The miR-561-5p/CX3CL1 Signaling Axis Regulates Pulmonary Metastasis in Hepatocellular Carcinoma Involving CX3CR1+ Natural Killer Cells Infiltration

Natural killer (NK) cell can inhibit tumor initiation and regulates metastatic dissemination, acting as key mediators of the innate immune response. Intrinsic factors modulating NK cells infiltration and its anticancer activity remain poorly characterized. We investigated the roles of dysregulation of micro(mi)RNAs and NK cells in progression of hepatocellular carcinoma (HCC). Methods: Small RNA sequencing were used to detect the miRNA profiles of tumor tissues from HCC patients with (n=14) or without (n=13) pulmonary metastasis and HCC cell lines with different pulmonary metastatic potentials. Chemokine expression profiling and bioinformatics were used to detect the downstream target of candidate target. In gain- and loss-of-function assays were used to investigate the role of miRNA in HCC progression. Different subsets of NK cells were isolated and used for chemotaxis and functional assays in vivo and in vitro. In situ hybridization and immunohistochemical analyses were performed to detect the expression of miRNA in tumor tissues from 242 HCC patients undergoing curative resection from 2010. Results: Three miRNAs (miR-137, miR-149-5p, and miR-561-5p) were identified to be associated with pulmonary metastasis in patients with HCC. miR-561-5p was most highly overexpressed in metastatic HCC tissues and high-metastatic-potential HCC cell lines. In gain- and loss-of-function assays in a murine model, miR-561-5p promoted tumor growth and spread to the lungs. Yet, miR-561-5p did not appear to affect cellular proliferation and migration in vitro. Bioinformatics and chemokine expression profiling identified chemokine (C-X3-C motif) ligand 1 (CX3CL1) as a potential target of miR-561-5p. Furthermore, miR-561-5p promoted tumorigenesis and metastasis via CX3CL1-dependent regulation of CX3CR1+ NK cell infiltration and function. CX3CR1+ NK cells demonstrated stronger in vivo anti-metastatic activity relative to CX3CR1- NK cells. CX3CL1 stimulated chemotactic migration and cytotoxicity in CX3CR1+ NK cells via STAT3 signaling. Blockade of CX3CL1, CX3CR1, or of pSTAT3 signaling pathways attenuated the antitumor responses. Clinical samples exhibited a negative correlation between miR-561-5p expression and levels of CX3CL1 and CX3CR1+ NK cells. High miR-561-5p abundance, low CX3CL1 levels, and low numbers of CX3CR1+ NK cells were associated with adverse prognosis. Conclusion: We delineated a miR-561-5p/CX3CL1/NK cell axis that drives HCC metastasis and demonstrated that CX3CR1+ NK cells serve as potent antitumor therapeutic effectors.


