CD61 + LDNs drive cancer metastasis by upregulating CCDC25 expression via DNA-TLR9-NF-kB axis in HCC

CD61 + LDNs drive cancer metastasis by upregulating CCDC25 expression via DNA-TLR9-NF-kB axis in HCC | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article CD61 + LDNs drive cancer metastasis by upregulating CCDC25 expression via DNA-TLR9-NF-kB axis in HCC Xiangqian Guan, Yuyan Lu, Chuanzheng wang, Ping Zhan, Zhigao Chen This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3868978/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background A subset of neutrophils isolated from peripheral blood mononuclear cells (PBMC) has recently been described in cancer patients. Methods Double gradient centrifugation was used to separate neutrophil subset. Western blot assay was performed to assess CCDC25 expression level. Results In this study, we found that low density neutrophils(LDNs) were highly enriched in metastatic hepatocellular carcinoma (HCC) patients compared to non-metastatic HCC patients. Then, we showed that the most prominent marker on LDNs was CD61, compared to high density neutrophils(HDNs). The CD61 + LDNs subset displayed an increased ability in triggering metastasis, producing free DNA, and synthesizing cytokine CCL5, and a decreased ability in forming NETs, synthesizing cytokine TNF-α and producing ROS, as compared with CD61 − LDNs or HDNs. Transcriptomic analysis revealed that CD61 + LDNs displayed distinct gene transcribe from CD61 − LDNs and HDNs. The abundance of circulating CD61 + LDNs was negatively correlated with disease prognosis, and positively correlated with the expression of CCDC25 within tumor. These CD61 + LDNs increased the invasion of HCC cells by upregulating CCDC25. Mechanistically, the CD61 + LDN-derived free DNA, excluding the NETs-DNA, enhanced the invasiveness of HCC cells and triggered their metastatic potential, which was mediated by TLR9-NF-κB-CCDC25 signaling. Blocking this signaling reversed the invasion of the CD61 + LDNs-evoked HCC cells. In vivo, we consistently showed that CD61 + LDNs enhanced HCC metastasis to the lungs. Conclusions Overall, our findings showed that a subset of CD61 + LDNs has pro-metastatic effects on HCC, which might be used for targeting HCC in the clinical setting. Low density neutrophils CCDC25 Hepatocellular carcinoma Orthotopic model Tumor microenvironment Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Introduction Cancer-associated neutrophils retain functional plasticity and can undergo alternative activation when exposed to different cues found in the tumor microenvironment (TME)[ 1 – 3 ]. Several studies have reported the presence of at least two distinct neutrophil phenotypes in the disease setting, including LDNs and HDNs[ 4 ]. LDNs are different from HDNs, and they have a density below 1.077 g/mL and precipitate in the PBMC layer after density gradient centrifugation of whole blood[ 4 – 5 ]. Morphologically, LDNs have a ring-shaped nucleus, whereas HDNs have a segmented nucleus[ 6 – 7 ]. LDNs consist of granulocyte myeloid-derived suppressor cells (G-MDSCs) and mature large neutrophils[ 8 ]. The phenotype of LDNs may vary depending on the context of the disease[ 9 ]. In sepsis, LDNs proliferate and exhibit characteristics of G-MDSCs[ 10 ]. The LDNs in systemic lupus erythematosus (SLE) trigger rapid cell death via the immune complex, and this cell death results in the extracellular release of nuclear DNA and oxidized mitochondrial DNA[ 11 ]. In patients with rheumatoid arthritis (RA), LDNs occur as an immature subset with prolonged survival but low response to cytokine stimulation[ 12 ]. In cancer patients, several differences occur between these two neutrophil subsets, such as the ability to inhibit the activation of CD8 + T cells[ 4 ][ 7 ]. In the breast cancer metastasis model, immature LDNs, but not mature LDNs, were found to accumulate and acquire a higher proportion in metastatic sites and peripheral blood at a later stage of liver metastasis[ 2 ]. Additionally, the transition of HDNs to LDNs was found to occur spontaneously in several tumor models[ 2 ][ 5 ]. Tumor growth factor-β (TGF-β) might act as a major regulator in inducing this transition from HDNs to mature LDNs in a dose-dependent manner[ 2 ]. In another study, we found that HCC-associated neutrophils, especially those from metastatic patients, were more sensitized to the NETosis process, which promoted the invasion of HCC cells in intro. The CCDC25 protein is a 25 kDa coiled protein present in various mammalian cells. It is a membrane-bound protein identified as a receptor of neutrophil extracellular traps (NETs) DNA. Thus, the CCDC25 protein is considered to be a precise navigator for NETs-mediated metastasis of cancers, and it forms a link between neutrophils and cancer metastasis[ 13 ]. Several studies showed that CCDC25 is expressed in cancer cells as a transmembrane protein and correlated with poor disease prognosis[ 14 – 15 ]. Eosinophilic extracellular traps activate pulmonary neuroendocrine via the CCDC25-ILK-PKCα-CRTC1 pathway, and inhibition of CCDC25 alleviates allergic inflammation[ 16 ]. However, the association between LDNs and CCDC25 during the metastasis of HCC remains largely unknown. In this study, we determined the correlation between the abundance of LDNs and tumor metastasis in HCC. We found that a subset of CD61 + LDNs can act as an important pro-metastatic factor, and the CD61 + LDNs promoted HCC metastasis by upregulating the expression of CCDC25 in tumor cells. Next, we showed that CD61 + LDNs can promote cancer metastasis via the TLR9-NF-κB-CCDC25 signaling pathway. The findings of various experiments revealed the pro-metastatic effect of the subset of CD61 + LDNs, which might act as a suitable target for the treatment of HCC in the clinical setting. Materials and methods Patients and blood Samples We collected anti-coagulant peripheral blood samples on postoperative day-1 from 199 HCC patients in our institute for conducting this study. Approximately 10 ml of blood was collected from each patient. The blood samples were gently loaded onto the double-gradient separation solution in less than 2 h. After separation, the cells from LDF and high-density fraction (HDF) were extracted for the subsequent experiments. We recruited 70 healthy participants whose basic conditions were similar to those of the participants with HCC and used their data as the control for comparison. Cell culture The HCC cell lines Huh7 and HepG2 were grown in our laboratory and stored in liquid nitrogen to ensure that the cells were passaged for less than one month before the experiments. The cell lines were authenticated by comparing them with the cell lines in the STR database. The cells were maintained in high glucose Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37°C with 5% CO 2 in pathogen-free air at the First Affiliated Hospital of USTC, University of Science and Technology of China. All experiments were performed with mycoplasma-free cells. Transwell assay For conducting the Transwell assay, approximately 2 × 10 5 species-matched CD61 + LDNs, CD61 − LDNs or HDNs were mixed in 500 µL of serum-free DMEM medium, seeded onto the bottom chamber, and supplemented with 10% fetal bovine serum (FBS). Then, 5 × 10 5 HCC cells in 200 µL of serum-free DMEM were seeded onto a polycarbonate membrane (pore size: 8 µm) with Matrigel in the upper chamber following the manufacturer’s instructions (Corning Incorporated; New York, NY, USA). For extracted DNA transwell co-culture, 1.5-2.0µg extracted DNA was mixed in 500 µL of serum-free DMEM medium, seeded onto the bottom chamber, and supplemented with 10% fetal bovine serum (FBS). Then, 4 × 10 5 HCC cells in 200 µL of serum-free DMEM were seeded onto a polycarbonate membrane (pore size: 8 µm) with Matrigel in the upper chamber following the manufacturer’s instructions (Corning Incorporated; New York, NY, USA). After incubating for 24 h, the non-invasive cells from the upper surface of the membrane were removed, and the cells on the bottom surface of the membrane were fixed with 4% paraformaldehyde (PFA) and stained with hematoxylin. The cells in 10 randomly selected views were assessed. Measurement of free DNA level We measured the level of free DNA in the cell supernatant by conducting fluorescence ELISA with some modifications. Briefly, 100 µL cell supernatant was added to each well, and 50 µL PicoGreen dsDNA Quantitation Reagent was subsequently added into each well and incubated at room temperature for 5 minutes, protected from light. For NETs marker MPO-DNA detection, anti-MPO monoclonal anti-body was coated to 96-well plates overnight at 4°C. Then, 100 µL cell supernatant was added per well and incubated at room temperature on a shaking device for 2h. After washing three times with PBS, PicoGreen dsDNA Quantitation Reagent was added following the manufacturer’s instructions. The values were then recorded using a fluorescence measuring instrument with a filter setting of 480 nm/520 nm excitation/emission and semi-quantitatively standardized to the healthy donor or control group. Immunoblot Analysis Cells were lysed using the radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime; Beijing, China). The protein concentration in the lysates was measured with a BCA Protein Assay Kit (ThermoFisher). Protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then, they were transferred onto polyvinylidene difluoride (PVDF) membranes(Millipore). The membranes were blocked with 5% non-fat milk (Bio-Rad) at room temperature for 1 h, and then, they were incubated with anti-CCDC25 (1:900, 21209-1-AP, Proteintech) antibody or anti-P-NF-κB (P65) (1:1000, 3033S, Cell signaling Technology) at 4°C overnight, followed by incubation with anti-rabbit or anti-mouse secondary antibodies conjugated to horseradish peroxidase (HRP, Jackson). The immunoreactive proteins were subsequently visualized using the enhanced chemiluminescence detection system (Millipore). Animal studies Male BALB/c nude mice (5–6 weeks old) were used for the in vivo experiments. All mice were fed in the animal center for specific pathogen-free experiments at University of Science and Technology of China. To establish spontaneous lung metastasis model, Huh7-derived conditional medium was infused into circulation blood every day at 10 days before HCC cells orthotopic implantation. Huh7 cells (1 × 10 6 ) were resuspended in 40 µL of DMEM and injected into the liver of 5–6 weeks old nude mice. Mices were sacrified by 30 days after HCC cells implantation. Metastatic burden was assessed by calculating the area percentage of lung tissue covered by the tumor. In the H&E-stained histological sections, the tumor-occupied area was quantitatively assessed by Image J software. All animal experiments were approved by the Animal Care and Use Committee of University of Science and Technology of China. Blood neutrophil separation All participants provided informed consent. 