IL-2 Increases Tams-Derived Exosomal Mir-375 to Ameliorate Hepatocellular Carcinoma Development and Progression

Background: Interleukin-2 (IL-2) is proved to play an irreplaceable role in anti-tumor regulation in numerous experimental and clinical trials. Tumor-associated macrophages (TAMs) is able to release exosomes to promote the development and progression of hepatocellular carcinoma (HCC) as part of microenvironment. Methods: 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 liver cancer tissues. The exosomes from the TAMs treated with IL-2 (Exo IL2-TAM ) or not (Exo TAM ) were identied and used to treat HCC cells. The HCC cells proliferation, apoptosis and metastasis were measured in vivo and in vitro. The changes of miR-375 in exosomes was explored to clarify whether it is reponsible to the anti-apoptotic effects of IL-2. Results: Both decrease of cell proliferation and metastasis and increase of apoptosis were observed with Exo IL2-TAM treatment compared with Exo TAM in vivo and in vitro. miR-375 was obviously abundant in Exo IL2-TAM . Enriched miR-375 could be transmitted between TAMs and HCC cells via exosomes and was responbible for the increased apoptosis of HCC cells. Conclusions: Taken together, IL-2 increases exosomal miR-375 from TAMs to attenuate hepatocellular carcinoma development. This study provides a new perspective to explain the mechanism how IL-2 inhibits hepatocellular carcinoma and implys the potential clinical value of exosomal miR-375 released by TAMs.


Background
Primary hepatocellular carcinoma (HCC) is the third most common malignant tumor in cancer mortality [1]. The treatment of HCC is mainly surgical resection and liver transplantation [2]. Because of its high degree of malignancy, the ve-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 liver cancer [3]. The 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 con rmed 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].Cells can secrete and widely exist in various body uids. Exosomes carries RNA and protein components of parental cells and are involved in signal transduction and immune escape of tumor and the diagnosis and treatment of some 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 macrophages [17,18]. IL-2 have been used in lots of clinical trials for the treatment of HCC [2]. However, it remains unkown whether IL-2 regulates the exosomes released from TAMs. Given the emerging role of IL-2 in cute 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.

Exosomes and TAMs cells observed by transmission electron microscopy
The samples were xed 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. The RNA was then analysed using real-time qPCR with SYBR Green PCR Master mix (Roche Applied Science, Mannheim, Germany). The relative gene expression was normalized to U6.

Quanti cation of apoptosis by ow cytometry
Apoptosis was determined using an Annexin V-FITC/PI apoptosis detection kit (eBioscience, USA). For both approaches cells were assessed via ow cytometry.

CCK8 assay
HepG2 cells were added to 96-well plates and cultured for 24, 48, 72, 96, 120 or 144 hours. Then, CCK-8 reagent was added to cells in serum-free medium for 2 hours, followed by measurements of absorbance at 450 nm.

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

Migration assay
Cell invasion assays were conducted on 24-well Transwell cell culture chambers with 8-μm sized pores without precoated Matrigel (Corning, USA). HepG2 cells were suspended in 500 μl of medium and added to the upper inserts. After 24 hour of incubation, the cells remaining in the upper chamber were removed, and the cells on the lower surface of the chamber were xed with 4% paraformaldehyde and stained with 0.5% crystal violet.

Scratch test
A scratch assay was performed to assess cell migration in vitro. First, HepG2 cells were seeded in 6-well plates until a con uent monolayer was formed. Then, upon con uence, cells were scratched with a 10 μL sterile pipette tip. Pictures were then taken of the scratch at different time-points under the microscope.
The cell migration rate was calculated as (width at 0 hours-width at 24 hours)/width at 0 hours.

Experimental animals
Xenograft mouse models All animal experiments were performed in accordance with 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 ) was performed BALB/c nude mice (4-6 weeks-old, n = 6 per group) accompanied with exosomes (40 ug/mL) injection via the tail vein every 2 days for 7 times. Tumor volumes were measured after 5 days every 10 days until mice were sacri ced 30 days later.

The liver and lung metastasis experiment
The 6-8 weeks old nude mice were divided into three randomized groups (n = 12 per group), and QJY-7703 (5 × 10 5 ) alone or with Exo TAM (40 ug/mL) or with Exo TAM-IL2 (40 ug/mL) in 100 ul were injected into the mice via tail vein every 2 days for 7 times. 30 days after cell injection, the mice were euthanized and were necropsied to assess metastatic burden. The tumor tissues, liver and lung tissues of mice were further examined by H&E and IHC staining.

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

Bioinformatics analysis
The Cancer Genome Atlas (TCGA) databse and UALCAN database which is built on PERL-CGI with high quality graphics using javascript and CSS were used to explore the expression of miR-375 in HCC patients.