Introduction
Hepatocellular carcinoma (HCC), the most prevalent liver malignancy, is the third leading cause of cancer-related deaths. Most of the HCC cases and deaths, exceeding half a million annually, occur in developing countries, with China accounting for 50% of the cases [1]. Various viral infections (including hepatitis B and C) and nonviral etiological factors, such as fatty liver disease, usually lead to diverse genetic change and eventually drive HCC development. Surgical resection and liver transplantation are common treatment options for HCC, the 5-year metastasis and recurrence rate after resection still exceeds 50% [2].
To metastasize, HCC cells must firstly fulfill certain tumorigenic functions and then move away from the primary site and enter the bloodstream through the vasculature. These malignant cells need to escape the killing of multiple immune cells, survive in the circulatory system and eventually form secondary cancer tissues in distant organs, such as lung, bone, and brain. Notably, lung is the most common metastatic organ. The tumor microenvironment contains a large number of cellular components and non-cellular components. Cellular components include tumor cells, immune cells, endothelial cells, fibroblasts and so on, while the non-cellular components include the extracellular matrix, cytokines, and chemokines [3]. Lymphocytes within the tumor environment include T cells, B cells, and natural killer (NK) cells. Much research has focused on examining and attempting to reverse defects in CD8 + T cells. However, NK cells, as key mediators of the innate immune response [4], represent one of the first blockers of tumor initiation and regulators of metastatic dissemination [5,6]. To date, NK cell-based antitumor immunotherapy approaches have not been tested in a clinical setting. Intrinsic factors modulating NK cells infiltration and anticancer activity remain poorly characterized. Understanding of these mechanisms can contribute to approaches that enhance NK cells-mediated tumor cell clearance.
Chemokines are associated with tumor-related inflammation. Chemokine signaling modulates various events associated with tumor progression, such as leukocyte recruitment, neo-angiogenesis, tumor cell proliferation, and metastasis. Fractalkine, also known as CX3C chemokine ligand 1(CX 3 CL1), is predominantly produced by the endothelium and binds only to the cognate chemokine (C-X 3 -C motif) receptor 1 (CX 3 CR1). CX 3 CR1 is a G-protein-coupled receptor expressed by CD8 + T lymphocytes [7], NK cells [8], and monocytes. The CX 3 CL1/CX 3 CR1 axis controls leukocyte migration [9], mediating chemotaxis and CX 3 CR1 + cell adherence. Because of its role in carcinogenesis and tumor progression, the CX 3 CL1/CX 3 CR1 axis has been explored as a candidate therapeutic target [10]. However, CX 3 CL1 function in HCC and in NK cells-mediated eradication of cancer cells remains largely uncharacterized.
MicroRNAs (miRNAs) are small non-coding RNAs that participate in post-transcriptional regulation of gene expression [11]. Expression of specific miRNAs, which can control a wide range of target genes, is often dysregulated in cancer cells [12,13]. Investigations of miRNAs in HCC metastasis can identify new prognostic biomarkers and therapeutic targets [14][15][16]. Thus, we employed small RNA sequencing (smRNA-seq) to investigate the roles of miRNAs in HCC metastasis. We observed that elevated miR-561-5p levels are associated with pulmonary metastasis and a poor prognosis in HCC patients undergoing liver resection. Further studies revealed that miR-561-5p promoted HCC xenograft outgrowth and lung metastasis via the regulation of CX3CR1 + NK cells infiltration. Mechanistically, miR-561-5p down-regulated CX 3 CL1 messenger RNA (mRNA) to reduce chemotaxis and function of CX 3 CR1 + NK cells, but not of CX 3 CR1 -NK cells. The CX 3 CR1 + NK subset reduced the rate of pulmonary metastasis in a mouse model. Overall, our results demonstrated how the crosstalk between cancer cells and immune cells modulates HCC metastasis.

Human Subjects and Patient Evaluation
A total of 325 HCC patients were enrolled in this study. Samples collected between March and December of 2010 from 14 primary HCC patients with postoperative pulmonary metastasis (one sample excluded from analysis due to poor RNA quality) and 14 HCC patients without postoperative pulmonary metastasis were used for smRNA-seq. All samples were collected immediately after surgical resection, placed into liquid nitrogen and stored at -80°C. We also collected paired adjacent liver tissues of 13 primary specimens for smRNA-seq (three samples were not obtained, and one sample excluded from analysis due to poor RNA quality). Detailed clinicopathological features are listed in Supplementary  Table S1. Samples from additional 55 HCC patients undergoing curative resection were collected for validation of associations between the candidate miRNA expression and prognosis.
Using SPSS software, we randomly selected 242 HCC patients undergoing curative resection in 2010 in the absence of pre-surgery anticancer therapy to construct tissue microarrays for expression analysis of miR-561-5p, CX3CL1, CX 3 CR1, and CD56. These patients were post-surgically monitored until July 7, 2015, with a median follow-up of 54 months (range: 1-66 months). The follow-up procedures were performed as described in our previous reports [17]. The patients did not exhibit signs of distant metastasis.
Tumors were assessed using the World Health Organization histological classification, with differentiation graded using the Edmondson-Steiner system. Liver function was assessed using the Child-Pugh classification. The Barcelona-Clinic Liver Cancer and the tumor-node-metastasis classification systems were used to determine the disease stage. Overall survival (OS) was determined as the time that elapsed between surgery and death or the last observation point. Data of surviving patients were censored at the last follow-up. Time elapsing between the dates of the surgery and of any diagnosed intrahepatic or extrahepatic relapse was defined as time to recurrence (TTR). The protocols for the use of human subjects in this study were approved by the Research Ethics Committee of Zhongshan Hospital. Informed consent was obtained from all subjects.

Cell Lines
The human hepatoma MHCC97L, MHCC97H, and HCCLM3 cell lines, demonstrating stepwise pulmonary metastatic potential, were previously developed at our institute. Low-metastatic-potential HepG2, PLC/PRF/5, SMCC-7721, and Huh7 cell lines were purchased from the Institute of Biochemistry and Cell Biology (Chinese Academy of Sciences, Shanghai, China). All cell lines were routinely maintained in Dulbecco's Modified Eagle Medium (GIBCO, NY, USA). Vectors used in this study, cell transfection procedures, NK cell isolation, chemotaxis assays, NK cytotoxicity assays, cell proliferation, and migration assays are described in the Supplementary Materials and Methods.