10ml anti-coagulant blood samples were collected from healthy donors or HCC patients. The samples were transported to the laboratory for analysis within 2 h after blood was collected. A double gradient was formed by layering an equal volume of Histopaque-1077 over Histopaque-1119. Next, anti-coagulated whole blood was carefully layered onto the upper Histopaque-1077 medium. During centrifugation, erythrocytes were aggregated by polysucrose and rapidly precipitated. Granulocytes were found at the lower Histopaque-1077/1119 interface, whereas lymphocytes and other mononuclear cells were found at the upper plasma/Histopaque-1077 interface. Immunohistochemistry staining Briefly, tissues were fixed with 10% neutral formalin, embedded in paraffin, and then, cut into thin sections (4 µm thick). The sections were deparaffinized, hydrated, and soaked in 3% H 2 O 2 for 1.5 h, and then, blocked with 5% BSA for 5 h at room temperature. The sections were incubated with anti-CCDC25 (1:200, 21209–1-AP, Proteintech) antibodies at 4°C overnight. The slides were incubated with biotinylated antibodies for 1 h and stained with diaminobenzidine (DAB; Zhongshangoldenbridge (ZSGB) Biotechnology, Beijing, China), followed by counterstaining with hematoxylin (Maixin Biotechnology). Transfections To generate TLR9, P65 and CCDC25 knockdown stable cell lines, a target set of shRNA sequence directed against human TLR9, P65 and CCDC25 was used. Human Lenti-shCCDC25, Lenti-shTLR9, Lenti-shP65 and Lenti-sh-control were designed and purchased from GeneChem Technologies (Shanghai, China). Transfection was performed according to the manufacturer’s protocol. Statistical analysis All statistical analyses were performed using the GraphPad Prism7 software (version 7). The data were presented as the standard error of the mean (SEM) and analyzed by unpaired t-test or variance analysis. The log-rank (Mantel-Cox) test was conducted for prognosis analysis of the follow-up data. All differences were considered to be statistically significant at P < 0.05. Results Production of LDNs in HCC To determine whether LDNs are produced during HCC, whole blood samples were collected from 199 HCC patients(Table S1 ). We used dual markers CD15 and CD66b to identify neutrophils. While purifying neutrophils on a density gradient, a significant proportion of CD15 + CD66b + cells was co-purified with the low-density mononuclear layer, besides the expected high-density granulocytic fraction. Although LDNs were rare in non-extrahepatic-metastatic HCC patients, their prevalence in the low-density fraction was significantly higher in metastatic HCC patients, especially in individuals with extra-hepatic metastasis(Figs. 1 ). Several studies have reported that LDNs are a unique neutrophil population[ 17 – 18 ], and differential expression intensity of markers, such as CD36, CD41, CD61, CD226, and Lox-1, was reported between LDNs and HDNs in lung cancer[ 19 ]. To confirm this difference between LDNs and HDNs under HCC context, we purified peripheral circulation LDNs and HDNs from 20 HCC patients, and performed fluorescence quantitative PCR assay for markers expression analysis. Our results showed that the CD61 displayed the most significant up-regulation in LDNs, compared with HDNs(Figs. 2 A). Immunofluorescence assay consistently indicated a stronger CD61 expression in LDNs, compared with HDNs(Figs. 2 B). We further performed transcriptomic analysis for CD61 − LDNs, CD61 + LDNs and HDNs purified from peripheral circulation of 38 HCC patients. Our results showed that the three neutrophil subsets had distinct gene-expression profile. Comparisons indicated various genes that were expressed differentially by CD61 + LDNs. In detail, as compared to CD61 − LDNs or HDNs, CD61 + LDNs displayed high level of genes encoding for neutrophil granule proteins(MPO, LL-37, DEFA4 and ELANE), cell cycle regulator(TOP2A), transcription factor(CEBPE) and surface marker(CEACAM8), and were characterized by low expression of inflammation response gene(TNF, IL-1β, OASL and COX2). Indeed, these analysis substantiated the proposal that CD61 + LDNs displayed a unique phenotype distinct from HDNs and CD61 − LDNs(Fig. 2 C). Morphologically, by DAPI staining, we observed the homogeneous segmented nuclear of HDNs, yet circulating LDNs subset mainly displayed the feature of banded nuclear(Figs. 2 D). Overall, our results highlight the differences between LDNs and HDNs, and a positive correlation between the abundance of CD61 + LDNs subset and tumor metastasis was determined in HCC. Functional properties of CD61 + LDNs We next perform ex vivo experiments to assess reactive oxygen species(ROS) production, neutrophil extracellular traps(NETs) formation and cytokine synthesis by LDNs subset isolated from blood of HCC patients. As compared to those of HDNs, CD61 + LDNs displayed a weaker respiratory burst upon treatment with LPS. CD61 + LDNs also showed a lower production of TNF-α and a higher production of chemokine CCL5 under treatment with cancer cell condition medium. NETosis is a neutrophil-associated function important for antibacterial and tumor metastasis. We showed that CD61 − LDNs, HDNs and CD61 + LDNs displayed a weak spontaneous NETs formation with no statistical difference. However, under the stimulation of PMA, CD61 − LDNs underwent a strongest NETs formation, while CD61 + LDNs displayed the weakest ability of forming NETs. Interestingly, as compared to those of CD61 − LDNs or HDNs, CD61 + LDNs spontaneously released a significantly higher level of free DNA molecular into extracellular space(Fig. 3 ). We next determined whether CD61 + LDNs facilitated the invasion of HCC cells in vitro. Circulating CD61 + LDNs, CD61 – LDNs, and HDNs were isolated from HCC patients. We cultured HCC cells alone and with CD61 + LDNs, CD61 – LDNs, or HDNs using the transwell system. Our results showed that CD61 + LDNs displayed significantly enhanced pro-metastatic effect, as compared to CD61 − LDNs or HDNs(Figs. 4 A). In addition, we also found that a higher circulation CD61 + LDNs abundance indicated the poorer disease prognosis in HCC patients(Figs. 4 B)(Table S2). Collectively, these results showed that CD61 + LDNs represented a unique neutrophil subtype, promoted HCC invasion, and correlated with disease prognosis. CD61 + LDNs promoted the invasion of HCC cells by upregulating CCDC25 To elucidate the mechanism by which CD61 + LDNs facilitate the metastasis of HCC, we performed RNA-seq using the Huh7 and HepG2 cells under the stimulation of CD61 + LDNs. Among 124 genes, CCDC25 displayed a marked trend of up-regulation after CD61 + LDNs stimulation(Figs. 5 A). In another study, we performed an immunohistochemistry assay and found that the abundance of circulating CD61 + LDNs was positively correlated with the expression of CCDC25 in tumors(Figs. 5 B). To determine whether CCDC25 promotes CD61 + LDNs-induced invasion of HCC cells, we first transfected Huh7 and HepG2 cell lines with short hairpin RNA (shRNA) using lentivirus vectors to silence the expression of CCDC25. Our results showed that the downregulation of CCDC25 strongly suppressed the invasion of HCC cells compared to the changes in the control conditions(Figs. 6 A). We also found that higher level of CCDC25 was expressed in cancer tissue than paracancerous region(Fig. 6 B). Then, we performed co-culture assays using the transwell system to determine whether CD61 + LDNs can affect the level of the CCDC25 protein in HCC cells. After 12 h, the CCDC25 protein level was significantly higher in the HCC cells in the presence of CD61 + LDNs compared to the CCDC25 levels in the control(Figs. 6 C). To examine whether CCDC25 was responsible for the pro-metastatic effect of CD61 + LDNs, we performed co-culture assays using the transwell system under knocking down CCDC25. The downregulation of CCDC25 inhibited CD61 + LDN-induced invasion of HCC cells(Figs. 6 D). These results indicated that CD61 + LDNs promoted the invasion of HCC cells by upregulating CCDC25 levels through indirect interactions. CD61 + LDNs upregulated the expression of CCDC25 via TLR9-NF-kB axis To further explore the mechanism by which CD61 + LDNs up-regulated CCDC25 in HCC cells, we performed the Gene Ontology Biological Process (GOBP) analysis based on RNA-seq, suggesting that the activation of TLR9 signaling in HCC cells was prominently triggered by the stimulation of CD61 + LDNs(Figs. 7 A). Western blot assays indicated an adequate TLR9 receptor expression in HCC cells(Fig. 7 B). Then, we intended to determine whether CD61 + LDNs caused the upregulation of CCDC25 via TLR9 signaling. For this, we used shRNA to silence TLR9 or P65 expression in HCC cells. Our results showed that CD61 + LDNs could not induce the phosphorylation of P65 and the expression of CCDC25 when either TLR9 or P65 was downregulated, compared to the changes in the control group(Figs. 8 A). Additionally, CD61 + LDNs lost the pro-metastatic effect on HCC cells when TLR9 or P65 was downregulated(Figs. 8 B). Furthermore, by performing transwell assay, we found a similar effect in raising CCDC25 mRNA level in HCC cells between CD61 + LDNs and specific TLR9 agonist ODN 2216, supporting the notion that CD61 + LDNs activated TLR9 pathway to up-regulate CCDC25 transcribe and led to the subsequent protein expression(Figs. 8 C). Thus, these results suggested that CD61 + LDNs upregulated the expression of CCDC25 and promoted the invasion of HCC cells in a TLR9-NF-κB signaling pathway-dependent manner. CD61 + LDN-derived free DNA upregulated the expression of CCDC25 by activating TLR9-NF-kB axis The mechanism by which CD61 + LDNs induce the upregulation of CCDC25 remains largely unknown. However, intracellular DNA is the critical ligand for the TLR9 receptor[ 22 – 23 ]. We found that CD61 + LDNs spontaneously released significantly higher level of DNA into extracellular space than CD61 − LDNs or HDNs. To assess whether CD61 + LDNs-derived DNA played a pathogenic role in the CD61 + LDNs-trggered upregulation of CCDC25, we isolated CD61 + LDNs from the peripheral blood of HCC patients and dynamically evaluated DNA level in the supernatant. The isolated CD61 + LDNs were incubated in vitro within RPMI 1640 medium for 8 hours, and DNA level was detected at timepoint of 2h, 4h, 6h and 8h. The results showed that CD61 + LDNs spontaneously released abundant DNA into the extracellular space with a persistent trend(Fig. 9 A). NETs formation is an important neutrophil function with intracellular DNA extrusion into extracellular space. A weak and spontaneous NETosis process was detected in CD61 − LDNs, CD61 + LDNs and HDNs in vitro. To remove NETs-DNA, we pre-treated the supernatant of CD61 + LDNs with solid-phase coating anti-MPO. To digest DNA, we pre-treated the supernatant of CD61 + LDNs with DNaseI. Our results indicated that the anti-MPO-treated supernatant of CD61 + LDNs upregulated the expression of CCDC25 in HCC cells, which was effectively inhibited by DNaseI digestion(Figs. 9 B). We next extracted DNA from the anti-MPO-treated supernatant of CD61 + LDNs and co-cultured them with HCC cells. We consistently found