blot was used to further confirm that the isolated small spheres were exosomes by detecting CD63 and not Calnexin derived from TAMs (Figure 1(c)). To determine the effects of exosomes on HCC cells, we examined whether exosomes could be taken up by HCCs. An immunofluorescence assay was performed by using exosomes labelled with PKH26, a red fluorescence dye. Red fluorescence was clearly observed in HCC cells around nuclei using the confocal microscope (Figure 1(d)).

e Effects of the Exosomes on HCC Development and
Progression In Vitro. First, the effects of the exosomes on the proliferation of HepG2 cells were explored. e results revealed that, when compared with Con group, the cell proliferation abilities were increased in the Exo TAM group. ese effects were ameliorated in the Exo IL2-TAM group compared with the Exo TAM group (Figures 2(a)-2(e)). en, the effects of the exosomes on the apoptosis of HepG2 cells were explored with flow cytometry. HCC cells' apoptotic rate was decreased in the Exo TAM group companied with Con group. Exo IL2-TAM partly reversed these effects compared with the Exo TAM group (Figures 2(f ) and 2(g)). In line with these findings, the protein levels of the antiapoptosis molecule Bcl-2 increased and the proapoptosis molecule Bax decreased in the Exo TAM group compared with Con group, and these changes were partly reversed in the Exo IL2-TAM group (Figures 2(h)-2(j)).
Last, the effects of the exosomes on the migration of HepG2 cells were measured. Transwell assay and scratch test were used to explore whether IL-2 treatment affects TAMsderived exosomes to influence the migration abilities of HepG2 cells. e cell migration abilities were increased in the Exo TAM group compared to Con group; these abilities were reduced in the Exo IL2-TAM group compared to the Exo TAM group (Figures 3(a)-3(d)). Western blot analysis also revealed that the activation of EMT by the Exo TAM treatment was restored in the Exo IL2-TAM group (Figures 3(e)-3(i)).
Collectively, our findings indicated that the effects of exosomes from TAMs promoting proliferative and migration behaviors and inhibiting apoptosis were partly reversed by IL-2 treatment in vitro.

e Effects of the Exosomes on HCC Development and Progression
In Vivo. To test the above results in vitro, the in vivo xenograft model was used in nude mice. QJY-7703 cells were injected subcutaneously into the flanks of nude mice with treatment next. e tumors produced by injection of QJY-7703 in Exo TAM group were significantly larger and heavier than those produced by QJY-7703 cells alone. However, the tumors were smaller and lighter in the Exo IL2-TAM group compared to the Exo TAM group (Figures 4(a)-4(c)). e tumors were collected and the apoptosis of tumor tissues was detected by the TUNEL staining and western blot. e results showed that decreased positive proportion of TUNEL and Bcl-2 protein levels and increased Bax protein levels were found in the Exo TAM group. However, positive proportion of TUNEL and Bcl-2 protein levels increased and Bax protein levels decreased in the Exo IL2-TAM group compared to the Exo TAM group (Figures 4(d)-4(h)). Meanwhile, the protein levels of PCNA and cyclin D1 to proliferation were reduced in the Exo IL2-TAM group (Figures 4(i)-4(k)).
To explore the effect of exosomes on tumor metastasis in vivo, we injected QJY-7703 cells treated with Exo TAM or with Exo IL2-TAM into the nude mice via the tail vein. Quantitation of metastasis of the livers and lungs of mice in each group revealed that higher rates of hepatic and pulmonary metastases were found in the Exo TAM group compared with Con group, and the rates were lower in the Exo IL2-TAM group (Figures 5(a) and 5(b)). Furthermore, the IHC staining revealed that Vimentin was significantly elevated in the Exo TAM group, accompanied by the increase of MMP2, MMP9, and N-cadherin and the decrease of E-cadherin. However, Vimentin, MMP2, MMP9, and N-cadherin were downregulated, and E-cadherin was upregulated in the Exo IL2-TAM group compared to the Exo TAM group (Figures 5(c)-5(h)). ese experiments indicated that the in vivo results are consistent with the in vitro results and IL-2 treatment ameliorates hepatocellular carcinoma development which was mediated by exosomes from TAMs.
e exosomal miRNAs were regarded as an important mechanism of crosstalk between cancer cells and TAMs [19]. It was reported that some miRNAs were reduced in TAMs compared with the macrophages from normal tissues and they are able to regulate the apoptosis of tumor cells [3]. It has been well documented that miRNAs, such as miR-1205, miR-143-3p, miR-375, and miR-125a, may inhibit HCC [20][21][22][23]. As shown in Figures 6(a) and 6(b), among four miRNAs, miR-375 was obviously increased in exosomes derived from TAMs with IL-2 treatment and miR-375 was increased in Exo IL2-TAM treated HCCs compared with that in Exo IL2-TAM treated HCCs. We analyzed miR-375 expression in HCC patients in TCGA database and UAL-CAN database to explore the role of miR-375 in HCC. e results of Welch's t-test showed that the expression of miR-375 in Tumor group was lower than that in Normal group, and the difference between the two groups was −1.949, and the difference was statistically significant (Figure 6(c)). e paired-samples t-test results showed that the expression of miR-375 in Tumor groupwas higher than that in Normal group, and the difference was statistically   Journal of Oncology significant ( Figure 6(d)). At the same time, we can also see that the expression of miR-375 in different stages of cancer was lower than that in normal tissues (Figure 6(e)). We can conclude that the prognosis of the low miR-375 expression group was much more severe than that of the high miR-375 expression group, suggesting that the absence of miR-375 served as unfavorable factors to the patients (Figure 6(f )). In predicting Tumor and Normal outcomes, the predictive ability of the variable hsa-miR-375 was with certain accuracy (Figure 6(g)). It was reported that most mature TAMs tend to M2 phenotype and function, including promoting angiogenesis, participating in tissue repair and reconstruction, and regulating inflammatory mediators response and adaptive immunity. In a variety of malignant solid tumors, TAMs infiltration can promote tumor growth, angiogenesis, invasion, and metastasis and resist immune damage [25].