Quantitative Polymerase Chain Reaction (qRT-PCR)
RNA isolation, reverse-transcription, and qRT-PCR are described in the Supplementary Materials and Methods.

RNA and protein detection
Western blotting, enzyme-linked immunosorbent assays (ELISAs), immunohistochemistry procedures, and analysis, as well as in situ hybridization, are described in the Supplementary Materials and Methods.

Animal Subjects
The study utilized 4-to 6-week-old male athymic BALB/c nu/nu mice (Shanghai Institute of Material Medicine, Chinese Academy of Sciences). The animals were maintained under specific-pathogen-free conditions. Humane animal care protocols were established according to the National Research Council Guide for the Care and Use of Laboratory Animals.

smRNA-seq
Total RNA was isolated from approximately 10 6 HepG2, PLC/PRF/5, MHCC97H, or HCCLM3 cells or from clinical samples using the TRIzol reagent (Invitrogen, Grand Island, NY). RNA quality and quantity were assessed using the 2100Bioanalyzer System (Agilent Technologies, Santa Clare, CA). The TruSeq Small RNA Library Prep Kit (Illumina) was used according to the manufacturer's protocol to prepare the smRNA-seq library using 1 μg of highquality total RNA as the starting material. Sequencing was performed using the HiSeq2500 platform (Illumina) at GenergyBiotechnology (Shanghai) Co.Ltd. The FASTX Toolkit (Version 0.0.14) was used to filter out the adapter sequences. We quantified the reads mapping to known or novel miRNAs using miRBase v21 or miRDeep 2, respectively, and used the transcripts per kilobase million method to determine the normalized expression of each miRNA.

Tumor Growth and Pulmonary Metastasis In Vivo Assays
Wild-type, knockdown, and overexpression (HepG2, HepG2-miR-561-5p, HepG2-shCX 3 CL1, Hep G2-miR-561-5p-CX 3 CL1, HCCLM3, HCCLM3-anti-miR-561-5p, HCCLM3-CX 3 CL1, HCCLM3-anti-miR-561-5p-shCX 3 CL1) cells (5 × 10 6 ) were suspended in 100 μL of a 1:1 mixture of serum-free Dulbecco's Modified Eagle Medium and Matrigel (BD Bioscience). The cell suspensions were injected subcutaneously into nude mice at the upper left flank region. After 4 weeks' injection, when subcutaneous tumors reached approximately 1cm in length, all tumors were peeled, sheared into 1mm 3 volume and inserted into the livers of nude mice. Seven days following inoculation, animals in the NK depletion experimental group were intravenously injected with anti-Asialo-monosialotetrahexosylganglioside (GM1) antibody twice weekly. All animals were observed weekly and sacrificed 6 weeks post-inoculation. The IVIS Lumina K Series III system (PerkinElmer) was utilized to perform bioluminescence imaging, with radiance values normalized using the Living Image software. We observed the mice over 5 weeks for tumor formation. Tumor volume was calculated as follows: V=ab 2 /2, where V is the tumor volume in cm 3 , and a and b are the largest and smallest tumor diameters measured during necropsy, respectively. Following lung removal and embedding in paraffin, microscopy was used to determine the number of metastases per lung. For evaluating different function of CX 3 CR1and CX 3 CR1 + NK cells in vivo, tail vein metastasis models of ten mice in each group were used. 1×10 6 HCC cells were injected into mice, and then CX 3 CR1or CX 3 CR1 + NK cells were injected into mice. CX 3 CR1or CX 3 CR1 + NK cells were injected into the mice biweekly for 5 consecutive weeks after inoculation. The metastases were classified as grade I through grade IV based on the presence of ≤20, 20-50, 50-100, or >100 tumor cells at the maximal section of each metastatic lesion, respectively. CX3CR1 + or CX 3 CR1human allogenic NK cells (5 × 10 6 ), purified from peripheral blood mononuclear cells from healthy donors using fluorescence-activated cell sorting, were injected intravenously.

