Lipopolysaccharides increase the risk of colorectal cancer recurrence and metastasis due to the induction of neutrophil extracellular traps after curative resection.

Intra-abdominal infection after curative surgery for colorectal cancer is a serious complication associated with an increased risk of recurrence. Lipopolysaccharides (LPS)-an essential component of the cell wall of Gram-negative bacteria-were found to exert a protumorigenic effect by stimulating the inflammatory pathology and formation of neutrophil extracellular traps (NETs). This study was conducted to test whether LPS-induced formation of NETs promotes the development of cancer and metastasis. The clinical characteristics, incidence of relapse, and serum myeloperoxidase-DNA complexes of 40 patients with infection and 40 patients without infection after curative surgery were analyzed. The effects of LPS on the induction of NETs were evaluated in a mouse model of colorectal cancer and liver metastasis. The toll-like receptor 9 (TLR9)-a DNA receptor-was knocked down to assess its effect on the mitogen-activated protein kinase pathway and activities implicated in the formation of NETs. Analysis of the clinical data obtained from these patients showed the significant relation of the formation of NETs and incidence of metastasis and survival rates. Subsequent in vitro experiments revealed an increased level of citrullinated-histone H3 and myeloperoxidase-DNA in LPS-injected mice with colorectal cancer. In the mimic metastatic model, injection of LPS enhanced the metastatic capacity, which was then attenuated by DNase I. This suggested that the formation of NETs was activated by LPS. Injection of TLR9-knockdown HCT116 cells in mice, followed by induction through LPS, mitigated the level of citrullinated-histone H3 and myeloperoxidase-DNA. This finding implied that the formation of NETs was suppressed. These findings shed light on the mechanism underlying the relationship between the elevated rate of colorectal cancer recurrence in patients who underwent surgery and the incidence of infection. This mechanism involves the protumorigenic activities of LPS-induced formation of NETs. The NETs which could be mediated by the TLR9 and the mitogen-activated protein kinase signaling pathway.

Wang WW ，Wu L ，Lu W ，Chen W ，Yan W ，Qi C ，Xuan S ，Shang A ... -  《-》

Neutrophil Extracellular Traps Promote T Cell Exhaustion in the Tumor Microenvironment.

While neutrophil extracellular traps (NETs) are important for directly promoting cancer growth, little is known about their impact on immune cells within the tumor microenvironment (TME). We hypothesize that NETs can directly interact with infiltrating T cells to promote an immunosuppressive TME. Herein, to induce a NET-rich TME, we performed liver Ischemia/Reperfusion (I/R) in an established cancer metastasis model or directly injected NETs in subcutaneous tumors. In this NET-rich TME, the majority of CD4+ and CD8+ tumor infiltrating lymphocytes expressed multiple inhibitory receptors, in addition these cells showed a functional and metabolic exhausted phenotype. Targeting of NETs in vivo by treating mice with DNAse lead to decreased tumor growth, decreased NET formation and higher levels of functioning T cells. In vitro, NETs contained the immunosuppressive ligand PD-L1 responsible for T cell exhaustion and dysfunction; an effect abrogated by using PD-L1 KO NETs or culturing NETs with PD-1 KO T cells. Furthermore, we found elevated levels of sPDL-1 and MPO-DNA, a NET marker, in the serum of patients undergoing surgery for colorectal liver metastases resection. Neutrophils isolated from patients after surgery were primed to form NETs and induced exhaustion and dysfunction of human CD4+ and CD8+ T cells. We next targeted PD-L1 in vivo by injecting a blocking antibody during liver I/R. A single dose of anti-PD-L1 during surgery lead to diminished tumors at 3 weeks and functional T cells in the TME. Our data thus reveal that NETs have the capability of suppressing T cell responses through metabolic and functional exhaustion and thereby promote tumor growth. Furthermore, targeting of PD-L1 containing NETs at time of surgery with DNAse or anti-PD-L1 lead to diminished tumor growth, which represents a novel and viable strategy for sustaining immune competence within the TME.

Kaltenmeier C ，Yazdani HO ，Morder K ，Geller DA ，Simmons RL ，Tohme S ... -  《Frontiers in Immunology》

Targeting neutrophil extracellular traps with thrombomodulin prevents pancreatic cancer metastasis.

Surgery is the only curative treatment option for pancreatic cancer, but patients often develop postoperative recurrence. Surgical invasiveness might be involved in the mechanism of recurrence. The associations among inflammation caused by surgery, neutrophils, and cancer metastasis were investigated. At first, neutrophil extracellular traps (NETs) were examined in clinical specimens, and NETs were observed around metastatic tumors. To explore how NETs were induced, neutrophils were cultured with pancreatic cancer or in cancer-conditioned medium. Neutrophils formed NETs when they were cultured with pancreatic cancer or even its conditioned medium. The effects of NETs on cancer cells were further investigated in vitro and in vivo. NETs induced the epithelial to mesenchymal transition in cancer cells and thereby promoted their migration and invasion. HMGB1 derived from NETs appeared to potentiate the malignancy of cancer cells. In a mouse model of liver metastasis with inflammation, NETs participated in the metastatic process by enhancing extravasation. Interestingly, thrombomodulin degraded HMGB1 and consequently inhibited the induction of NETs, thereby preventing pancreatic cancer metastasis to the liver. In conclusion, NETs interact reciprocally with pancreatic cancer cells, which play a pivotal role in inflammation-associated metastasis. Targeting NETs with thrombomodulin can be a novel strategy to improve the surgical outcome of pancreatic cancer patients.

Kajioka H ，Kagawa S ，Ito A ，Yoshimoto M ，Sakamoto S ，Kikuchi S ，Kuroda S ，Yoshida R ，Umeda Y ，Noma K ，Tazawa H ，Fujiwara T ... -  《-》

Neutrophil Extracellular Traps Promote Metastases of Colorectal Cancers through Activation of ERK Signaling by Releasing Neutrophil Elastase.

Okamoto M ，Mizuno R ，Kawada K ，Itatani Y ，Kiyasu Y ，Hanada K ，Hirata W ，Nishikawa Y ，Masui H ，Sugimoto N ，Tamura T ，Inamoto S ，Sakai Y ，Obama K ... -  《INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES》

Fusobacterium nucleatum-triggered neutrophil extracellular traps facilitate colorectal carcinoma progression.

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. 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. 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. 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.

Kong X ，Zhang Y ，Xiang L ，You Y ，Duan Y ，Zhao Y ，Li S ，Wu R ，Zhang J ，Zhou L ，Duan L ... -  《JOURNAL OF EXPERIMENTAL & CLINICAL CANCER RESEARCH》