Statistical Analysis
Data are 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. Value of P<0.05 was considered to be signi cant.

Phenotypic characteristics of TAMs
To obtain exosomes, we collected HCC tissues to Isolate and culture TAMs. The macrophages were identi ed by F4/80 as surface marker (Fig. 1A). After isolation of TAMs from HCC tissues, we rst performed immuno uorescence to identify macrophage surface maker (F4/80) of TAMs (Fig. 1A). We treated TAMs with IL-2 (20ng/ml) for 24 hours, the morphology of macrophages changed, with increased volume and extended pseudopodia in IL-2 treated TAMs compared to Con TAMs under electron microscope (Fig. 1B).

The internalization of exosomes by HCC cells
The exosomes were isolated from macrophage supernatants with ultracentrifugation and were obersved by transmission electron microscope (TEM) which were hemisphere with one side depression with typical characteristics ( Fig.2A). The diameter of exosomes ranging from 30 to 150 nm, as shown in Figure 2B. Western blot was used to further con rm that the isolated small spheres were exosomes by detecting CD63 and not Calnexin derived from TAMs (Fig. 2C). To determine the effects of exosomes on HCC cells, we examined whether exosomes could be taken up by HCCs. An immuno uorescence assay was performed by using exosomes labeled with PKH26, a red uorescence dye. Red uorescence was clearly observed in HCC cells around nuclei using the confocal microscope (Fig. 2D).
The effects of the exosmes on HCC development and progressionin vitro First, to evaluate the effects of exosomes on the HCC cells, Huh7, HepG2 and QJY-7703 cell were treated with PBS, Exo TAM and Exo IL2-TAM and cell survival ability was assayed by MTT. The results showed that Exo TAM promoted survival ability. However, Exo IL2-TAM reduced the cell survival ability compared to Exo TAM (Fig. 2E).
Then, the effects of the exosmes on the proliferation and apoptosis of HpG2 cells were explored. The results revealed that when compared with the control (Con) group, the cell proliferation abilities were increased, whereas the apoptotic rate was decreased in the Exo TAM group companied with related proteins. Exo IL2-TAM reversed these effects compared with Exo TAM group (Figures 3A-3J).
Last, the effects of the exosmes on the migration and invasion of HpG2 cells were measured. Transwell assay and scratch test were used to explore whether IL-2 treatment affects TAMs derived exosomes to in uence the migratory and invasive abilities of hepG2 cells. The cell migrative and invasive abilities were increased in Exo TAM group compared to Con group, these abilites were reduced in Exo IL2-TAM group compared to Exo TAM group (Figure 4).
Taken together, our ndings indicated that the effects that exosomes fromTAMs promote proliferative, migratory and invasive behaviors and inhibited apopotosis were reversed by IL-2 treatment in vitro.
The effects of the exosmes 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 anks of nude mice with exosomes injection via the tail vein. The tumors produced by co-injection of QJY-7703 and Exo TAM were signi cantly larger and heavier than those produced by QJY-7703 cells alone. However, the tumors were smaller and lighter in Exo IL2-TAM group compared to Exo TAM group ( Figure 5A-C). The tumors were collected and the apoptosis of tumor tissues were detected by the TUNEL staining and western blot. The results showed that decreased positive expression of TUNEL and Bcl2 protein levels and increased Bax protein levels were found in Exo TAM group. However, positive expression of TUNEL and Bcl2 protein levels increased and Bax protein levels decreased in Exo IL2-TAM group compared to Exo TAM group( Figure 5D-H). Meanwhile, the protein levels of PCNA and cyclin D1 were reduced in Exo IL2-TAM group( Figure 5I-K).
To explore the effect of exosomes on tumor metastasis in vivo, we injected QJY-7703 cells alone, or with Exo TAM , or with Exo IL2-TAM into 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 Exo TAM group compared with Con group, and the rates were lower in Exo IL2-TAM group (Fig. 6A-B).
Furthermore, the IHC staining revealed that Vinmentin were signi cantly elevated in the Exo TAM group, accompanied by increased protein levels related to EMT, but Vinmentin staining and protein levels related to EMT were reduced in Exo IL2-TAM group compared to Exo TAM group (Fig. 6C-H).
These experiments indicated that in vivo results are consistent with in vitro results and IL-2 treatment ameliorates hepatocellular carcinoma development mediated by exosomes from TAMs.
The exosomal miR-375 was increased from IL-2 treated TAMs and promotes HCC cell apoptosis The exosomal miRNAs was regarded as an important mechanism of Crosstalk between cancer cells and TAMs [19]. It was reportrd that miRNAs were reduced in TAMs compared with the macrophages from normal tissues and it is able to regulate the apoptosis of tumor cells [3].
It has been well documented that miRNAs, such as miR-120, miR-494, miR-143-3p, miR-375, miR-125a and miR-9-5p may inhibit cancer. As shown in Figure 7A, among six miRNAs miR-375 was obviously increased in exosomes derived from TAMs with IL-2 treatment. We analyzed miR-375 expression in HCC patients in the TCGA database and UALCAN database to explore the role of miR-375 in HCC. miR-375 was signi cantly elevated in HCC tissues compared with the control and the levels of miR-375 were signi cantly lower in patients with later stage than those with the early stage( Figure 7B-E).
The transport function of exosomes in HCC cells was analyzed with co-culture system. The TAMs transfected with FITC-miR-375 mimics were in apical chamber and the HCC cells were in the basolateral chamber. After 24-h co-culture, green uorescence could be found in HCC cells ( Figure 8A). To further con rm that exosomes released from TAMs is able to transfer miR-375 to HCC cells ( Figure 8B for a schematic design), we collected the conditioned media from TAMs to treat HCC cells. We found that miR-375 was increased in Group3. However, this increase was reversed by GW4869 in Group4 ( Figure 7C).
Last, to study the function of exosomal miR-375, we inhibited miR-375 by antagomir and overexpressed miR-375 using the agomir in TAMs. HCC cells were treated with exosomes derived from the agomir and antagomir transfected TAMs treated with IL-2 or not respectively. The upregulation of miR-375 signi cantly reduced the rate of apoptosis. Consistently, decreased miR-375 caused increased the rate of apoptosis ( Figure 8D). These results re ected that the inhibited apoptosis by exosomes from IL-2 treated TAMs is partly mediated by miR-375.