Overexpression of miR-561-5p is associated with HCC and HCC metastasis
Lung metastases rarely occur in HCC patients that have undergone surgical resection. To identify miRNAs associated with HCC lung metastasis, we performed next-generation smRNA-seq to compare miRNA abundance patterns in primary human HCC with and without metastasis. We also performed smRNA-seq using high-metastatic-potential (MHCC 97H and HCCLM3) and low-metastatic-potential (HepG2 and PLC/PRF/5) human HCC cell lines. Analysis of the clinical samples identified 57 differentially expressed (fold-change > 2, P < 0.01) miRNAs ( Figure 1A). Moreover, 284 miRNAs demonstrated metastatic potential-dependent abundance levels in the HCC cell lines. Further analysis of tumors and adjacent non-tumor tissues identified 321 differentially expressed miRNAs (Supplementary Figure S1A, Supplementary Excel 1). Combination of these bioinformatics analyses suggested that expression of miR-137, miR-149-5p, and miR-561-5p, which was confirmed by qRT-PCR, correlates with HCC progression and lung metastasis ( Figure 1B). As miR-561-5p demonstrated the most substantial changes in abundance ( Figure 1C-E, and Supplementary Figure S1B-C), we subsequently sought to explore the role of miR-561-5p in HCC lung metastasis.
First, we evaluated the relationship between endogenous miR-561-5p levels and the metastatic potential of HCC lines. Cell lines with highmetastatic-potential exhibited elevated expression of miR-561-5p relative to non-metastatic cell lines, indicating the miR-561-5p were positively correlated with increasing metastatic potential ( Figure 1F). Next, using qRT-PCR, we measured the expression of miR-561-5p in paired tumor and adjacent tissue samples from HCC cohorts with (n=23) and without metastasis (n=32). Highest miR-561-5p levels were observed in tumors with metastasis, whereas lowest levels were detected in the adjacent non-tumor tissues ( Figure 1G-H). Taken together, these findings prompted us to investigate the mechanisms by which miR-561-5p contributes to HCC metastasis.

miR-561-5p promotes tumor progression and lung metastasis in vivo, without affecting HCC cell proliferation and invasion in vitro
To further determine the effects of miR-561-5p in HCC, miR-561-5p levels in HCC cell lines were modulated using miR-561-5p or anti-miR-561-5p lentiviral vectors. Altered miR-561-5p levels were confirmed via qRT-PCR (Supplementary Figure S2A). Down-regulation of miR-561-5p in HCCLM3 and MHCC97H cells did not affect proliferation rates. Proliferation rates of miR-561-5p-overexpressing HepG2 and PLC/PRF/5 cells were similar to those of control cells (Supplementary Figure S2B). Modulation of miR-561-5p levels also had no effect on migration and invasion of HCC cells (Supplementary Figure  S2C-D).
To assess whether miR-561-5p affects tumor growth and metastasis in vivo, HCC cells harboring different miR-561-5p levels were injected into nude mice. The tumor size of the HCCLM3-derived xenografts (5.12 ± 0.73 cm 3 ) was significantly larger than that of xenografts derived from HCCLM3-anti-miR-561-5p cells (3.09 ± 0.69 cm 3 ). Similarly, the average tumor size in the HepG2-miR-561-5p group was significantly greater than that of the HepG2-derived tumors (3.58 ± 0.85 cm 3 vs 1.95 ± 0.54 cm 3 ; Figure 2A). These results were confirmed by bioluminescence imaging ( Figure 2B). Whereas pulmonary metastases were observed in all animals in the HCCLM3 group, <20% of the mice (1/6, P=0.015) exhibited metastases in the HCCLM3-anti-miR-561-5p group. Animals in the HCCLM3 group also harbored more metastatic nodules of each grade ( Figure 2C). Likewise, pulmonary metastasis was observed in 83.3% (5/6) of the animals in the HepG2-miR-561-5p group, but was not detected in the HepG2 group ( Figure 2C, P=0.015). These finding demonstrated that miR-561-5p promotes HCC growth and pulmonary metastasis in vivo.