Discussion
TAMs and HCC cells interact to promote the progress of liver cancer: on the one hand, liver cancer cells secrete cytokines and chemokines, recruit macrophages to gather, and constantly adjust their own characteristics [26,27]. On the other hand, mature TAMs participate in the progress of liver cancer through a variety of mechanisms, including promoting angiogenesis to obtain sufficient nutrients for liver cancer cells in the state of rapid growth, participating in the degradation of basement membrane and matrix remodeling to prepare for local invasion and distant metastasis of tumor, and negatively regulating the antihepatoma immune response to escape the immune system, and provide a "safe haven" for tumor development [28]. erefore, the study of TAMs may help to find a molecular targeted drug that can effectively block the crosstalk between cancer cells and TAMs.
It is reported that exosomes mediate the intercellular information exchange which plays an important role in the occurrence and development of hepatocellular carcinoma [4][5][6]. Although the research on the relationship between cancers and exosomes from macrophages has become more and more popular in recent years, there is limited information on the exosomes derived from the cell lines [29][30][31][32]. Few studies have been carried out on the TAMs extracted directly from the tissues of patients [7][8][9]. Most of the studies on exosomes and their signaling pathways are only in the early stage, and many studies are limited to cell experiments and the results need to be further verified in animal models [31,33]. We directly collected HCC tissues and isolated TAMs which retain some of their biological characteristics in vivo. More importantly, the effects of exosomes in vivo were explored.
Recent research has shown that IL-2 is a cytokine produced by activating T cells whose antitumor mechanism lies in stimulating and activating its effector cells and then plays an antitumor role [33,34]. Recent studies have shown that IL-2 can promote the synthesis and secretion of IFN-c, and IFN-c regulates macrophage polarization to M1 type [35]. LPS induces macrophage M1 polarization and the M1 macrophages release exosomes to potentiate the anticancer efficacy [10]. However, LPS is not an approach for human therapy. e miRNAs play a key role in the occurrence and development of human malignant tumors [36,37]. In many malignant tumors, the expression levels of miRNA were changed to varying degrees compared with the surrounding normal tissues [11,12]. More and more in vitro experiments show that the changes of miRNAs can effectively affect the proliferation and metastasis of tumor cells [19,38]. It is reported that miR-375 inhibits HCC development and progression [21,39,40]. In this study, we found that miRNAs inhibiting HCC were upregulated in exosomes derived from TAMs treated with IL-2.

Conclusions
is study was the first to explore the effects of exosomes from IL-2 treated TAMs on HCC development and the possible mechanisms. We have found that exosomes derived from TAMs promote the development and progression of hepatocellular carcinoma in vivo and in vitro and these effects of exosomes were partly reversed by IL-2 treatment for TAMs. IL-2 increased the exosomal miRNAs which may be responsible for the HCC development.
ese findings provide a new perspective to explain the mechanism by which IL-2 inhibits HCC and provide basis for the clinical use of IL-2.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare that there are no conflicts of interest regarding the publication of this study.