Discussion
The malignant behavior of tumor is not only determined by the characteristics of tumor cells, but also affected and regulated by various components in tumor microenvironment.TAMs, as the most abundant immune cells in tumor immune microenvironment, plays an important role in bridging the in ammatory mediators and tumors. Studies have shown that the degree of TAMs in ltration in HCC is negatively correlated with the prognosis of patients [20].
It was reported that most mature TAMs tend to M2 phenotype and function, including promoting angiogenesis, participating in tissue repair and reconstruction, regulating in ammatory mediators response and adaptive immunity. In a variety of malignant solid tumors, TAMs in ltration can promote tumor growth, angiogenesis, invasion and metastasis, and resist immune damage [21].
TAMs and HCC cells interact and interact to promote the progress of liver cancer: on the one hand, liver cancer cells secrete cytokines and chemokines, recruit macrophages to gather, domesticate and induce, and constantly adjust their own characteristics [22,23].On the other hand, mature TAMs participates in the progress of liver cancer through a variety of mechanisms, including: promoting angiogenesis to obtain su cient 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; negatively regulating the anti-hepatoma immune response to escape the immune system and provides a "safe haven" for tumor development [24].Therefore, the study of TAMs may help to nd 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 [25][26][27][28]. 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 veri ed in animal models [27,29].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 anti-tumor mechanism lies in stimulating and activating its effector cells, and then plays an anti-tumor role [29,30].Recent studies have shown that IL-2 can promote the synthesis and secretion of IFN -γ, and IFNγ regulates macrophage polarization to M1 type. [31] LPS induces macrophage M1 polarization and the M1 macrophages release exosomes to potentiate the anticancer e cacy [10]. However, LPS is not an approach for human therapy.
miRNAs plays a key role in the occurrence and development of human malignant tumors [32,33]. 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 [19,34].
It is reported that miR-375 inhibits human liver cancer development and progession [35][36][37]. In this study, we found that miR-375 is up-regulated in exosomes derived from TAMs treated with IL-2. Furthermore, to study the function of exosomal miR-375, we inhibited miR-375 by antagomir and overexpressed miR-375 using the agomir in HCC cells. The inhibition of miR-375 resulted in a decrease of apoptosis in HCC cells, while the overexpression by agomir resulted in an increase of apoptosis in HCC cells

Conclusions
Taken together, we have found that extracellular vesicles derived from TAMs promote the development and progression of hepatocellular carcinoma in vivo and in vitro and these effects of extracellular vesicles were reversed by IL-2 treatment. IL-2 increased the exosomal miR-375 which may be responsible for the increase of HCC cells apoptosis. These ndings provides a new perspective to explain the mechanism that IL-2 inhibits HCC and provide basis for the clinical use of IL-2 .    treated with PBS, ExoTAM and ExoIL2-TAM for 0 to 6 days and cell survival ability was assayed by CCK8 assay. Error bars, SD. *P < 0.05; **P < 0.01.  (D)Quali cation of invasive cells in scratch test; (E-I) Western blot analysis of MMP2, MMP9, N-cadherin and E-cadherin expression. Error bars, SD. *P < 0.05; **P < 0.01; ***P < 0.001.