CX 3 CL1 is a direct downstream target of miR-561-5p
The discrepancy between the in vitro and in vivo results indicated that a cell nonautonomous mechanism may be responsible for miR-561-5p effects. One such mechanism involves the regulation of cytokines to modulate the tumor microenvironment [18]. Thus, we assessed the effect of miR-561-5p on cytokine expression by testing HCCLM3 and MHCC97H cells transfected with anti-miR-561-5p and HepG2 and PLC/PRF/5 cells transfected with miR-561-5p. CX 3 CL1 was the only chemokine that demonstrated consistent inverse correlation with miR-561-5p in all experimental groups ( Figure 3A, Supplementary Table S2,). ELISA confirmed elevated CX 3 CL1 levels following miR-561-5p knockdown in HCCLM3 and MHCC97H cells. In contrast, overexpression of miR-561-5p markedly reduced CX 3 CL1 expression in HepG2 and PLC/PRF/5 cells ( Figure 3B).We also observed that basal levels of CX 3 CL1 negatively correlated with miR-561-5p levels in eight HCC cell lines ( Figure 3C).Moreover, the lowest levels of CX 3 CL1 expression were observed in HCC with metastasis, whereas the highest levels were detected in the paired non-tumor tissues ( Figure 3D-E).
TargetScan (http://www.targetscan.org) and miRDB (http://mirdb.org) bioinformatics analyses predicted CX 3 CL1 as a potential target of miR-561-5p. The putative binding sequence of miR-561-5p was predicted to be located within the 3' untranslated region (UTR) of the CX 3 CL1 mRNA ( Figure 3F). Expression of miR-561-5p effectively reduced luciferase activity of the CX 3 CL1 reporter construct. This effect was abrogated via seed sequence mutations in predicted miR-561-5p binding sites. Taken together, these experiments confirmed a direct interaction between miR-561-5p and the 3'-UTR of CX3CXL1 mRNA in vitro and in vivo. (H) qRT-PCR revealed that the expression of miR-561-5p in HCCs was significantly increased in the tumor (T), when compared to corresponding adjacent nontumor tissues (N). The data of (F, G, H) was generated from eight HCC cell lines, and another cohort including 23 paired metastatic tumor and adjacent tissue sample (n=23), and cancer samples without metastasis (n=32)). Data shown are mean±SD from three independent experiments, each performed in triplicate. (*P < 0.05, **P<0.01, ***P < 0.001, ****P < 0.0001; Student's t-tests).

CX 3 CL1 triggers chemotactic migration and cytotoxicity of NK cells via signal transducer and activator of transcription 3 (STAT3) signaling.
CX 3 CL1 can recruit CX 3 CR1 + inflammatory cells [19]. Therefore, we tested the effect of recombinant CX 3 CL1 on the migration of monocytes, T cells, dendritic cells, and NK cells isolated from HCC patients.
Till now, it is still not clarified which pathways were activated in CX 3 CR1 + NK cells. In our study, we investigated some critical classical pathways for NK cells. We first performed western blotting analysis of NK cells in the presence or absence of 100 nM CX3CL1 to assess potential pathways associated with the CX 3 CL1-mediated effects on NK cells. We observed that CX 3 CL1 led to the increased phosphorylation of STAT3, but did not affect p65, FAK, JNK, STAT1, AKT, p38-MAPK, or ERK phosphorylation in CX 3 CR1 + NK cells. Furthermore, blocking CX 3 CL1 or CX 3 CR1 by anti-CX 3 CL1 or anti-CX 3 CR1 antibody in CX 3 CR1 + NK cells caused a significant decrease in the phosphorylation levels of STAT3 compared with CX 3 CL1 group. These results revealed that the effect of CX 3 CL1 on STAT3 phosphorylation in CX 3 CR1 + NK cells was dependent on the CX 3 CL1 receptor, CX 3 CR1 ( Figure 4C, Supplementary Figure S3B).
Next, we assessed whether HCC cells use CX 3 CL1 to recruit NK cells in vitro. Conditioned medium from high CX 3 CL1 expressers (HCCLM3-CX 3 CL1 and HepG2-Control) significantly increased the migration of NK cells relative to that from low CX 3 CL1 expressers (HCCLM3-Control and HepG2-shCX 3 CL1; Figure 4D). Anti-CX 3 CL1 antibodies eliminated the chemotactic migratory effect of the conditioned medium. Then we assessed the effect of CX 3 CL1 levels on cytolytic activity of NK cells co-cultured with different HCC cell lines. Co-culture with HCCLM3-CX 3 CL1 cells enhanced the cytolytic activity of NK cells, whereas the presence of HepG2-shCX 3 CL1 cells reduced the cytolytic activities of NK cells ( Figure 4E). These data indicated that CX 3 CL1 can directly chemoattract NK cells and enhance their cytotoxicity via STAT3 phosphorylation.