a prominent effect of the extracted DNA in upregulating the CCDC25 protein level in HCC cells(Figs. 9 C). Furthermore, we performed transwell co-culture assays to determine the pro-metastatic ability of extracted DNA. Our results showed that extracted DNA efficiently promoted HCC cell invasion, which could be abrogated by CCDC25 downregulation(Figs. 9 D). In order to explore whether the extracted DNA upregulated CCDC25 via activating TLR9-NF-κB signaling, we silenced TLR9 or P65 using shRNA in HCC cells. Consistently, we found that the extracted DNA could not induce the phosphorylation of P65 and the expression of CCDC25, and then the HCC cells invasion, when TLR9 or P65 was downregulated, compared to the changes in the control group(Figs. 9 E, F). Taken together, our results indicated that CD61 + LDN-derived DNA, but not NETs-DNA, upregulated CCDC25 to enhance HCC cells invasion potential in a TLR9-NF-κB signaling-dependent manner. CD61 + LDNs promoted the metastasis of HCC cells in vivo To elucidate the pro-metastatic effect of CD61 + LDNs in vivo, we isolated circulating CD61 + LDNs and CD61 − LDNs from patients with HCC. Then, we implanted Huh7 cells alone, with CD61 + LDNs or CD61 − LDNs into the liver of mice. In line with vitro experiments, our results showed that the combinational implantation of Huh7 cells with CD61 + LDNs exhibited the strongest pro-metastatic ability, as compared to CD61 − LDNs, although infusion of CD61 − LDNs exhibited an effective pro-metastatic efficiency(Figs. 10 A). The infusion of CD61 + LDNs led to an increase in the expression of CCDC25 in mouse liver tumors(Figs. 10 B). Overall, these results indicated that CD61 + LDNs subset promoted HCC lung metastasis in vivo. Thus, CD61 + LDNs might be a novel and practical target for preventing HCC metastasis. Discussion Metastasis is a complex multistep cascade which is related to the biological features of tumor cells and non-malignant tumor stroma[ 24 – 26 ]. Several studies have suggested that the subset of LDNs might act as a link between the host and cancer cells and affect cancer metastasis[ 27 ]. However, the specific functions of LDNs in the metastasis of HCC need to be illustrated. In this study, we elucidated the mechanism underlying the link between LDNs and the progression of HCC. Initially, we evaluated the abundance of LDNs in patients and found that the abundance of circulating LDNs was positively related to the metastasis of HCC. Then, we found a close association between LDNs and the level of CCDC25 within the tumor. The LDNs promoted metastasis through the DNA-TLR9-CCDC25 axis. The subset of LDNs acquired heterogeneous characteristics[ 28 ]. Hence, we assessed the phenotype of LDNs that contributed the most to cancer metastasis. We found that the marker CD61 was prominently expressed on LDNs. CD61 + LDNs displayed different phenotype of secreting cytokine and forming NETs, as compared to CD61 − LDNs and HDNs. Next, we compared the pro-metastatic ability of CD61 + LDNs with the pro-metastatic ability of CD61 – LDNs and HDNs. Although both CD61 – LDNs and HDNs strongly promoted the invasion of HCC cells, CD61 + LDNs exhibited the strongest ability to promote the invasion of HCC cells. A previous study have shown that neutrophils can facilitate cancer metastasis via the NETs-CCDC25 pathway, and CCDC25 can strongly promote cancer metastasis[ 13 ]. Several studies have also shown that serum CCDC25 might be a potential diagnostic biomarker[ 14 ]. Hence, we investigated whether CD61 + LDNs can facilitate cancer metastasis via the action of CCDC25. Our results indicated that the pro-invasion effect of CD61 + LDNs was effectively reversed by knocking down CCDC25. The CCDC25 protein is membrane-bound, and several studies have shown that this protein is correlated with the infiltration of immune cells, cancer prognosis, and ferroptosis in the microenvironment. However, studies on the interaction between immune cells and CCDC25 are lacking. In this study, we used siRNA to downregulate CCDC25 in HCC cells and found that the downregulation of CCDC25 reduced the invasion of HCC cells. We also found that CD61 + LDNs increased the expression of CCDC25, and the downregulation of CCDC25 strongly inhibited the invasion of HCC cells triggered by CD61 + LDNs. We showed that the subset of CD61 + LDNs promoted the metastasis of HCC through the upregulation of CCDC25. Song et al. found that CCDC25 can act as a NETs-DNA receptor on cancer cells that recognize extracellular DNA, and then, they can activate the ILK-β-parvin pathway to enhance cell motility[ 13 ]. To determine whether free DNA promotes the upregulation of CCDC25 in HCC cells after the stimulation of CD61 + LDNs, we first evaluated the level of free DNA in the supernatant of CD61 + LDNs. Then, we performed RNA sequencing and GOBP analysis to determine whether CD61 + LDNs can trigger the activation of TLR9 signaling in HCC cells. The CD61 + LDNs spontaneously and persistently released DNA into the extracellular space. Upon further examination, we found that the CD61 + LDN-derived DNA led to the upregulation of CCDC25. However, whether the TLR9 signaling pathway is essential in the CD61 + LDNs-triggered upregulation of CCDC25 needs to be elucidated. We used Si-RNA to silence TLR9 and P65, and found that the CD61 + LDN-triggered upregulation of CCDC25 was strongly inhibited by silencing either TLR9 or P65, which indicated that CD61 + LDN-induced upregulation of CCDC25 depended on the initiation of TLR9-NF-κB signaling. The results of the GOBP analysis showed that other signaling pathways besides the TLR9 pathway were simultaneously initiated under the stimulation of CD61 + LDNs. Further studies might provide new insights into the specific effects of those signaling pathways under this condition. The CD61 protein participates in reprogramming tumor metabolism, shaping the stromal and immune microenvironment, facilitating epithelial to mesenchymal transition (EMT) and endothelial to mesenchymal transition (End-MT)[ 29 – 30 ], and it is highly expressed by tumor-associated LDNs. In this study, we performed ex vitro experiments to show that CD61 + LDNs could release abundant DNA into extracellular space. However, the information on the effect of the CD61 receptor during spontaneous DNA release and the pro-metastatic effect of CD61 + LDNs are limited. Several studies suggest that pulmonary metastasis was increased when HCC cells were co-injected with tumor-associated neutrophils (TANs), which provided direct evidence that TANs have a pro-metastatic effect[ 31 – 33 ]. In this study, to determine whether CD61 + LDNs promoted the metastasis of HCC in vivo, we transferred Huh7 cells with or without CD61 + LDNs into the liver of nude mice. Obviously, the infusion of CD61 + LDNs greatly increased pulmonary metastasis, as compared to CD61 − LDNs infusion or Huh7 alone. To summarize, in this study, we determined a cellular, molecular, and clinical network associated with LDNs, involving CCDC25, free DNA, TLR9 signaling, and CD61. CD61 + LDNs were identified as a unique neutrophil subtype under HCC context. This network showed a bridge between LDNs subset and tumor progression. Our findings suggested that this network might be a promising target for treating HCC. Declarations Ethics Approval and consent to participate Samples were collected after approval by the medical ethics committee of The First Affiliated Hospital of University of Science and Technology of China and written informed consents were obtained from each patient. All animal experiments were approved by the animal Ethics committee of University of Science and Technology of China, and all institutional and national guidelines for the care and use of laboratory animals were followed. All authors consent for publication. Availability of data and materials All data generated or analyzed during this study are included in the this published article. Conflict of Interest All authors declare that they have no competing interest on this manuscript. Funding None Author contributions X. Q. Guan performed the study, acquisition and analysis of data, and drafted the manuscript. Y. Y. Lu and C. Z. Wang offered assistance with flow cytometry analysis and participated in animal studies. Z. G. Chen and P. Zhan provided assistance for flow cytometry analysis and cellular studies. X. Q. Guan started the study and particaped in experimental design. X. Q. Guan approved for the final version and submission. 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Uhel F, Azzaoui I, Grégoire M, Pangault C, Dulong J, Tadié JM, Gacouin A, Camus C, Cynober L, Fest T, Le Tulzo Y, Roussel M, Tarte K. Early Expansion of Circulating Granulocytic Myeloid-derived Suppressor Cells Predicts Development of Nosocomial Infections in Patients with Sepsis. Am J Respir Crit Care Med. 2017 Aug 1;196(3):315-327. Lood C, Blanco LP, Purmalek MM, Carmona-Rivera C, De Ravin SS, Smith CK, Malech HL, Ledbetter JA, Elkon KB, Kaplan MJ. Neutrophil extracellular traps enriched in oxidized mitochondrial DNA are interferogenic and contribute to lupus-like disease. Nat Med. 2016 Feb;22(2):146-53. Cornish AL, Campbell IK, McKenzie BS, Chatfield S, Wicks IP. G-CSF and GM-CSF as therapeutic targets in rheumatoid arthritis. Nat Rev Rheumatol. 2009 Oct;5(10):554-9. Yang L, Liu Q, Zhang X, Liu X, Zhou B, Chen J, Huang D, Li J, Li H, Chen F, Liu J, Xing Y, Chen X, Su S, Song E. DNA of neutrophil extracellular traps promotes cancer metastasis via CCDC25. Nature. 2020 Jul;583(7814):133-138. Deng H, Zhang J, Zheng Y, Li J, Xiao Q, Wei F, Han W, Xu X, Zhang Y. CCDC25 may be a potential diagnostic and prognostic marker of hepatocellular carcinoma: Results from microarray analysis. Front Surg. 2022 Sep 23;9:878648. Proungvitaya S, Klinthong W, Proungvitaya T, Limpaiboon T, Jearanaikoon P, Roytrakul S, Wongkham C, Nimboriboonporn A, Wongkham S. High expression of CCDC25 in cholangiocarcinoma tissue samples. Oncol Lett. 2017 Aug;14(2):2566-2572. Lu Y, Huang Y, Li J, Huang J, Zhang L, Feng J, Li J, Xia Q, Zhao Q, Huang L, Jiang S, Su S. Eosinophil extracellular traps drive asthma progression through neuro-immune signals. Nat Cell Biol. 2021 Oct;23(10):1060-1072. Blanco-Camarillo C, Alemán OR, Rosales C. Low-Density Neutrophils in Healthy Individuals Display a Mature Primed Phenotype. Front Immunol. 2021 Jul 2;12:672520. Cho Y, Bukong TN, Tornai D, Babuta M, Vlachos IS, Kanata E, Catalano D, Szabo G. Neutrophil extracellular traps contribute to liver damage and increase defective low-density neutrophils in alcohol-associated hepatitis. J Hepatol. 2023 Jan;78(1):28-44. Valadez-Cosmes P, Maitz K, Kindler O, Raftopoulou S, Kienzl M, Santiso A, Mihalic ZN, Brcic L, Lindenmann J, Fediuk M, Pichler M, Schicho R, Houghton AM, Heinemann A, Kargl J. Identification of Novel Low-Density Neutrophil Markers Through Unbiased High-Dimensional Flow Cytometry Screening in Non-Small Cell Lung Cancer Patients. Front Immunol. 2021 Aug 13;12:703846. Wigerblad G, Kaplan MJ. Neutrophil extracellular traps in systemic autoimmune and autoinflammatory diseases. Nat Rev Immunol. 