Background: Neutrophil extracellular traps (NETs) are considered significant contributors to cancer progression, especially metastasis. However, it is still unclear whether NETs are involved in hepatitis B virus (HBV)‐related hepatocarcinogenesis and have potential clinical significance during evaluation and management for hepatocellular carcinoma (HCC). In this study, we aimed to investigate the functional mechanism of NETs in HBV‐related hepatocarcinogenesis and their clinical significance. Methods: A total of 175 HCC patients with and without HBV infection and 58 healthy controls were enrolled in this study. NETs were measured in tissue specimens, freshly isolated neutrophils and blood serum from these patients, and the correlation of circulating serum NETs levels with malignancy was evaluated. The mechanism by which HBV modulates NETs formation was explored using cell‐based studies. In addition, in vitro and in vivo experiments were further performed to clarify the functional mechanism of NETs on the growth and metastasis of HCC. Results: We observed an elevated level of NETs in blood serum and tissue specimens from HCC patients, especially those infected with HBV. NETs facilitated the growth and metastasis of HCC both in vitro and in vivo, which were mainly dominated by increased angiogenesis, epithelial‐mesenchymal transition (EMT)‐related cell migration, matrix metalloproteinases (MMPs)‐induced extracellular matrix (ECM) degradation and NETs‐mediated cell trapping. Inhibition of NETs generation by DNase 1 effectively abrogated the NETs‐aroused HCC growth and metastasis. In addition, HBV‐induced S100A9 accelerated the generation of NETs, which was mediated by activation of toll‐like receptor (TLR4)/receptor for advanced glycation end products (RAGE)‐reactive oxygen species (ROS) signaling. Further, circulatory NETs were found to correlate with viral load, TNM stage and metastasis status in HBV‐related HCC, and the identified NETs could predict extrahepatic metastasis, with an area under the ROC curve (AUC) of 0.83 and 90.3% sensitivity and 62.8% specificity at a cutoff value of 0.32. Conclusions: Our findings indicated that activation of RAGE/TLR4‐ROS signaling by HBV‐induced S100A9 resulted in abundant NETs formation, which subsequently facilitated the growth and metastasis of HCC cells. More importantly, the identified circulatory NETs exhibited potential as an alternative biomarker for predicting extrahepatic metastasis in HBV‐related HCC.

Fusobacterium nucleatum (Fn) has been considered as a significant contributor in promoting colorectal carcinoma (CRC) development by suppressing host anti-tumor immunity. Recent studies demonstrated that the aggregation of M2 macrophage (Mφ) was involved in CRC progress driven by Fn infection. However, the underlying molecular mechanisms are poorly characterized. Here, we investigated the role of Fn in Mφ polarization as well as its effect on CRC malignancy. Fn infection facilitated differentiation of Mφ into the M2-like Mφ phenotype by in vitro study. Histological observation from Fn-positive CRC tissues confirmed the abundance of tumor-infiltrating M2-like Mφ. Fn-induced M2-like Mφ polarization was weakened once inhibiting a highly expressed damage-associated molecular pattern (DAMP) molecule S100A9 mainly derived from Fn-challenged Mφ and CRC cells. In addition, Fn-challenged M2-like Mφ conferred CRC cells a more malignant phenotype, showing stronger proliferation and migration characteristics in vitro and significantly enhanced tumor growth in vivo, all of which were partially inhibited when S100A9 was lost. Mechanistic studies further demonstrated that activation of TLR4/NF-κB signaling pathway mediated Fn-induced S100A9 expression and subsequent M2-like Mφ activation. Collectively, these findings indicate that elevated S100A9 in Fn-infected CRC microenvironment participates in M2-like Mφ polarization, thereby facilitating CRC malignancy. Furthermore, targeting TLR4/NF-κB/S100A9 cascade may serve as promising immunotherapeutic strategy for Fn-associated CRC.

Myeloid-derived suppressor cells (MDSCs) are a major component of the immunosuppressive tumor microenvironment (TME) and have been recognized as a contributing factor to inflammation-related cancers. However, the molecular mechanisms of MDSCs accumulation and activation remain elusive. We previously showed that the proinflammatory molecule S100A9 in TME exerts a tumor-promoting effect in colorectal carcinoma (CRC). In this report, we investigated the effect and molecular mechanisms of S100A9 on the accumulation and immunosuppressive function of MDSCs in CRC. Elevated S100A9 and MDSCs were found in tumor tissue and peripheral blood from CRC patients. Circulating S100A9 and MDSCs were positively associated to each other, and both S100A9 and MDSCs were correlated to neoplastic progression. Using a CRC cell line LoVo-induced MDSCs model, we found that S100A9 stimulated chemotaxis and activation but not viability of MDSCs. Mechanistic studies demonstrated that activation of RAGE-mediated p38 MAPK and TLR4-mediated NF-κB signaling pathways were involved in S100A9-induced chemotaxis and MDSCs activation, respectively. Furthermore, ROC analysis showed that combination detection of S100A9 and MDSCs was superior to individual detection of these two factors for diagnosing CRC patients with advanced staging and lymphatic metastasis, which yielded an area under the ROC curve (AUC) of 0.92 with 86.7% sensitivity and 86.4% specificity, and an AUC of 0.82 with 75% sensitivity and 77.1% specificity, respectively. Collectively, our study suggests that the S100A9 plays a pivotal role in immunosuppressive TME by stimulating MDSCs chemotaxis and activation, and combination detection of S100A9 and MDSCs may serve as a potential marker for diagnosis of CRC progression.