Overexpression of miR-561-5p down-regulates CX 3 CL1 and promotes tumor growth and metastasis via suppression of NK cells infiltration
We next assessed whether the effect of miR-561-5p on HCC progression is negatively correlated with CX 3 CL1-induced NK cells infiltration. Knockdown of miR-561-5p or overexpression of CX 3 CL1 enhanced the migratory effect of the HCCLM3 cell conditioned medium on NK cells. Secondary knockdown of CX 3 CL1 eliminated the anti-miR-561-5p effect. Overexpression of miR-561-5p or CX3CL1 knockdown reduced chemotaxis of NK cells in HepG2 cell conditioned medium. However, CX 3 CL1 overexpression reversed the miR-561-5p effects ( Figure  5A-B). These findings indicated that HCC cells use miR-561-5p to inhibit NK cell migration via down-regulation of CX 3 CL1.
To further assess the inhibitory function of CX 3 CL1 in HCC development, we modulated CX 3 CL1 levels in HCC cells. In a manner similar to miR-561-5p modulation, manipulation of CX 3 CL1 levels did not affect the phenotype of HCC cells in vitro (data not shown). However, in vivo, CX 3 CL1 reduced tumorigenesis and pulmonary metastasis, with CX 3 CL1 suppression promoting tumor development ( Figure  5C-D). NK cells infiltrated the HCCLM3-derived xenografts at a rate lower than the HCCLM3-CX 3 CL1 xenografts. Likewise, HepG2-derived xenografts contained higher numbers of infiltrating NK cells relative to the HepG2-shCX 3 CL1 group ( Figure 5C-D). On the other hand, there was no difference in the numbers of monocyte and myeloid-derived suppressor cells (Supplementary Figure S4A-B).
We subsequently assessed how the miR-561-5p/ CX3CL1/NK cells axis regulates HCC progression and pulmonary metastasis. CD56 is a specific surface marker of NK cells. We counted the number of positive staining cell with CD56 in each group. CX 3 CL1 overexpression or miR-561-5p knockdown in HCCLM3 cells significantly suppressed tumor volume, reduced the rate of lung metastasis, and enhanced NK cells infiltration. Knockdown of CX 3 CL1 countered the effects of anti-miR-561-5p. Furthermore, overexpression of CX 3 CL1 rescued the HepG2-miR-561-5p phenotype ( Figure 5D), indicating that miR-561-5p drives HCC development by suppressing CX 3 CL1-induced NK cells infiltration.

CX 3 CR1 + NK cells suppress HCC progression in a murine model
Based on the above results, we hypothesized that miR-561-5p suppresses CX3CL1 levels, thus reducing the infiltration of NK cells. CX 3 CR1 + NK cells, harboring a CX 3 C chemokine receptor, play a critical role in CX 3 CL1-induced NK cell migration. Using flow cytometry, we assessed the prevalence of CX 3 CR1 + NK cells in 20 fresh HCC patients' peripheral blood and 20 fresh healthy control samples ( Figure  6A). The results showed CX 3 CR1 + NK cells are abundant in healthy donors compared with HCC patients.
To investigate the effect of CX 3 CR1 + NK cells on HCC growth and metastasis in vivo, we administered the NK cell-depleting anti-Asialo-GM1 antibody to nude xenograft mice and determined their response to CX 3 CR1 + NK cells. The mice were treated with either CX 3 CR1 + or CX 3 CR1 -NK cells twice a week for three weeks ( Figure 6B). The average tumor size and pulmonary metastasis rate were decreased in mice treated with CX 3 CR1 + NK cells alone relative to those treated with CX 3 CR1 + NK cells combined with an anti-CX 3 CL1 antibody or an anti-CX 3 CR1 antibody ( Figure 6C-E). Likewise, the pulmonary metastasis rate in mice treated with CX 3 CR1 + NK cells was markedly decreased relative to that in mice treated with CX 3 CR1 -NK cells ( Figure 6F). To compare the functional difference on circulating HCC cells metastasis between CX 3 CR1 + NK cells group and CX 3 CR1 -NK cells group, we designed new experiments. We injected HCC cells and then we injected CX 3 CR1 + or CX 3 CR1 -NK cells in ten mice each group by tail vein injection lung metastasis model. In vivo study showed that injection of CX 3 CL1 + NK following HCC cells resulted in significantly decreased pulmonary metastasis compared HCC cells with injection of CX 3 CR1 -NK cells (1/10 vs 6/10) (P<0.05, Figure 6G). The results suggested that the CX 3 CL1/CX 3 CR1 axis might be responsible for the negligible therapeutic effect against HCC in nude mice, perhaps due to enhanced cytotoxicity of CX 3 CR1 + NK cells.

