Emerging evidence indicate that cancer-associated fibroblasts (CAFs) affect tumor progression by reshaping the tumor microenvironment. Neutrophils are prominent components of solid tumors and important in cancer progression. Whether the phenotype and function of neutrophils in hepatocellular carcinoma (HCC) are influenced by CAFs is not well understood. Herein, we investigated the effect of HCC-derived CAFs (HCC-CAFs) on the neutrophils and explored the biological role of this effect. We found that HCC-CAFs induced chemotaxis of neutrophils and protected them from spontaneous apoptosis. Neutrophils were activated by the conditioned medium from HCC-CAFs with increased expression of CD66b, PDL1, IL8, TNFa, and CCL2, and with decreased expression of CD62L. HCC-CAF-primed neutrophils impaired T-cell function through the PD1/PDL1 signaling pathway. We revealed that HCC-CAFs induced the activation of STAT3 pathways in neutrophils, which are essential for the survival and function of activated neutrophils. In addition, we demonstrated that HCC-CAF-derived IL6 was responsible for the STAT3 activation of neutrophils. Collectively, our results suggest that HCC-CAFs regulate the survival, activation, and function of neutrophils within HCC through an IL6–STAT3–PDL1 signaling cascade, which presents a novel mechanism for the role of CAFs in remodeling the cancer niche and provides a potential target for HCC therapy.

Background Regulatory B (Breg) cells represent one of the B cell subsets that infiltrate solid tumors and exhibit distinct phenotypes in different tumor microenvironments. However, the phenotype, function and clinical relevance of Breg cells in human hepatocellular carcinoma (HCC) are presently unknown. Methods Flow cytometry analyses were performed to determine the levels, phenotypes and functions of TIM-1⁺Breg cells in samples from 51 patients with HCC. Kaplan-Meier plots for overall survival and disease-free survival were generated using the log-rank test. TIM-1⁺Breg cells and CD8⁺ T cells were isolated, stimulated and/or cultured in vitro for functional assays. Exosomes and B cells were isolated and cultured in vitro for TIM-1⁺Breg cell expansion assays. Results Patients with HCC showed a significantly higher TIM-1⁺Breg cell infiltration in their tumor tissue compared with the paired peritumoral tissue. The infiltrating TIM-1⁺Breg cells showed a CD5^highCD24⁻CD27^−/+CD38^+/high phenotype, expressed high levels of the immunosuppressive cytokine IL-10 and exhibited strong suppressive activity against CD8⁺ T cells. B cells activated by tumor-derived exosomes strongly expressed TIM-1 protein and were equipped with suppressive activity against CD8⁺ T cells similar to TIM-1⁺Breg cells isolated from HCC tumor tissue. Moreover, the accumulation of TIM-1⁺Breg cells in tumors was associated with advanced disease stage, predicted early recurrence in HCC and reduced HCC patient survival. Exosome-derived HMGB1 activated B cells and promoted TIM-1⁺Breg cell expansion via the Toll like receptor (TLR) 2/4 and mitogen-activated protein kinase (MAPK) signaling pathways. Conclusions Our results illuminate a novel mechanism of TIM-1⁺Breg cell-mediated immune escape in HCC and provide functional evidence for the use of these novel exosomal HMGB1-TLR2/4-MAPK pathways to prevent and to treat this immune tolerance feature of HCC.

There is increasing evidence that circular RNA (circRNA) are involved in cancer development, but the regulation and function of human circRNA remain largely unknown. In this study, we demonstrated that ZKSCAN1, a zinc finger family gene, is expressed in both linear and circular (circZKSCAN1) forms of RNA in human hepatocellular carcinoma (HCC) tissues and cell lines. Here, we analyzed a cohort of 102 patients and found that expression of both ZKSCAN1mRNA and circZKSCAN1 was significantly lower (P < 0.05) in the HCC samples compared with that in matched adjacent nontumorous tissues by reverse transcription PCR (RT‐PCR). The low expression level of ZKSCAN1 was only associated with tumor size (P = 0.032), while the cirZKSCAN1 levels varied in patients with different tumor numbers (P < 0.01), cirrhosis (P = 0.031), vascular invasion (P = 0.002), or microscopic vascular invasion (P = 0.002), as well as with the tumor grade (P < 0.001). Silencing both ZKSCAN1mRNA and circZKSCAN1 promoted cell proliferation, migration, and invasion. In contrast, overexpression of both forms of RNA repressed HCC progression in vivo and in vitro. Silencing or overexpression of both forms of RNA did not interfere with each other. RNA‐seq revealed a very different molecular basis for the observed effects; ZKSCAN1mRNA mainly regulated cellular metabolism, while circZKSCAN1 mediated several cancer‐related signaling pathways, suggesting a nonredundant role for ZKSCAN1mRNA and circRNA. In conclusion, our results revealed two post‐translational products (ZKSCAN1mRNA and circZKSCAN1) that cooperated closely with one another to inhibit growth, migration, and invasion of HCC. cirZKSCAN1 might be a useful marker for the diagnosis of HCC.