Background: Fusobacterium nucleatum (Fn) acts as a procarcinogenic bacterium in colorectal carcinoma (CRC) by regulating the inflammatory tumor microenvironment (TME). Neutrophil extracellular traps (NETs), which can be generated by persistent inflammation, have been recently considered to be significant contributors in promoting cancer progression. However, whether NETs are implicated in Fn-related carcinogenesis is still poorly characterized. Here, we explored the role of NETs in Fn-related CRC as well as their potential clinical significance. Methods: Fn was measured in tissue specimens and feces samples from CRC patients. The expression of NET markers were also detected in tissue specimens, freshly isolated neutrophils and blood serum from CRC patients, and the correlation of circulating NETs levels with Fn was evaluated. Cell-based experiments were conducted to investigate the mechanism by which Fn modulates NETs formation. In addition, we clarified the functional mechanism of Fn-induced NETs on the growth and metastasis of CRC in vitro and in vivo experiments. Results: Tissue and blood samples from CRC patients, particularly those from Fn-infected CRC patients, exhibited greater neutrophil infiltration and higher NETs levels. Fn infection induced abundant NETs production in in vitro studies. Subsequently, we demonstrated that Fn-induced NETs indirectly accelerated malignant tumor growth through angiopoiesis, and facilitated tumor metastasis, as manifested by epithelial-mesenchymal transition (EMT)-related cell migration, matrix metalloproteinase (MMP)-mediated basement membrane protein degradation, and trapping of CRC cells. Mechanistically, the Toll-like receptor (TLR4)-reactive oxygen species (ROS) signaling pathway and NOD-like receptor (NOD1/2)-dependent signaling were responsible for Fn-stimulated NETs formation. More importantly, circulating NETs combined with carcinoembryonic antigen (CEA) could predict CRC occurrence and metastasis, with areas under the ROC curves (AUCs) of 0.92 and 0.85, respectively. Conclusions: Our findings indicated that Fn-induced NETs abundance by activating TLR4-ROS and NOD1/2 signalings in neutrophils facilitated CRC progression. The combination of circulating NETs and CEA was identified as a novel screening strategy for predicting CRC occurrence and metastasis.

NLRP3 inflammasome-dependent pyroptosis has been implicated in liver fibrosis progression. However, the definite intrahepatic cell types that undergo pyroptosis and the underlying mechanism as well as the clinical importance remain unclear. Here, augmented levels of pyroptosis-related indicators GSDMD, IL-1β, and IL-18 were verified in both liver fibrosis patients and CCl4-induced fibrotic mouse model. Confocal imaging of NLRP3 with albumin, F4/80 or α-SMA revealed that enhanced NLRP3 was mainly localized to kupffer cells (KCs), indicating that KCs are major cell types that undergo pyroptosis. Targeting pyroptosis by inhibitor MCC950 attenuated the severity and ameliorated liver function in fibrosis models. In addition, elevated S100A8 in liver fibrosis patients was correlated with pyroptosis-related indicators. S100A8 stimulated pyroptotic death of macrophages, which resulted in activation of human hepatic stellate cell line LX-2 cells and increased collagen deposition. Mechanistically, S100A8 activated TLR4/NF-κB signaling and upregulated its target genes NLRP3, pro-IL-1β, and pro-IL-18 expression, and induced reactive oxygen (ROS) abundance to activate NLRP3 inflammasome, finally leading to pyroptotic cell death in macrophages. More importantly, circulating GSDMD had the optimal predicting value for liver fibrosis progression. In conclusion, S100A8-mediated NLRP3 inflammasome-dependent pyroptosis by TLR4/NF-κB activation and ROS production in macrophages facilitates liver fibrosis progression. The identified GSDMD has the potential to be a biomarker for liver fibrosis evaluation.

Absent in melanoma 2 (AIM2) inflammasome-dependent pyroptosis and neutrophil extracellular traps (NETs) have been implicated in chronic liver disease (CLD). However, the specific intrahepatic cell type that undergoes AIM2 inflammasome-dependent pyroptosis and how their interaction augments hepatic inflammation/fibrosis remains unclear. The expression and correlation of AIM2 inflammasome-dependent pyroptosis-related indicators and NETs were analyzed in biopsy tissue and blood specimens from chronic hepatitis patients (CHs). Cell-based experiments were conducted to investigate their interaction. In vitro and in vivo experiments were used to analyze their effects on the progression of hepatic inflammation/fibrosis as well as their clinical importance. Elevated levels of AIM2 inflammasome-dependent pyroptosis indicators and NETs were detected in biopsy tissue and blood specimens. Circulating NETs were positively correlated with pyroptosis-related indicators, and both were related with disease severity. Confocal imaging revealed that AIM2 was mainly localized to hepatic macrophages, indicating that hepatic macrophages were the major cell type that underwent pyroptosis. NETs were directly engulfed by macrophages and then stimulated AIM2 inflammasome-dependent macrophage pyroptosis in vitro, which amplified the activation of hepatic stellate cells (HSCs) and increased collagen deposition. Administration of the NETs degradation agent DNase I or the AIM2 inflammasome activation inhibitor ODN A151 effectively alleviated chronic liver inflammation/fibrosis progression in vivo. NETs-induced AIM2 inflammasome-dependent pyroptosis in macrophages facilitates liver inflammation/fibrosis progression. The identified NET–AIM2 inflammasome cascade could serve as a novel therapeutic target for hepatic inflammation/fibrosis progression.