miR-561-5p, CX 3 CL1, and CX 3 CR1 + NK cells predict HCC patient prognosis
Colonization of the lung is a crucial step in HCC pulmonary metastasis. The above results indicated that the miR-561-5p/CX 3 CL1/NK cell axis promotes HCC growth and lung metastasis. To further explore the clinical relevance of this axis, we performed in situ hybridization to determine miR-561-5p localization, in combination with immuno-staining of CX 3 CR1 + NK cells, on serial sections of clinical samples from primary cancers and paired lung metastases. Tumor cells and surrounding fibroblasts exhibited miR-561-5p expression ( Figure 7A). We observed miR-561-5p expression was higher in the metastatic lung niche than that in primary cancers, with fewer CX 3 CR1 + NK cells detected in the metastatic lung niche ( Figure 7A). To confirm the expression of miR-561-5p in HCC tissue and lung metastatic tissue sample, RT-PCR revealed that the expression of miR-561-5p in significantly decreased in primary tumors compared to the corresponding lung metastatic tissues ( Figure  7B). These data underscored the hypothesis that high levels of miR-561-5p in primary HCC and metastases suppress NK cells-mediated eradication of cancer cells and promote tumor dissemination and colonization of the lung.
To evaluate the clinical prognostic significance of miR-561-5p, CX3CL1, and CX3CR1 + NK cells, we assessed the expression of miR-561-5p, CX 3 CL1, CX 3 CR1, and CD56 in primary tumors from a distinct 242-patient HCC cohort ( Figure 7C-D). Levels of miR-561-5p inversely correlated with survival rates, as determined using the Kaplan-Meier analysis. The high-level miR-561-5p HCC patients demonstrated the poorest prognosis at 1, 3, and 5 years and exhibited higher cumulative recurrence rates than low-level miR-561-5p patients ( Figure 7E-F). In contrast, CX3CL1 levels positively correlated with survival rates and negatively correlated with recurrence rates (Figure 7E-F). Detection of CX 3 CR1 + NK cells exhibited a positive correlation with OS (P<0.029) and TTR (P<0.001). Moreover, low-level miR-561-5p expression correlated with higher CX 3 CL1 abundance and more NK cells infiltration in HCC patients ( Figure 7C-D). To assess the combined prognostic value of these factors, we assigned patients to the following three groups: group I comprised patients with low miR-561-5p, high CX3CL1, and high CX 3 CR1 + NK cells levels; group III comprised patients with high miR-561-5p, low CX 3 CL1, and low CX 3 CR1 + NK cells levels; group II comprised the remaining patients. Group I OS values were higher than those of group II, with the lowest values observed for group III (Figure 7E-F). Group I cumulative recurrence rates were substantially lower than those observed for individuals in groups II and III ( Figure 7F). Overall, miR-561-5p levels, CX3CL1 abundance, CX 3 CR1 + NK cells infiltration, and the co-index were found to be independent prognostic factors of HCC OS and TTR (Table 1).