2023 May;23(5):274-288. Mousset A, Lecorgne E, Bourget I, Lopez P, Jenovai K, Cherfils-Vicini J, Dominici C, Rios G, Girard-Riboulleau C, Liu B, Spector DL, Ehmsen S, Renault S, Hego C, Mechta-Grigoriou F, Bidard FC, Terp MG, Egeblad M, Gaggioli C, Albrengues J. Neutrophil extracellular traps formed during chemotherapy confer treatment resistance via TGF-β activation. Cancer Cell. 2023 Apr 10;41(4):757-775.e10. Zhang SW, Wu CR, Liao H. DNA sensing in the pathological process of ischemic stroke. Eur J Neurosci. 2023 Apr;57(8):1432-1441. Albuquerque-Souza E, Crump KE, Rattanaprukskul K, Li Y, Shelling B, Xia-Juan X, Jiang M, Sahingur SE. TLR9 Mediates Periodontal Aging by Fostering Senescence and Inflammaging. J Dent Res. 2022 Dec;101(13):1628-1636. Gerstberger S, Jiang Q, Ganesh K. Metastasis. Cell. 2023 Apr 13;186(8):1564-1579. Fares J, Fares MY, Khachfe HH, Salhab HA, Fares Y. Molecular principles of metastasis: a hallmark of cancer revisited. Signal Transduct Target Ther. 2020 Mar 12;5(1):28. Wu M, Ma M, Tan Z, Zheng H, Liu X. Neutrophil: A New Player in Metastatic Cancers. Front Immunol. 2020 Sep 24;11:565165. Hassani M, Hellebrekers P, Chen N, van Aalst C, Bongers S, Hietbrink F, Koenderman L, Vrisekoop N. On the origin of low-density neutrophils. J Leukoc Biol. 2020 May;107(5):809-818. Zhu C, Kong Z, Wang B, Cheng W, Wu A, Meng X. ITGB3/CD61: a hub modulator and target in the tumor microenvironment. Am J Transl Res. 2019 Dec 15;11(12):7195-7208. Zarbock A, Polanowska-Grabowska RK, Ley K. Platelet-neutrophil-interactions: linking hemostasis and inflammation. Blood Rev. 2007 Mar;21(2):99-111. Arvanitakis K, Mitroulis I, Germanidis G. Tumor-Associated Neutrophils in Hepatocellular Carcinoma Pathogenesis, Prognosis, and Therapy. Cancers (Basel). 2021 Jun 10;13(12):2899. Guan X, Lu Y, Zhu H, Yu S, Zhao W, Chi X, Xie C, Yin Z. The Crosstalk Between Cancer Cells and Neutrophils Enhances Hepatocellular Carcinoma Metastasis via Neutrophil Extracellular Traps-Associated Cathepsin G Component: A Potential Therapeutic Target. J Hepatocell Carcinoma. 2021 May 20;8:451-465. Zhou SL, Zhou ZJ, Hu ZQ, Huang XW, Wang Z, Chen EB, Fan J, Cao Y, Dai Z, Zhou J. Tumor-Associated Neutrophils Recruit Macrophages and T-Regulatory Cells to Promote Progression of Hepatocellular Carcinoma and Resistance to Sorafenib. Gastroenterology. 2016 Jun;150(7):1646-1658.e17. Zhou SL, Yin D, Hu ZQ, Luo CB, Zhou ZJ, Xin HY, Yang XR, Shi YH, Wang Z, Huang XW, Cao Y, Fan J, Zhou J. A Positive Feedback Loop Between Cancer Stem-Like Cells and Tumor-Associated Neutrophils Controls Hepatocellular Carcinoma Progression. Hepatology. 2019 Oct;70(4):1214-1230. Additional Declarations No competing interests reported. Supplementary Files Supplementalmaterials.docx Table S1. Clinicopathological characteristicsof HCC patients for blood LDNs abundance detection. The Clinicopathological characteristics, such as extra-hepatic metastasis, sex, age, HBV-DNA, Liver cirrhosis, Tumor size and portal vein tumor thrombosis, were presented. Table S2. Clinicopathological characteristics of HCC patients for prognosis assess. The Clinicopathological characteristics, such as extra-hepatic metastasis, sex, age, serum AFP level, HBV-DNA, Tumor size and portal vein tumor thrombosis, were presented. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3868978","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":267773025,"identity":"1293a75e-425c-4aa5-8b2f-f5d86a7f27e8","order_by":0,"name":"Xiangqian Guan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA10lEQVRIiWNgGAWjYBACeWaGxAcfKiTk+PmbDxCnxbCd4bHhjDM2xpIzjiUQac15xmfSnG1piRsacgyI08HYzJxszMB2mHEDw5mPN94w2MnpNhDQws7Mlvi4gOcwszlz72bLOQzJxmYHCNrCk2w8Q+Iwm2XD2W3SPAwHErcR0sJwmP+bNI/BYR6DAznPiNXCkCbNk5AmAdTCRpwWw2aGZMMZB2wMgIFsbDnHgAi/yPMfSHzw8Z9EfT9/88Mbbyrs5AhqQQESPERGDbIWUnWMglEwCkbBiAAAkohEN1RR3b0AAAAASUVORK5CYII=","orcid":"","institution":"University of Science and Technology of China","correspondingAuthor":true,"prefix":"","firstName":"Xiangqian","middleName":"","lastName":"Guan","suffix":""},{"id":267773026,"identity":"8b1c1706-d0c8-459c-997c-a52a87940389","order_by":1,"name":"Yuyan Lu","email":"","orcid":"","institution":"Fujian Medical university","correspondingAuthor":false,"prefix":"","firstName":"Yuyan","middleName":"","lastName":"Lu","suffix":""},{"id":267773027,"identity":"7a7f190c-e337-4518-b752-39e1290f307e","order_by":2,"name":"Chuanzheng wang","email":"","orcid":"","institution":"Fujian Medical university","correspondingAuthor":false,"prefix":"","firstName":"Chuanzheng","middleName":"","lastName":"wang","suffix":""},{"id":267773028,"identity":"a5079ac1-46f9-44e3-b268-738f33725a97","order_by":3,"name":"Ping Zhan","email":"","orcid":"","institution":"Fujian Medical university","correspondingAuthor":false,"prefix":"","firstName":"Ping","middleName":"","lastName":"Zhan","suffix":""},{"id":267773029,"identity":"09946fa8-469f-49a5-aa6d-dd51abd088e2","order_by":4,"name":"Zhigao Chen","email":"","orcid":"","institution":"Fujian Medical university","correspondingAuthor":false,"prefix":"","firstName":"Zhigao","middleName":"","lastName":"Chen","suffix":""}],"badges":[],"createdAt":"2024-01-16 06:29:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3868978/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3868978/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":49896167,"identity":"be588d0b-ea5e-42e2-bb78-acc1a71ed444","added_by":"auto","created_at":"2024-01-19 21:54:03","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":13743950,"visible":true,"origin":"","legend":"\u003cp\u003eThe abundance of LDNs increased in metastatic HCC. Flow-cytometric analysis for CD15\u003csup\u003e+\u003c/sup\u003eCD66b\u003csup\u003e+\u003c/sup\u003e cells in low density layer isolated from peripheral blood of HCC patients(n=199) or healthy donor(n=70). ***, P\u0026lt;0.001; *, P\u0026lt;0.05; ns, P\u0026gt;0.05.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-3868978/v1/c383226d8b008830285d9791.png"},{"id":49896044,"identity":"b97f1d34-05c8-4cad-83d2-5b8f42fed0dd","added_by":"auto","created_at":"2024-01-19 21:46:04","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":4173602,"visible":true,"origin":"","legend":"\u003cp\u003eCD61\u003csup\u003e+\u003c/sup\u003eLDNs displayed a unique phenotype. A, RNA detection for markers in LDNs and HDNs. B, Immunofluorescence assay for CD61 expression in LDNs and HDNs. C, RNA-seq analysis for the differential gene expression between CD61\u003csup\u003e+\u003c/sup\u003eLDNs, CD61\u003csup\u003e-\u003c/sup\u003eLDNs and HDNs. D, DAPI staining for the nuclear morphology between LDNs and HDNs.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-3868978/v1/6c3ff5f0b7ddc95958b01d8a.png"},{"id":49896165,"identity":"46b9e618-8b7e-498e-b5a9-fcb387b479d3","added_by":"auto","created_at":"2024-01-19 21:54:03","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1365709,"visible":true,"origin":"","legend":"\u003cp\u003eA different ability of CD61\u003csup\u003e+\u003c/sup\u003eLDNs in NETs formation, ROS production and cytokine synthesis. Fluorescence ELISA analysis for the level of NETs marker MPO-DNA and free DNA in supernatant. ELISA assay for cytokine TNF-α and CCL5 level in supernatant. Fluorescence probe reactive oxygen species assay for ROS production. ***, P\u0026lt;0.001; **，P\u0026lt;0.01; *, P\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-3868978/v1/65878d5077dd72a41d55a142.png"},{"id":49896036,"identity":"58570d18-db3b-4470-b173-96fc9255768a","added_by":"auto","created_at":"2024-01-19 21:46:03","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1602668,"visible":true,"origin":"","legend":"\u003cp\u003eCD61\u003csup\u003e+\u003c/sup\u003eLDNs promote HCC cells invasion in vitro. A, Transwell analysis for the effects of the three neutrophil subsets on HCC cells invasion. B, Follow-up data of HCC patients with different CD61\u003csup\u003e+\u003c/sup\u003eLDNs abundance for survival ratio analyses(n=40). ***, P\u0026lt;0.001; *, P\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-3868978/v1/d88a17d18eae83c8c3df8ed4.png"},{"id":49896042,"identity":"e2cece53-0bd9-48d5-8dc5-847c2dc7cf07","added_by":"auto","created_at":"2024-01-19 21:46:03","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2029386,"visible":true,"origin":"","legend":"\u003cp\u003eCD61\u003csup\u003e+\u003c/sup\u003eLDNs trigger CCDC25 up-regulation on HCC cells. A, RNA-seq analysis for transcriptomic alteration in Huh7 and HepG2 cells under CD61\u003csup\u003e+\u003c/sup\u003eLDNs stimulation. B, Pearson correlation analysis of CD61\u003csup\u003e+\u003c/sup\u003eLDNs abundance and intratumor CCDC25 expression in HCC patients(n=20).\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-3868978/v1/455d505b4aac6ca2b75ce5a1.png"},{"id":49896040,"identity":"a6bb1446-ee1f-4633-9ee6-013480b63042","added_by":"auto","created_at":"2024-01-19 21:46:03","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":5831792,"visible":true,"origin":"","legend":"\u003cp\u003eCD61\u003csup\u003e+\u003c/sup\u003eLDNs promote HCC cells invasion by upregulating CCDC25. A, Western blot and Q-PCR assay for the CCDC25 knockdown, and control HCC cells. Transwell assay for the invasion ability of HCC cells under CCDC25 knockdown. The cells were counted in five randomly selected single vision fields. B, CCDC25 protein in cancer tissues and paracancerous region was detected by western blot assay, respectively. C, Co-culture assays for CD61\u003csup\u003e+\u003c/sup\u003eLDNs and HCC cells, and CCDC25 in HCC cells was detected by western blot assay. D, Transwell assays for the effects of CD61\u003csup\u003e+\u003c/sup\u003eLDNs on HCC cells invasion under CCDC25 knockdown. Representative images were shown. **，P\u0026lt;0.01; *, P\u0026lt;0.05; ns, P\u0026gt;0.05.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-3868978/v1/2cfc72d542d5978ce7c58c88.png"},{"id":49896038,"identity":"8383ac8e-03af-445e-b72d-55e3d757bd5c","added_by":"auto","created_at":"2024-01-19 21:46:03","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2683398,"visible":true,"origin":"","legend":"\u003cp\u003eTLR9 signaling was initiated in response to CD61\u003csup\u003e+\u003c/sup\u003eLDNs. A, RNA-sequence transcriptome and GO biological process analysis for HCC cells under CD61\u003csup\u003e+\u003c/sup\u003eLDNs stimulation. B, Western blot assay for TLR9 expression in Huh7 and HepG2 cells.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-3868978/v1/348dd8c27b7d2f5d97b8bf46.png"},{"id":49896045,"identity":"ee8823f6-ee00-4b07-b7c8-8df11272c1ed","added_by":"auto","created_at":"2024-01-19 21:46:04","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":3410241,"visible":true,"origin":"","legend":"\u003cp\u003eCD61\u003csup\u003e+\u003c/sup\u003eLDNs regulate CCDC25 expression via TLR9-NF-κB axis. A, Western blot assay for the effect of CD61\u003csup\u003e+\u003c/sup\u003eLDNs on CCDC25 expression and phosphorylation of P65 in HCC cells under TLR9 or P65 silence. B, Transwell assay for the effects of CD61\u003csup\u003e+\u003c/sup\u003eLDNs on HCC cells invasion under TLR9 or P65 silence. C, Transwell assay for the co-culture of HCC cells with CD61\u003csup\u003e+\u003c/sup\u003eLDNs or TLR9 agonist ODN 2216, and CCDC25 mRNA was detected using qPCR. **，P\u0026lt;0.01; ***, P\u0026lt;0.001; ns, P\u0026gt;0.05.