Role of CD61+ low-density neutrophils in promoting hepatocellular carcinoma metastasis through CCDC25 upregulation.

BACKGROUND: A subset of neutrophils isolated from the peripheral blood mononuclear cells (PBMC) layer has recently been described in cancer patients. METHODS: Double-gradient centrifugation was used to separate the neutrophil subsets. Western blotting and immunohistochemical assays were performed to assess CCDC25 expression levels. RESULTS: In this study, we found that low-density neutrophils (LDNs) were more highly enriched in metastatic hepatocellular carcinoma (HCC) patients than in non-metastatic HCC patients. We then showed a CD61+ LDNs subset, which displayed distinct functions and gene expression, when compared with high-density neutrophils (HDNs) and CD61- LDNs. Transcriptomic analysis revealed that the CD61+ LDNs were predominantly enhanced in the transcription of glycolysis and angiogenesis associated gene, HMGB1 associated gene and granulation protein gene. These CD61+ LDNs displayed a prominent ability to trigger metastasis, compared with HDNs and CD61- LDNs. Specifically, CD61+ LDN-derived HMGB1 protein increased the invasion of HCC cells by upregulating CCDC25. Mechanistically, the CD61+ LDN-derived HMGB1 protein enhanced the invasiveness of HCC cells and triggered their metastatic potential, which was mediated by TLR9-NF-κB-CCDC25 signaling. Blocking this signaling pathway reversed the invasion of the CD61+ LDN-induced HCC cells. In vivo, we consistently showed that CD61+ LDN-derived HMGB1 enhances HCC metastasis to the lungs. CONCLUSIONS: Overall, our findings showed that a subset of CD61+ LDNs has pro-metastatic effects on HCC, and may be used to target HCC in the clinical setting.

Tumor-associated NK cells facilitate tumor growth via NKp46 in immunocompetent murine hepatocellular carcinoma.

Natural killer(NK) cells comprise one subset of the innate lymphoid cells family. Despite reported anti-tumor activity of NK cells, their tangible contribution to tumor control remains controversial. This is due to the incomplete understanding of NK alterations within tumor microenvironment(TME). Here we showed, using murine hepatocellular carcinoma(HCC) model, that early NK cells deletion markedly attenuated tumor growth in a CD8+T cells dependent manner. This effect was accompanied by an enhanced CD8+T cells effector function in tumor rather than circulating blood. Then, we demonstrated that abundant NKp46+ NK subset, but not NKp46- NK, were recruited towards tumor microenvironment during tumor progression. Frequency of intratumor NKP46+ NK cells were inversely related to CD8+T cells activation, and positively correlated with tumor growth. Intratumor NKp46+ NK cells exhibited dysfunction and increased expression of inhibitory receptors, when compared with NKp46- NK cells. Blockade of NK cells-associated NKp46 effectively attenuated HCC growth. Infusion of tumor-derived NKp46+ NK cells markedly enhanced HCC growth in vivo, in contrast to tumor cells inoculation alone. The further mechanistic investigations unveiled that NK cells boosted tumor growth by NKp46-mediated impairment of CD8+T cells effector function. Overall, this work supported a previously unappreciated regulatory property of tumor-associated NK cells in HCC, and NKp46 as a potential target against HCC in clinical setting.

The Crosstalk Between Cancer Cells and Neutrophils Enhances Hepatocellular Carcinoma Metastasis via Neutrophil Extracellular Traps-Associated Cathepsin G Component: A Potential Therapeutic Target.

BACKGROUND: Emerging evidences have highlighted the roles of neutrophils, as the major host microenvironment component, in the development of hepatocellular carcinoma (HCC). Neutrophils extracellular traps (NETs) produced in the infection can strengthen the behavior of cancer metastasis. Here, we investigated the roles of NETs in HCC metastasis and further explore the underlying mechanism of how NETs interact with cancer. METHODS: The neutrophils were isolated from whole blood of HCC patients and used to evaluate the formation of NETs. NET markers were detected in tissue samples, plasma and cell climbing slice. Mouse models were used to evaluate the roles of NETs in HCC metastasis in vivo, and the corresponding mechanisms were explored using in vivo and in vitro assays. RESULTS: An increase in the release of NETs in patients with HCC, particularly those with portal vein tumor thrombosis (PVTT). The presence of NETs in HCC tumor tissues closely correlated with a poor prognosis. Functionally, the invasion ability of HCC cells was enhanced by co-culture with HCC neutrophils, through NETs formation, while the neutrophils from a healthy donor (HD) exhibited the inhibition of the invasion ability. Furthermore, we observed an enhanced ability of forming NETs in neutrophils from HCC patients in vitro, especially patients with PVTT or extra-hepatic metastasis. An in-vivo animal study demonstrated that neutrophils of HCC facilitated the metastatic behavior towards the lung. The further mechanistic investigation unveiled that HCC cells-derived cytokine IL-8 triggered NETs formation in an NADPH oxidase-dependent manner, and NETs-associated cathepsin G (cG) promoted HCC metastasis in vitro as well as vivo. Clinically, the expression of the cG protein in tumor tissues displayed a close correlation with the disease prognosis of HCC patients. CONCLUSION: Our findings implicated that the induction of NETs by HCC cells is a critical metastasis-supporting cancer-host interaction and that NETs may serve as an immune-based potential therapeutic target against HCC progression.