Discussion
The interplay between tumor cells and their microenvironment influences a variety of processes, including tumor invasion, metastasis, and response to therapy. NK cells, a major component of the tumor microenvironment, constitute 30-40% of all intrahepatic lymphocytes and exhibit antitumor functions [20]. We identified a novel signaling pathway in which miR-561-5p alters the tumor microenvironment by suppressing its target CX3CL1 and consequently blocking the recruitment and infiltration of NK cells, thereby allowing HCC cells to evade NK cell cytotoxicity, proliferate, and metastasize to the lungs (Figure 8).
To our knowledge, this is the first work to report a positive correlation between miR-561-5p levels and pulmonary metastasis, establishing miR-561-5p as both a risk factor and a prognostic marker in HCC metastasis. Overexpression of miR-561-5p in HCC patients corresponded to lung metastasis and poor prognosis, underscoring its cancer-promoting role in HCC progression. In contrast to other miRNAs implicated in HCC progression, miR-561-5p promoted tumor progression by influencing tumor microenvironment rather than tumor cell intrinsic activity, and stimulated tumor growth and pulmonary metastasis in animal models. A study of 242 patients revealed that the level of miR-561-5p represents a new and meaningful molecular marker to predict HCC patient prognosis. This pro-metastatic role of miR-561-5p was found to be based on its regulation of CX3CR1 + NK cell function via its target CX 3 CL1.
Identification of miR-561-5p was somewhat surprising, as very little is known about its role in cancer [21].To determine the role of miR-561-5p, we speculated that it can modulate cytokine or chemokine levels to reshape the tumor microenvironment to favor metastasis [22]. CX3CL1 mRNA was identified as the direct downstream target of miR-561-5p, with miR-561-5p overexpression in HCC patients reducing CX 3 CL1 levels, indicating that the crosstalk between cancer cells and their associated stroma cells is potentially regulated by CX 3 CL1. Furthermore, our results confirmed that the main resource of CX 3 CL1 is tumor cells, not endothelial cells or other stromal cells (Supplementary Figure  S5A-B). A dual luciferase reporter assay and rescue experiments confirmed CX 3 CL1-dependent regulation of HCC progression by miR-561-5p. CX 3 CL1, which signals through CX 3 CR1, can recruit and activate NK cells via upregulation of several key proteins. However, to date, the function of this chemokine in tumorigenesis has remained poorly characterized. CX 3 CL1 has been shown to robustly affect the chemotactic movement of NK cells [23,24]. Presently, we demonstrated that CX 3 CL1 stimulates CX 3 CR1 + NK cell chemotactic migration and cytotoxicity, a response abolished by anti-CX 3 CL1 or anti-CX 3 CR1 antibodies. The antitumor and anti-metastatic effects of CX 3 CL1 were driven by NK cell recruitment and infiltration, as revealed by in vivo studies. Considering that CX 3 CR1 is expressed in T cells as well as NK cells in intact humans and mice, T cells may be responding to the chemokine CX 3 CL1. Our in vitro experiments only observed the low chemotactic effects of CX3CL1 on T cells, compared with those on NK cells. In addition, we immune-stained CD8+T cells in the TMA and found that the level of miR-561-5p was not associated with the number of CD8+T cells (P=0.621, Supplementary Figure S6A-B). Aoyama et al [25] described CX3CL1-CX 3 CR1 interaction could prevent liver inflammation and fibrosis. This observation, which contradicts our observation, may stem from the difference between the specimens and the isolated cells. In his study, NK cells were not isolated, so the role of CX3CR1 in NK cells remained unknown. We isolated the CX3CR1 + NK cells from human HCC tissues to better reflect the real tumor environment. Our results Together, these findings suggested that loss of CX 3 CL1 contributes to the absence of CX 3 CR1 + NK cells during HCC development.
We also demonstrated that these CX 3 CL1mediated effects likely involve the activation of STAT3 signaling in NK cells. Although a growing body of literature has linked NK cell dysfunction and cancer [26][27][28][29], the regulation of NK cell migration to the tumor nest has remained poorly understood. The tumor microenvironment can contain a diverse repertoire of chemokines and cytokines, with tumors capable of transforming their chemokine patterns to hinder NK recruitment and infiltration. While a variety of cells are recruited to tumor sites [30][31][32], NK cells are particularly suppressed during tumor development [33]. Basic research and clinical studies have indicated that the tumor microenvironment is often configured to block NK cell recruitment and function. However, if not blocked, NK cells can function as robust effectors against tumors [34][35][36]. Presently, we isolated CX3CR1 + NK cells from HCC tissues and used murine models to confirm their vital role in HCC progression. We also validated these cells as integral components of the miR-561-5p/CX 3 CL1 signaling regulation of pulmonary metastasis. These experiments demonstrated that the tumor microenvironment can be altered through a series of events initiated by changes in miR-561-5p expression.
Our evidence of HCC-specific expression patterns and pro-metastatic function nominated miR-561-5p as an independent prognostic biomarker and a potential intervention target. We also observed that miR-561-5p, CX3CL1, and CX 3 CR1 + NK cells levels can serve as independent indicators of HCC prognosis, with the combination of these factors providing a more sensitive independent prognostic measure. Thus, it is important to further explore the antitumor effects of these factors in vivo. Accumulating evidence has indicated the contribution of the CX3CL1-CX3CR1 axis to the pathogenesis of many disorders including cancer [37,38], inflammatory [39] and diabetes [40]. We cannot investigate all the CX 3 CR1 expressing immune cells in the tumor.
In summary, this study elucidated a miR-561-5p/CX 3 CL1/CX 3 CR1 + NK cells axis that delineates mechanisms underlying HCC pulmonary metastasis. In addition, miR-561-5p, CX 3 CL1, and CX 3 CR1 + NK cells can be used to gauge the prognosis of HCC patients undergoing curative resection. Moreover, components of this signaling axis represent potential immunotherapy targets to combat immune escape strategies in HCC.