\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-3868978/v1/0d716cd3028832d6326676d2.png"},{"id":49896166,"identity":"17c5702d-207a-4e94-a9e6-669148273790","added_by":"auto","created_at":"2024-01-19 21:54:03","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":1742786,"visible":true,"origin":"","legend":"\u003cp\u003eCD61\u003csup\u003e+\u003c/sup\u003eLDN-derived DNA upregulated the expression of CCDC25 by activating TLR9-NF-κB axis. A, Fluorescence ELISA assay for supernatant free DNA level of neutrophil subsets(CD61\u003csup\u003e+\u003c/sup\u003eLDNs, CD61\u003csup\u003e-\u003c/sup\u003eLDNs and HDNs). B, Western blot assay for the effect of anti-MPO-treated supernatant on CCDC25 expression in HCC cells under DNaseI digestion. C, DNA was extracted from anti-MPO-treated CD61\u003csup\u003e+\u003c/sup\u003eLDNs supernatant, and the extracted DNA was co-cultured with HCC cells. CCDC25 in HCC cells was detected by western blot. HCC cells were co-cultured with extracted DNA for 6 h. D, Transwell assay for the effects of extracted DNA on HCC cells invasion under CCDC25 silence. E, Western blot assay for the effects of extracted DNA on CCDC25 expression and P65 phosphorylation in HCC cells under TLR9 or P65 silence. F, Transwell assay for the effects of extracted DNA on HCC cells invasion under TLR9 or P65 silence.**，P\u0026lt;0.01; *, P\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"Figure9.png","url":"https://assets-eu.researchsquare.com/files/rs-3868978/v1/06ceb0aeb3a5ac0178427516.png"},{"id":49896037,"identity":"c2ef7ce2-c116-470f-9658-82bc3b1d30d2","added_by":"auto","created_at":"2024-01-19 21:46:03","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":4129255,"visible":true,"origin":"","legend":"\u003cp\u003eCD61\u003csup\u003e+\u003c/sup\u003eLDNs promoted the metastasis of HCC cells in vivo. A, HCC cells were co-injected into mouse livers alone, with CD61\u003csup\u003e+\u003c/sup\u003eLDNs or CD61\u003csup\u003e-\u003c/sup\u003eLDNs. Lung metastasis burden was assessed by H\u0026amp;E staining. B, Western blot assay for CCDC25 expression in mouse liver tumors under the infusion of CD61\u003csup\u003e+\u003c/sup\u003eLDNs and CD61\u003csup\u003e-\u003c/sup\u003eLDNs(n=6 each group). ***, P\u0026lt;0.001; **，P\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"Figure10.png","url":"https://assets-eu.researchsquare.com/files/rs-3868978/v1/856c0f8ce3b0e478905e1868.png"},{"id":49971170,"identity":"e74ee875-54cb-4ba7-bff3-9c9fb55a97c6","added_by":"auto","created_at":"2024-01-22 13:31:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3180356,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3868978/v1/5cd1f5c0-fe82-4833-8a0a-87a0ee32e354.pdf"},{"id":49896034,"identity":"4fc9136c-a2c5-43d7-a587-e8665b22b4b9","added_by":"auto","created_at":"2024-01-19 21:46:03","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":21608,"visible":true,"origin":"","legend":"\u003cp\u003eTable S1. Clinicopathological characteristicsof HCC patients for blood LDNs abundance detection. The Clinicopathological characteristics, such as extra-hepatic metastasis, sex, age, HBV-DNA, Liver cirrhosis, Tumor size and portal vein tumor thrombosis, were presented.\u003c/p\u003e\n\u003cp\u003eTable S2. Clinicopathological characteristics of HCC patients for prognosis assess. The Clinicopathological characteristics, such as extra-hepatic metastasis, sex, age, serum AFP level, HBV-DNA, Tumor size and portal vein tumor thrombosis, were presented.\u003c/p\u003e","description":"","filename":"Supplementalmaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-3868978/v1/215473f2844b6bd6dedb19d4.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"CD61 + LDNs drive cancer metastasis by upregulating CCDC25 expression via DNA-TLR9-NF-kB axis in HCC","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCancer-associated neutrophils retain functional plasticity and can undergo alternative activation when exposed to different cues found in the tumor microenvironment (TME)[\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Several studies have reported the presence of at least two distinct neutrophil phenotypes in the disease setting, including LDNs and HDNs[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eLDNs are different from HDNs, and they have a density below 1.077 g/mL and precipitate in the PBMC layer after density gradient centrifugation of whole blood[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Morphologically, LDNs have a ring-shaped nucleus, whereas HDNs have a segmented nucleus[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. LDNs consist of granulocyte myeloid-derived suppressor cells (G-MDSCs) and mature large neutrophils[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The phenotype of LDNs may vary depending on the context of the disease[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In sepsis, LDNs proliferate and exhibit characteristics of G-MDSCs[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The LDNs in systemic lupus erythematosus (SLE) trigger rapid cell death via the immune complex, and this cell death results in the extracellular release of nuclear DNA and oxidized mitochondrial DNA[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. In patients with rheumatoid arthritis (RA), LDNs occur as an immature subset with prolonged survival but low response to cytokine stimulation[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn cancer patients, several differences occur between these two neutrophil subsets, such as the ability to inhibit the activation of CD8\u003csup\u003e+\u003c/sup\u003e T cells[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e][\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. In the breast cancer metastasis model, immature LDNs, but not mature LDNs, were found to accumulate and acquire a higher proportion in metastatic sites and peripheral blood at a later stage of liver metastasis[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Additionally, the transition of HDNs to LDNs was found to occur spontaneously in several tumor models[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e][\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Tumor growth factor-β (TGF-β) might act as a major regulator in inducing this transition from HDNs to mature LDNs in a dose-dependent manner[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. In another study, we found that HCC-associated neutrophils, especially those from metastatic patients, were more sensitized to the NETosis process, which promoted the invasion of HCC cells in intro.\u003c/p\u003e \u003cp\u003eThe CCDC25 protein is a 25 kDa coiled protein present in various mammalian cells. It is a membrane-bound protein identified as a receptor of neutrophil extracellular traps (NETs) DNA. Thus, the CCDC25 protein is considered to be a precise navigator for NETs-mediated metastasis of cancers, and it forms a link between neutrophils and cancer metastasis[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Several studies showed that CCDC25 is expressed in cancer cells as a transmembrane protein and correlated with poor disease prognosis[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Eosinophilic extracellular traps activate pulmonary neuroendocrine via the CCDC25-ILK-PKCα-CRTC1 pathway, and inhibition of CCDC25 alleviates allergic inflammation[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. However, the association between LDNs and CCDC25 during the metastasis of HCC remains largely unknown.\u003c/p\u003e \u003cp\u003eIn this study, we determined the correlation between the abundance of LDNs and tumor metastasis in HCC. We found that a subset of CD61\u003csup\u003e+\u003c/sup\u003eLDNs can act as an important pro-metastatic factor, and the CD61\u003csup\u003e+\u003c/sup\u003eLDNs promoted HCC metastasis by upregulating the expression of CCDC25 in tumor cells. Next, we showed that CD61\u003csup\u003e+\u003c/sup\u003eLDNs can promote cancer metastasis via the TLR9-NF-κB-CCDC25 signaling pathway. The findings of various experiments revealed the pro-metastatic effect of the subset of CD61\u003csup\u003e+\u003c/sup\u003eLDNs, which might act as a suitable target for the treatment of HCC in the clinical setting.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePatients and blood Samples\u003c/h2\u003e \u003cp\u003eWe collected anti-coagulant peripheral blood samples on postoperative day-1 from 199 HCC patients in our institute for conducting this study. Approximately 10 ml of blood was collected from each patient. The blood samples were gently loaded onto the double-gradient separation solution in less than 2 h. After separation, the cells from LDF and high-density fraction (HDF) were extracted for the subsequent experiments. We recruited 70 healthy participants whose basic conditions were similar to those of the participants with HCC and used their data as the control for comparison.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eCell culture\u003c/h2\u003e \u003cp\u003eThe HCC cell lines Huh7 and HepG2 were grown in our laboratory and stored in liquid nitrogen to ensure that the cells were passaged for less than one month before the experiments. The cell lines were authenticated by comparing them with the cell lines in the STR database. The cells were maintained in high glucose Dulbecco\u0026rsquo;s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e in pathogen-free air at the First Affiliated Hospital of USTC, University of Science and Technology of China. All experiments were performed with mycoplasma-free cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eTranswell assay\u003c/h2\u003e \u003cp\u003eFor conducting the Transwell assay, approximately 2 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e species-matched CD61\u003csup\u003e+\u003c/sup\u003eLDNs, CD61\u003csup\u003e\u0026minus;\u003c/sup\u003eLDNs or HDNs were mixed in 500 \u0026micro;L of serum-free DMEM medium, seeded onto the bottom chamber, and supplemented with 10% fetal bovine serum (FBS). Then, 5 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e HCC cells in 200 \u0026micro;L of serum-free DMEM were seeded onto a polycarbonate membrane (pore size: 8 \u0026micro;m) with Matrigel in the upper chamber following the manufacturer\u0026rsquo;s instructions (Corning Incorporated; New York, NY, USA).\u003c/p\u003e \u003cp\u003eFor extracted DNA transwell co-culture, 1.5-2.0\u0026micro;g extracted DNA was mixed in 500 \u0026micro;L of serum-free DMEM medium, seeded onto the bottom chamber, and supplemented with 10% fetal bovine serum (FBS). Then, 4 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e HCC cells in 200 \u0026micro;L of serum-free DMEM were seeded onto a polycarbonate membrane (pore size: 8 \u0026micro;m) with Matrigel in the upper chamber following the manufacturer\u0026rsquo;s instructions (Corning Incorporated; New York, NY, USA).