Estrogen-related receptor alpha triggers the proliferation and migration of human non-small cell lung cancer via interleukin-6.

Human non-small cell lung cancer (NSCLC) is one of the leading causes of cancer deaths worldwide. Estrogenic signals have been suggested to be important for the growth and metastasis of NSCLC cells. Our present data showed that estrogen-related receptor alpha (ERRα), while not ERRß or ERRγ, was significantly elevated in NSCLC cell lines as compared with that in normal bronchial epithelial cell line BEAS-2B. The expression of ERRα in clinical NSCLC tissues was significantly greater than that in their matched normal adjacent tissues. Over expression of ERRα can trigger the proliferation, migration, and invasion of NSCLC cells, while si-ERRα or ERRα inhibitor showed opposite effects. ERRα can increase the mRNA and protein expression of IL-6, while not IL-8, IL-10, IL-22, VEGF, TGF-ß, or TNF-α, in NSCLC cells. Silence of IL-6 attenuated ERRα induced proliferation and cell invasion. Furthermore, our data revealed the inhibition of NF-κB, while not ERK1/2 or PI3K/Akt, abolished ERRα induced production of IL-6. This might be due to that overexpression of ERRα can increase the expression and nuclear translocation of p65 in NSCLC cells. Collectively, our data showed that activation of NF-κB/IL-6 is involved in ERRα induced migration and invasion of NSCLC cells. It suggested that ERRα might be a potential target for NSCLC treatment.

Sumoylation modulates oxidative stress relevant to the viability and functionality of pancreatic beta cells.

Sumoylation is an evolutionarily conserved regulatory mechanism to play an important role in various cellular processes through modulation of protein localization, stability and functionality. Recent studies including ours have consistently demonstrated that sumoylation provides protection for cells against oxidative stress. Given that pancreatic beta cells are a vulnerable target of oxidative stress, we thus in this minireview, updated the advancement of sumoylation in the regulation of ROS generation, and discussed its impact on several critical signaling pathways relevant to beta cells against oxidative stress and maintenance of functionality. Specifically, we bring together how sumoylation represses intracellular ROS formation, and protects beta cells against oxidative stress through regulating IκB/NFκB, JNK/c-Jun, and Maf/Nrf2 pathways. The tight implication of sumoylation in oxidative stress reflects that it could be an essential mechanism for beta cells to adapt to the detrimental cellular microenvironment.

[Construction and identification of mouse BTLA lentiviral expression vector].

OBJECTIVE: To construct and identify a lentiviral vector for mBTLA (mouse B and T lymphocyte attenuator) expression. METHODS: The entire coding sequence of mBTLA gene was amplified from pET-28a-mBTLA plasmid, and then mBTLA gene was transferred into pMD18-T plasmid before cloning into a lentiviral transfer vector. Liposome was used to package lentiviral particles in 293T cells. After lentiviral particles packaging, morphological changes of 293T cells were observed by fluorescence microscope. The recombinant plasmid was identified using RT-PCR and sequencing. Lentiviral titer was detected by 50% tissue culture infectious dose (TCID50;) assay. RT-PCR and Western blotting were used to detect mBTLA mRNA and protein expression. RESULTS: pMD18-T-mBTLA and pSL6-mBTLA plasmids were successfully constructed and digested for electrophoresis appearing a near 1 kb strip which matched the size of mBTLA coding sequence. Gene sequencing and alignment analysis further confirmed mBTLA coding sequence to be successfully integrated into the plasmid vector. Morphological observation and supernatant PCR amplification of 293T cells confirmed that Lenti-mBTLA lentiviral packaging was successful, and the Lenti-mBTLA lentiviral titer was 1.3×10(8); pfu/mL. RT-PCR and Western blotting demonstrated that the Lenti-mBTLA vector could effectively express mBTLA mRNA and protein. CONCLUSION: The lentiviral vector which can effectively express mBTLA mRNA and protein has been successfully constructed.