Mesenchymal stem cells (MSCs) have been reported to exert therapeutic effects on immunoregulation, tissue repair, and regeneration from the bench to the bedside. Increasing evidence demonstrates that extracellular vesicles (EVs) derived from MSCs could contribute to these effects and are considered as a potential replacement for stem cell‐based therapies. However, the efficacy and underlying mechanisms of EV‐based treatment in hepatic ischemia‐reperfusion injury (IRI) remain unclear. Here, we demonstrated that human umbilical cord MSC‐EVs (huc‐MSC‐EVs) could protect against IRI‐induced hepatic apoptosis by reducing the infiltration of neutrophils and alleviating oxidative stress in hepatic tissue in vivo . Meanwhile, huc‐MSC‐EVs reduced the respiratory burst of neutrophils and prevented hepatocytes from oxidative stress‐induced cell death in vitro . Interestingly, we found that the mitochondria‐located antioxidant enzyme, manganese superoxide dismutase (MnSOD), was encapsulated in huc‐MSC‐EVs and reduced oxidative stress in the hepatic IRI model. Knockdown of MnSOD in huc‐MSCs decreased the level of MnSOD in huc‐MSC‐EVs and attenuated the antiapoptotic and antioxidant capacities of huc‐MSC‐EVs, which could be partially rescued by MnSOD mimetic manganese (III) 5,10,15,20‐tetrakis (4‐benzoic acid) porphyrin (MnTBAP). In summary, these findings provide new clues to reveal the therapeutic effects of huc‐MSC‐EVs on hepatic IRI and evaluate their preclinical application.—Yao, J., Zheng, J., Cai, J., Zeng, K., Zhou, C., Zhang, J., Li, S., Li, H., Chen, L., He, L., Chen, H., Fu, H., Zhang, Q., Chen, G., Yang, Y., Zhang, Y. Extracellular vesicles derived from human umbilical cord mesenchymal stem cells alleviate rat hepatic ischemia‐reperfusion injury by suppressing oxidative stress and neutrophil inflammatory response. FASEB J. 33, 1695–1710 (2019). www.fasebj.org

Purpose: Elevated levels of neutrophils have been associated with poor survival in various cancers, but direct evidence supporting a role for neutrophils in the immunopathogenesis of human cancers is lacking. Experimental Design: A total of 573 patients with gastric cancer were enrolled in this study. Immunohistochemistry and real-time PCR were performed to analyze the distribution and clinical relevance of neutrophils in different microanatomic regions. The regulation and function of neutrophils were assessed both in vitro and in vivo. Results: Increased neutrophil counts in the peripheral blood were associated with poor prognosis in gastric cancer patients. In gastric cancer tissues, neutrophils were enriched predominantly in the invasive margin, and neutrophil levels were a powerful predictor of poor survival in patients with gastric cancer. IL17+ neutrophils constitute a large portion of IL17-producing cells in human gastric cancer. Proinflammatory IL17 is a critical mediator of the recruitment of neutrophils into the invasive margin by CXC chemokines. Moreover, neutrophils at the invasive margin were a major source of matrix metalloproteinase-9, a secreted protein that stimulates proangiogenic activity in gastric cancer cells. Accordingly, high levels of infiltrated neutrophils at the invasive margin were positively correlated with angiogenesis progression in patients with gastric cancer. Conclusions: These data provide direct evidence supporting the pivotal role of neutrophils in gastric cancer progression and reveal a novel immune escape mechanism involving fine-tuned collaborative action between cancer cells and immune cells in the distinct tumor microenvironment. Clin Cancer Res; 23(6); 1575–85. ©2016 AACR.