\u003c/p\u003e \u003cp\u003eAfter incubating for 24 h, the non-invasive cells from the upper surface of the membrane were removed, and the cells on the bottom surface of the membrane were fixed with 4% paraformaldehyde (PFA) and stained with hematoxylin. The cells in 10 randomly selected views were assessed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eMeasurement of free DNA level\u003c/h2\u003e \u003cp\u003eWe measured the level of free DNA in the cell supernatant by conducting fluorescence ELISA with some modifications. Briefly, 100 \u0026micro;L cell supernatant was added to each well, and 50 \u0026micro;L PicoGreen dsDNA Quantitation Reagent was subsequently added into each well and incubated at room temperature for 5 minutes, protected from light. For NETs marker MPO-DNA detection, anti-MPO monoclonal anti-body was coated to 96-well plates overnight at 4\u0026deg;C. Then, 100 \u0026micro;L cell supernatant was added per well and incubated at room temperature on a shaking device for 2h. After washing three times with PBS, PicoGreen dsDNA Quantitation Reagent was added following the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003cp\u003eThe values were then recorded using a fluorescence measuring instrument with a filter setting of 480 nm/520 nm excitation/emission and semi-quantitatively standardized to the healthy donor or control group.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eImmunoblot Analysis\u003c/h2\u003e \u003cp\u003eCells were lysed using the radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime; Beijing, China). The protein concentration in the lysates was measured with a BCA Protein Assay Kit (ThermoFisher). Protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then, they were transferred onto polyvinylidene difluoride (PVDF) membranes(Millipore). The membranes were blocked with 5% non-fat milk (Bio-Rad) at room temperature for 1 h, and then, they were incubated with anti-CCDC25 (1:900, 21209-1-AP, Proteintech) antibody or anti-P-NF-κB (P65) (1:1000, 3033S, Cell signaling Technology) at 4\u0026deg;C overnight, followed by incubation with anti-rabbit or anti-mouse secondary antibodies conjugated to horseradish peroxidase (HRP, Jackson). The immunoreactive proteins were subsequently visualized using the enhanced chemiluminescence detection system (Millipore).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eAnimal studies\u003c/h2\u003e \u003cp\u003eMale BALB/c nude mice (5\u0026ndash;6 weeks old) were used for the in vivo experiments. All mice were fed in the animal center for specific pathogen-free experiments at University of Science and Technology of China. To establish spontaneous lung metastasis model, Huh7-derived conditional medium was infused into circulation blood every day at 10 days before HCC cells orthotopic implantation. Huh7 cells (1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e) were resuspended in 40 \u0026micro;L of DMEM and injected into the liver of 5\u0026ndash;6 weeks old nude mice. Mices were sacrified by 30 days after HCC cells implantation. Metastatic burden was assessed by calculating the area percentage of lung tissue covered by the tumor. In the H\u0026amp;E-stained histological sections, the tumor-occupied area was quantitatively assessed by Image J software. All animal experiments were approved by the Animal Care and Use Committee of University of Science and Technology of China.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eBlood neutrophil separation\u003c/h2\u003e \u003cp\u003e All participants provided informed consent. 10ml anti-coagulant blood samples were collected from healthy donors or HCC patients. The samples were transported to the laboratory for analysis within 2 h after blood was collected. A double gradient was formed by layering an equal volume of Histopaque-1077 over Histopaque-1119. Next, anti-coagulated whole blood was carefully layered onto the upper Histopaque-1077 medium. During centrifugation, erythrocytes were aggregated by polysucrose and rapidly precipitated. Granulocytes were found at the lower Histopaque-1077/1119 interface, whereas lymphocytes and other mononuclear cells were found at the upper plasma/Histopaque-1077 interface.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry staining\u003c/h2\u003e \u003cp\u003eBriefly, tissues were fixed with 10% neutral formalin, embedded in paraffin, and then, cut into thin sections (4 \u0026micro;m thick). The sections were deparaffinized, hydrated, and soaked in 3% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 1.5 h, and then, blocked with 5% BSA for 5 h at room temperature. The sections were incubated with anti-CCDC25 (1:200, 21209\u0026ndash;1-AP, Proteintech) antibodies at 4\u0026deg;C overnight. The slides were incubated with biotinylated antibodies for 1 h and stained with diaminobenzidine (DAB; Zhongshangoldenbridge (ZSGB) Biotechnology, Beijing, China), followed by counterstaining with hematoxylin (Maixin Biotechnology).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eTransfections\u003c/h2\u003e \u003cp\u003eTo generate TLR9, P65 and CCDC25 knockdown stable cell lines, a target set of shRNA sequence directed against human TLR9, P65 and CCDC25 was used. Human Lenti-shCCDC25, Lenti-shTLR9, Lenti-shP65 and Lenti-sh-control were designed and purchased from GeneChem Technologies (Shanghai, China). Transfection was performed according to the manufacturer\u0026rsquo;s protocol.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll statistical analyses were performed using the GraphPad Prism7 software (version 7). The data were presented as the standard error of the mean (SEM) and analyzed by unpaired t-test or variance analysis. The log-rank (Mantel-Cox) test was conducted for prognosis analysis of the follow-up data. All differences were considered to be statistically significant at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eProduction of LDNs in HCC\u003c/h2\u003e \u003cp\u003eTo determine whether LDNs are produced during HCC, whole blood samples were collected from 199 HCC patients(Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). We used dual markers CD15 and CD66b to identify neutrophils. While purifying neutrophils on a density gradient, a significant proportion of CD15\u003csup\u003e+\u003c/sup\u003eCD66b\u003csup\u003e+\u003c/sup\u003e cells was co-purified with the low-density mononuclear layer, besides the expected high-density granulocytic fraction. Although LDNs were rare in non-extrahepatic-metastatic HCC patients, their prevalence in the low-density fraction was significantly higher in metastatic HCC patients, especially in individuals with extra-hepatic metastasis(Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSeveral studies have reported that LDNs are a unique neutrophil population[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], and differential expression intensity of markers, such as CD36, CD41, CD61, CD226, and Lox-1, was reported between LDNs and HDNs in lung cancer[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. To confirm this difference between LDNs and HDNs under HCC context, we purified peripheral circulation LDNs and HDNs from 20 HCC patients, and performed fluorescence quantitative PCR assay for markers expression analysis. Our results showed that the CD61 displayed the most significant up-regulation in LDNs, compared with HDNs(Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Immunofluorescence assay consistently indicated a stronger CD61 expression in LDNs, compared with HDNs(Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). We further performed transcriptomic analysis for CD61\u003csup\u003e\u0026minus;\u003c/sup\u003eLDNs, CD61\u003csup\u003e+\u003c/sup\u003eLDNs and HDNs purified from peripheral circulation of 38 HCC patients. Our results showed that the three neutrophil subsets had distinct gene-expression profile. Comparisons indicated various genes that were expressed differentially by CD61\u003csup\u003e+\u003c/sup\u003eLDNs. In detail, as compared to CD61\u003csup\u003e\u0026minus;\u003c/sup\u003eLDNs or HDNs, CD61\u003csup\u003e+\u003c/sup\u003eLDNs displayed high level of genes encoding for neutrophil granule proteins(MPO, LL-37, DEFA4 and ELANE), cell cycle regulator(TOP2A), transcription factor(CEBPE) and surface marker(CEACAM8), and were characterized by low expression of inflammation response gene(TNF, IL-1β, OASL and COX2). Indeed, these analysis substantiated the proposal that CD61\u003csup\u003e+\u003c/sup\u003eLDNs displayed a unique phenotype distinct from HDNs and CD61\u003csup\u003e\u0026minus;\u003c/sup\u003eLDNs(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Morphologically, by DAPI staining, we observed the homogeneous segmented nuclear of HDNs, yet circulating LDNs subset mainly displayed the feature of banded nuclear(Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOverall, our results highlight the differences between LDNs and HDNs, and a positive correlation between the abundance of CD61\u003csup\u003e+\u003c/sup\u003eLDNs subset and tumor metastasis was determined in HCC.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eFunctional properties of CD61\u003csup\u003e+\u003c/sup\u003eLDNs\u003c/h2\u003e \u003cp\u003eWe next perform ex vivo experiments to assess reactive oxygen species(ROS) production, neutrophil extracellular traps(NETs) formation and cytokine synthesis by LDNs subset isolated from blood of HCC patients. As compared to those of HDNs, CD61\u003csup\u003e+\u003c/sup\u003eLDNs displayed a weaker respiratory burst upon treatment with LPS. CD61\u003csup\u003e+\u003c/sup\u003eLDNs also showed a lower production of TNF-α and a higher production of chemokine CCL5 under treatment with cancer cell condition medium. NETosis is a neutrophil-associated function important for antibacterial and tumor metastasis. We showed that CD61\u003csup\u003e\u0026minus;\u003c/sup\u003eLDNs, HDNs and CD61\u003csup\u003e+\u003c/sup\u003eLDNs displayed a weak spontaneous NETs formation with no statistical difference. However, under the stimulation of PMA, CD61\u003csup\u003e\u0026minus;\u003c/sup\u003eLDNs underwent a strongest NETs formation, while CD61\u003csup\u003e+\u003c/sup\u003eLDNs displayed the weakest ability of forming NETs. Interestingly, as compared to those of CD61\u003csup\u003e\u0026minus;\u003c/sup\u003eLDNs or HDNs, CD61\u003csup\u003e+\u003c/sup\u003eLDNs spontaneously released a significantly higher level of free DNA molecular into extracellular space(Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe next determined whether CD61\u003csup\u003e+\u003c/sup\u003eLDNs facilitated the invasion of HCC cells in vitro. Circulating CD61\u003csup\u003e+\u003c/sup\u003eLDNs, CD61\u003csup\u003e\u0026ndash;\u003c/sup\u003eLDNs, and HDNs were isolated from HCC patients. We cultured HCC cells alone and with CD61\u003csup\u003e+\u003c/sup\u003eLDNs, CD61\u003csup\u003e\u0026ndash;\u003c/sup\u003eLDNs, or HDNs using the transwell system. Our results showed that CD61\u003csup\u003e+\u003c/sup\u003eLDNs displayed significantly enhanced pro-metastatic effect, as compared to CD61\u003csup\u003e\u0026minus;\u003c/sup\u003eLDNs or HDNs(Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). In addition, we also found that a higher circulation CD61\u003csup\u003e+\u003c/sup\u003eLDNs abundance indicated the poorer disease prognosis in HCC patients(Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB)(Table S2).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCollectively, these results showed that CD61\u003csup\u003e+\u003c/sup\u003eLDNs represented a unique neutrophil subtype, promoted HCC invasion, and correlated with disease prognosis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eCD61\u003csup\u003e+\u003c/sup\u003eLDNs promoted the invasion of HCC cells by upregulating CCDC25\u003c/h2\u003e \u003cp\u003eTo elucidate the mechanism by which CD61\u003csup\u003e+\u003c/sup\u003eLDNs facilitate the metastasis of HCC, we performed RNA-seq using the Huh7 and HepG2 cells under the stimulation of CD61\u003csup\u003e+\u003c/sup\u003eLDNs. Among 124 genes, CCDC25 displayed a marked trend of up-regulation after CD61\u003csup\u003e+\u003c/sup\u003eLDNs stimulation(Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). In another study, we performed an immunohistochemistry assay and found that the abundance of circulating CD61\u003csup\u003e+\u003c/sup\u003eLDNs was positively correlated with the expression of CCDC25 in tumors(Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). To determine whether CCDC25 promotes CD61\u003csup\u003e+\u003c/sup\u003eLDNs-induced invasion of HCC cells, we first transfected Huh7 and HepG2 cell lines with short hairpin RNA (shRNA) using lentivirus vectors to silence the expression of CCDC25. Our results showed that the downregulation of CCDC25 strongly suppressed the invasion of HCC cells compared to the changes in the control conditions(Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). We also found that higher level of CCDC25 was expressed in cancer tissue than paracancerous region(Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Then, we performed co-culture assays using the transwell system to determine whether CD61\u003csup\u003e+\u003c/sup\u003eLDNs can affect the level of the CCDC25 protein in HCC cells. After 12 h, the CCDC25 protein level was significantly higher in the HCC cells in the presence of CD61\u003csup\u003e+\u003c/sup\u003eLDNs compared to the CCDC25 levels in the control(Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo examine whether CCDC25 was responsible for the pro-metastatic effect of CD61\u003csup\u003e+\u003c/sup\u003eLDNs, we performed co-culture assays using the transwell system under knocking down CCDC25. The downregulation of CCDC25 inhibited CD61\u003csup\u003e+\u003c/sup\u003eLDN-induced invasion of HCC cells(Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eThese results indicated that CD61\u003csup\u003e+\u003c/sup\u003eLDNs promoted the invasion of HCC cells by upregulating CCDC25 levels through indirect interactions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eCD61\u003csup\u003e+\u003c/sup\u003eLDNs upregulated the expression of CCDC25 via TLR9-NF-kB axis\u003c/h2\u003e \u003cp\u003eTo further explore the mechanism by which CD61\u003csup\u003e+\u003c/sup\u003eLDNs up-regulated CCDC25 in HCC cells, we performed the Gene Ontology Biological Process (GOBP) analysis based on RNA-seq, suggesting that the activation of TLR9 signaling in HCC cells was prominently triggered by the stimulation of CD61\u003csup\u003e+\u003c/sup\u003eLDNs(Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Western blot assays indicated an adequate TLR9 receptor expression in HCC cells(Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). Then, we intended to determine whether CD61\u003csup\u003e+\u003c/sup\u003eLDNs caused the upregulation of CCDC25 via TLR9 signaling. For this, we used shRNA to silence TLR9 or P65 expression in HCC cells. Our results showed that CD61\u003csup\u003e+\u003c/sup\u003eLDNs could not induce the phosphorylation of P65 and the expression of CCDC25 when either TLR9 or P65 was downregulated, compared to the changes in the control group(Figs.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). Additionally, CD61\u003csup\u003e+\u003c/sup\u003eLDNs lost the pro-metastatic effect on HCC cells when TLR9 or P65 was downregulated(Figs.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB). Furthermore, by performing transwell assay, we found a similar effect in raising CCDC25 mRNA level in HCC cells between CD61\u003csup\u003e+\u003c/sup\u003eLDNs and specific TLR9 agonist ODN 2216, supporting the notion that CD61\u003csup\u003e+\u003c/sup\u003eLDNs activated TLR9 pathway to up-regulate CCDC25 transcribe and led to the subsequent protein expression(Figs.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThus, these results suggested that CD61\u003csup\u003e+\u003c/sup\u003eLDNs upregulated the expression of CCDC25 and promoted the invasion of HCC cells in a TLR9-NF-κB signaling pathway-dependent manner.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eCD61\u003csup\u003e+\u003c/sup\u003eLDN-derived free DNA upregulated the expression of CCDC25 by activating TLR9-NF-kB axis\u003c/h2\u003e \u003cp\u003eThe mechanism by which CD61\u003csup\u003e+\u003c/sup\u003eLDNs induce the upregulation of CCDC25 remains largely unknown. However, intracellular DNA is the critical ligand for the TLR9 receptor[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. We found that CD61\u003csup\u003e+\u003c/sup\u003eLDNs spontaneously released significantly higher level of DNA into extracellular space than CD61\u003csup\u003e\u0026minus;\u003c/sup\u003eLDNs or HDNs. To assess whether CD61\u003csup\u003e+\u003c/sup\u003eLDNs-derived DNA played a pathogenic role in the CD61\u003csup\u003e+\u003c/sup\u003eLDNs-trggered upregulation of CCDC25, we isolated CD61\u003csup\u003e+\u003c/sup\u003eLDNs from the peripheral blood of HCC patients and dynamically evaluated DNA level in the supernatant. The isolated CD61\u003csup\u003e+\u003c/sup\u003eLDNs were incubated in vitro within RPMI 1640 medium for 8 hours, and DNA level was detected at timepoint of 2h, 4h, 6h and 8h. The results showed that CD61\u003csup\u003e+\u003c/sup\u003eLDNs spontaneously released abundant DNA into the extracellular space with a persistent trend(Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNETs formation is an important neutrophil function with intracellular DNA extrusion into extracellular space. A weak and spontaneous NETosis process was detected in CD61\u003csup\u003e\u0026minus;\u003c/sup\u003eLDNs, CD61\u003csup\u003e+\u003c/sup\u003eLDNs and HDNs in vitro. To remove NETs-DNA, we pre-treated the supernatant of CD61\u003csup\u003e+\u003c/sup\u003eLDNs with solid-phase coating anti-MPO. To digest DNA, we pre-treated the supernatant of CD61\u003csup\u003e+\u003c/sup\u003eLDNs with DNaseI. Our results indicated that the anti-MPO-treated supernatant of CD61\u003csup\u003e+\u003c/sup\u003eLDNs upregulated the expression of CCDC25 in HCC cells, which was effectively inhibited by DNaseI digestion(Figs.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eB). We next extracted DNA from the anti-MPO-treated supernatant of CD61\u003csup\u003e+\u003c/sup\u003eLDNs and co-cultured them with HCC cells. We consistently found a prominent effect of the extracted DNA in upregulating the CCDC25 protein level in HCC cells(Figs.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eC). Furthermore, we performed transwell co-culture assays to determine the pro-metastatic ability of extracted DNA. Our results showed that extracted DNA efficiently promoted HCC cell invasion, which could be abrogated by CCDC25 downregulation(Figs.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eIn order to explore whether the extracted DNA upregulated CCDC25 via activating TLR9-NF-κB signaling, we silenced TLR9 or P65 using shRNA in HCC cells. Consistently, we found that the extracted DNA could not induce the phosphorylation of P65 and the expression of CCDC25, and then the HCC cells invasion, when TLR9 or P65 was downregulated, compared to the changes in the control group(Figs.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eE, F). Taken together, our results indicated that CD61\u003csup\u003e+\u003c/sup\u003eLDN-derived DNA, but not NETs-DNA, upregulated CCDC25 to enhance HCC cells invasion potential in a TLR9-NF-κB signaling-dependent manner.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eCD61\u003csup\u003e+\u003c/sup\u003eLDNs promoted the metastasis of HCC cells in vivo\u003c/h2\u003e \u003cp\u003eTo elucidate the pro-metastatic effect of CD61\u003csup\u003e+\u003c/sup\u003eLDNs in vivo, we isolated circulating CD61\u003csup\u003e+\u003c/sup\u003eLDNs and CD61\u003csup\u003e\u0026minus;\u003c/sup\u003eLDNs from patients with HCC. Then, we implanted Huh7 cells alone, with CD61\u003csup\u003e+\u003c/sup\u003eLDNs or CD61\u003csup\u003e\u0026minus;\u003c/sup\u003eLDNs into the liver of mice. In line with vitro experiments, our results showed that the combinational implantation of Huh7 cells with CD61\u003csup\u003e+\u003c/sup\u003eLDNs exhibited the strongest pro-metastatic ability, as compared to CD61\u003csup\u003e\u0026minus;\u003c/sup\u003eLDNs, although infusion of CD61\u003csup\u003e\u0026minus;\u003c/sup\u003eLDNs exhibited an effective pro-metastatic efficiency(Figs.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eA). The infusion of CD61\u003csup\u003e+\u003c/sup\u003eLDNs led to an increase in the expression of CCDC25 in mouse liver tumors(Figs.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOverall, these results indicated that CD61\u003csup\u003e+\u003c/sup\u003eLDNs subset promoted HCC lung metastasis in vivo. Thus, CD61\u003csup\u003e+\u003c/sup\u003eLDNs might be a novel and practical target for preventing HCC metastasis.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eMetastasis is a complex multistep cascade which is related to the biological features of tumor cells and non-malignant tumor stroma[\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Several studies have suggested that the subset of LDNs might act as a link between the host and cancer cells and affect cancer metastasis[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. However, the specific functions of LDNs in the metastasis of HCC need to be illustrated. In this study, we elucidated the mechanism underlying the link between LDNs and the progression of HCC. Initially, we evaluated the abundance of LDNs in patients and found that the abundance of circulating LDNs was positively related to the metastasis of HCC. Then, we found a close association between LDNs and the level of CCDC25 within the tumor. The LDNs promoted metastasis through the DNA-TLR9-CCDC25 axis.\u003c/p\u003e \u003cp\u003eThe subset of LDNs acquired heterogeneous characteristics[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Hence, we assessed the phenotype of LDNs that contributed the most to cancer metastasis. We found that the marker CD61 was prominently expressed on LDNs. CD61\u003csup\u003e+\u003c/sup\u003eLDNs displayed different phenotype of secreting cytokine and forming NETs, as compared to CD61\u003csup\u003e\u0026minus;\u003c/sup\u003eLDNs and HDNs. Next, we compared the pro-metastatic ability of CD61\u003csup\u003e+\u003c/sup\u003eLDNs with the pro-metastatic ability of CD61\u003csup\u003e\u0026ndash;\u003c/sup\u003eLDNs and HDNs. Although both CD61\u003csup\u003e\u0026ndash;\u003c/sup\u003eLDNs and HDNs strongly promoted the invasion of HCC cells, CD61\u003csup\u003e+\u003c/sup\u003eLDNs exhibited the strongest ability to promote the invasion of HCC cells. A previous study have shown that neutrophils can facilitate cancer metastasis via the NETs-CCDC25 pathway, and CCDC25 can strongly promote cancer metastasis[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Several studies have also shown that serum CCDC25 might be a potential diagnostic biomarker[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Hence, we investigated whether CD61\u003csup\u003e+\u003c/sup\u003eLDNs can facilitate cancer metastasis via the action of CCDC25. Our results indicated that the pro-invasion effect of CD61\u003csup\u003e+\u003c/sup\u003eLDNs was effectively reversed by knocking down CCDC25.\u003c/p\u003e \u003cp\u003eThe CCDC25 protein is membrane-bound, and several studies have shown that this protein is correlated with the infiltration of immune cells, cancer prognosis, and ferroptosis in the microenvironment. However, studies on the interaction between immune cells and CCDC25 are lacking. In this study, we used siRNA to downregulate CCDC25 in HCC cells and found that the downregulation of CCDC25 reduced the invasion of HCC cells. We also found that CD61\u003csup\u003e+\u003c/sup\u003eLDNs increased the expression of CCDC25, and the downregulation of CCDC25 strongly inhibited the invasion of HCC cells triggered by CD61\u003csup\u003e+\u003c/sup\u003eLDNs. We showed that the subset of CD61\u003csup\u003e+\u003c/sup\u003eLDNs promoted the metastasis of HCC through the upregulation of CCDC25. Song et al. found that CCDC25 can act as a NETs-DNA receptor on cancer cells that recognize extracellular DNA, and then, they can activate the ILK-β-parvin pathway to enhance cell motility[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. To determine whether free DNA promotes the upregulation of CCDC25 in HCC cells after the stimulation of CD61\u003csup\u003e+\u003c/sup\u003eLDNs, we first evaluated the level of free DNA in the supernatant of CD61\u003csup\u003e+\u003c/sup\u003eLDNs. Then, we performed RNA sequencing and GOBP analysis to determine whether CD61\u003csup\u003e+\u003c/sup\u003eLDNs can trigger the activation of TLR9 signaling in HCC cells. The CD61\u003csup\u003e+\u003c/sup\u003eLDNs spontaneously and persistently released DNA into the extracellular space. Upon further examination, we found that the CD61\u003csup\u003e+\u003c/sup\u003eLDN-derived DNA led to the upregulation of CCDC25. However, whether the TLR9 signaling pathway is essential in the CD61\u003csup\u003e+\u003c/sup\u003eLDNs-triggered upregulation of CCDC25 needs to be elucidated. We used Si-RNA to silence TLR9 and P65, and found that the CD61\u003csup\u003e+\u003c/sup\u003eLDN-triggered upregulation of CCDC25 was strongly inhibited by silencing either TLR9 or P65, which indicated that CD61\u003csup\u003e+\u003c/sup\u003eLDN-induced upregulation of CCDC25 depended on the initiation of TLR9-NF-κB signaling. The results of the GOBP analysis showed that other signaling pathways besides the TLR9 pathway were simultaneously initiated under the stimulation of CD61\u003csup\u003e+\u003c/sup\u003eLDNs. Further studies might provide new insights into the specific effects of those signaling pathways under this condition.\u003c/p\u003e \u003cp\u003eThe CD61 protein participates in reprogramming tumor metabolism, shaping the stromal and immune microenvironment, facilitating epithelial to mesenchymal transition (EMT) and endothelial to mesenchymal transition (End-MT)[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], and it is highly expressed by tumor-associated LDNs. In this study, we performed ex vitro experiments to show that CD61\u003csup\u003e+\u003c/sup\u003eLDNs could release abundant DNA into extracellular space. However, the information on the effect of the CD61 receptor during spontaneous DNA release and the pro-metastatic effect of CD61\u003csup\u003e+\u003c/sup\u003eLDNs are limited.\u003c/p\u003e \u003cp\u003eSeveral studies suggest that pulmonary metastasis was increased when HCC cells were co-injected with tumor-associated neutrophils (TANs), which provided direct evidence that TANs have a pro-metastatic effect[\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. In this study, to determine whether CD61\u003csup\u003e+\u003c/sup\u003eLDNs promoted the metastasis of HCC in vivo, we transferred Huh7 cells with or without CD61\u003csup\u003e+\u003c/sup\u003eLDNs into the liver of nude mice. Obviously, the infusion of CD61\u003csup\u003e+\u003c/sup\u003eLDNs greatly increased pulmonary metastasis, as compared to CD61\u003csup\u003e\u0026minus;\u003c/sup\u003eLDNs infusion or Huh7 alone.\u003c/p\u003e \u003cp\u003eTo summarize, in this study, we determined a cellular, molecular, and clinical network associated with LDNs, involving CCDC25, free DNA, TLR9 signaling, and CD61. CD61\u003csup\u003e+\u003c/sup\u003eLDNs were identified as a unique neutrophil subtype under HCC context. This network showed a bridge between LDNs subset and tumor progression. Our findings suggested that this network might be a promising target for treating HCC.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics Approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSamples were collected after approval by the medical ethics committee of The First Affiliated Hospital of University of Science and Technology of China and written informed consents were obtained from each patient. All animal experiments were approved by the animal Ethics committee of University of Science and Technology of China, and all institutional and national guidelines for the care and use of laboratory animals were followed. All authors consent for publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in the this published article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors declare that they have no competing interest on this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eX. Q. Guan performed the study, acquisition and analysis of data, and drafted the manuscript. Y. Y. Lu and C. Z. Wang offered assistance with flow cytometry analysis and participated in animal studies. Z. G. Chen and P. Zhan provided assistance for flow cytometry analysis and cellular studies. X. Q. Guan started the study and particaped in experimental design. X. Q. Guan approved for the final version and submission. All authors made a significant contribution to the work reported.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Fujian Provincial Key Laboratory of Chronic Liver Disease and Hepatocellular Carcinoma. We thank the research central of Anhui provincial hospital for providing the platform for animal study and cellular experiments.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSagiv JY, Michaeli J, Assi S, Mishalian I, Kisos H, Levy L, Damti P, Lumbroso D, Polyansky L, Sionov RV, Ariel A, Hovav AH, Henke E, Fridlender ZG, Granot Z. Phenotypic diversity and plasticity in circulating neutrophil subpopulations in cancer. 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Hepatology. 2019 Oct;70(4):1214-1230.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Low density neutrophils, CCDC25, Hepatocellular carcinoma, Orthotopic model, Tumor microenvironment","lastPublishedDoi":"10.21203/rs.3.rs-3868978/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3868978/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eA subset of neutrophils isolated from peripheral blood mononuclear cells (PBMC) has recently been described in cancer patients.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eDouble gradient centrifugation was used to separate neutrophil subset. Western blot assay was performed to assess CCDC25 expression level.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eIn this study, we found that low density neutrophils(LDNs) were highly enriched in metastatic hepatocellular carcinoma (HCC) patients compared to non-metastatic HCC patients. Then, we showed that the most prominent marker on LDNs was CD61, compared to high density neutrophils(HDNs). The CD61\u003csup\u003e+\u003c/sup\u003eLDNs subset displayed an increased ability in triggering metastasis, producing free DNA, and synthesizing cytokine CCL5, and a decreased ability in forming NETs, synthesizing cytokine TNF-α and producing ROS, as compared with CD61\u003csup\u003e\u0026minus;\u003c/sup\u003eLDNs or HDNs. Transcriptomic analysis revealed that CD61\u003csup\u003e+\u003c/sup\u003eLDNs displayed distinct gene transcribe from CD61\u003csup\u003e\u0026minus;\u003c/sup\u003eLDNs and HDNs. The abundance of circulating CD61\u003csup\u003e+\u003c/sup\u003eLDNs was negatively correlated with disease prognosis, and positively correlated with the expression of CCDC25 within tumor. These CD61\u003csup\u003e+\u003c/sup\u003eLDNs increased the invasion of HCC cells by upregulating CCDC25. Mechanistically, the CD61\u003csup\u003e+\u003c/sup\u003eLDN-derived free DNA, excluding the NETs-DNA, enhanced the invasiveness of HCC cells and triggered their metastatic potential, which was mediated by TLR9-NF-κB-CCDC25 signaling. Blocking this signaling reversed the invasion of the CD61\u003csup\u003e+\u003c/sup\u003eLDNs-evoked HCC cells. In vivo, we consistently showed that CD61\u003csup\u003e+\u003c/sup\u003eLDNs enhanced HCC metastasis to the lungs.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eOverall, our findings showed that a subset of CD61\u003csup\u003e+\u003c/sup\u003eLDNs has pro-metastatic effects on HCC, which might be used for targeting HCC in the clinical setting.\u003c/p\u003e","manuscriptTitle":"CD61 + LDNs drive cancer metastasis by upregulating CCDC25 expression via DNA-TLR9-NF-kB axis in HCC","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-19 21:45:58","doi":"10.21203/rs.3.rs-3868978/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"409b3e53-be01-4547-8938-0d003fa84d6d","owner":[],"postedDate":"January 19th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-01-22T13:23:48+00:00","versionOfRecord":[],"versionCreatedAt":"2024-01-19 21:45:58","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3868978","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3868978","identity":"rs-3868978","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}