The inflammatory response associated with immune cells infiltrating the tumor microenvironment (TME) is thought as an indispensable mediator leading to tumor progression and metastasis along with immunotherapy response [43,44,45,46]. Neutrophils are the predominant circulating leukocytes in humans and are often regarded as a central player in host responses to pathogens. As related studies have progressed, the proportion of neutrophils in the immune infiltration of a variety of tumors, including HCC [47], PDAC [48], CRC [49], and GC [50], has become increasingly evident. They can kill pathogens quickly through phagocytosis, but also by releasing their potent antimicrobial arsenal without phagocytosis, including oxidants [51], granular enzymes, and proteins, as well as NETs [52,53]. The existence of NETs has been identified in the TME of a wide range of solid tumors including GI cancers [54]. G-CSF release into the bloodstream has been reported to contribute to tumor recruitment of neutrophils to form NETs [55]. Furthermore, exocytosis by carcinoma cells, including GI carcinoma cells, triggers IL-8 generation and stimulates NETosis by neutrophils [56,57]. However, there is relatively little straightforward assessment of collaterals for NETs and other immune cells in the TME. NETs can inhibit the suppressive effect of immunity in the surrounding microenvironment on tumor cells by acting as a physical barrier and as a chemical that suppresses immune cells. It has become increasingly clear that the tumor-associated neutrophil (TAN) N1 type releases immunostimulatory or pro-inflammatory cytokines, comprising CXCL10, CCL3, and tumor necrosis factor (TNF)-α, which promote CD8+ T cell recruitment and activation. TGF-β in the tumor microenvironment invokes tumor-promoting N2-TAN and blocks the TGF-β-induced tumor suppressor N1-TAN [58]. TAN N1 has also been reported to produce NETs to surround malignant tumor cells, thereby protecting them from CD8+ T cell- and NK cell-mediated cytotoxicity [59]. Time-lapse confocal microscopy has shown that NETs block contact between cancer cells and cytotoxic immune cells in subcutaneous cancer in a mouse model. Furthermore, the effect of NETs may go beyond the physical barrier of cancer cells and may involve deleterious effects of mediators derived from neutrophils on NK cells and/or CTLs. However, N2-TAN is more common in the TME. Similarly to tumor-associated macrophages (TAM), N2 TAN can act by producing pro-tumor factors and impacting other cells of the immune system by immunosuppressive means, such as by triggering T cell tolerance [60]. Therefore, tumor growth and metastasis [61,62] can be suppressed and the level of immunosuppression in the TME can be reduced by depleting N2-polarized TANs, thereby increasing the activity of CD8 cytotoxic T-lymphocytes (CTLs) [20].

NETs can promote tumor growth by triggering the exhaustion of T cells. De Andrea et al. found a negative association between CD8+ T-cell density and the NET area in tissue microarrays of bladder and NSCLC cancer after adding CD8 dye to the multiplex panel [63]. Furthermore, a study conducted by Kaltenmeier revealed that a NET-rich environment induces and accelerates the growth of metastatic tumors and promotes an exhausted phenotype in T cells. Furthermore, it demonstrated that NETs can suppress T-cell responses via being metabolized and functionally depleted [64]. During hepatic ischemia-reperfusion injury (IRI), neutrophils can facilitate the recruitment and activation of CD8+ T cells via different cytokines and chemokines expressed in the chromatin of activated neutrophils [65]. NETs have been found to contain the immunomodulatory protein programmed death ligand 1 (PD-L1), which binds to PD-1 on activated T cells, rendering them inactive and depleted. These new findings may have clinical significance to overcome T-cell depletion and tumor progression by aiming at neutrophils and NETs [64,66].

NETs and cancer-associated fibroblasts (CAFs) promote each other and work together to support the occurrence of cancer. CAFs are not only the major component of the tumor microenvironment but also an important factor in GI tumor metastasis. They may maintain tumorigenesis by modulating the immune response to some extent [67,68].

There is a certain relationship between NETs and gastric cancer. The formation of NETs was first identified in the microenvironment of GC tissues by Yiyin Zhang et al. NETs levels and neutrophil buildup was reduced in peripheral blood (PB) from neoplastic to paraneoplastic tissues. Higher levels of peripheral blood NETs at baseline are related to poorer progression-free survival (PFS). Their study also showed that NETs have better tumor serodiagnostic capabilities than routine biomarkers, like carbohydrate antigen 19-9 (CA19-9) and carcinoembryonic antigen (CEA) in GC. These suggest that NETs have a significant role in oncogenesis and tumor growth in GC [74].

NETs may promote tumor development by inducing epithelial–mesenchymal transition (EMT) and promoting vascular remodeling. It has been observed that NETs can impair endothelial cells so that trapped neoplastic cells may disseminate and form neo-metastases following adhesion to activated endothelial cells [75]. Specifically, NETs may promote tumor spread and metastasis in two ways. The first way was revealed that NETs could promote GC cell metastasis via EMT. EMT is a critical stage in the pathogenesis of GC related to spreading and metastasis [76]. This study also demonstrated that NET formation was significantly upregulated in GC patients, and its increased levels were consistent with the increased tumor stage [77]. The detailed process might be that GC enables neutrophils to induce NETosis, and then NETs were deposited in GC tissue or adhered to epithelial cancer cells, specifically without interfering with cell proliferation and the cell cycle. Another way NETs stimulate tumor initiation and progression is by affecting the expression of the ANGPT2 gene in endothelial cells. Shifeng Yang et al. found that NETs promoted ANGPT2 overexpression in human umbilical vein endothelial cells. ANGPT2 is a member of the angiopoietin (Ang) family [78], which modulates vascular remodeling and tumor growth in many pathologic situations via its differential influence on TIE2 signaling. In this study, they found that ANGPT2 overexpression in GC is not only related to poor prognosis, but may also regulates multiple biological functions. Targeting neutrophils/NETs and ANGPT2 may be a future novel approach to antitumor therapy [79].

Furthermore, NETs may affect tumor development by regulating RNA expression. Numerous studies have revealed that RNA has emerged as a direct mechanism for the transformation of healthy cells into tumor cells, which has a critical function in cancer diagnosis and prognosis [80,81], especially in GC. For example, miR-96-5p has been revealed to promote GC cell growth by direct inhibition of FOXO3 expression [82]. A mechanism study revealed that NORAD overexpression could facilitate GC cell growth by modulating the miR-608/FOXO6 pathway [83]. A recent study comprehensively analyzed the genes that are differentially expressed in GC cells treated with NETs and verified the clinical implications of NEAT1-related signaling. RNA interference of NEAT1 was found to inhibit NETs-induced AGS cell invasion, suppress miR-3158-5p expression, and enhance RAB3B expression [84]. RAB3B was positively associated with tumor staging, and miR-3158-5p was negatively associated with tumor staging. However, more basic and clinically intensive experiments to uncover the modulation of NETs by ceRNA networks in GC are urgently needed. Apart from direct contact with tumor cells, NETs could be engaged in abnormal clotting in patients with gastric cancer. NETs levels have been observed to markedly correlate with D-dimer and thrombin–antithrombin complex levels in GC patients, indicating that NETs may be involved in facilitating blood coagulation in GC progression [85]. Venous thromboembolism (VTE) is a common complication among GC patients [86,87] and is associated with high mortality [88,89]. An increase in activated platelets may lead to increased thrombus formation [90]. A recent study found that NETs induced a hypercoagulable state of platelets by upregulating the expression of P-selectin and phosphatidylserine on the cells. Compared with control mice in the inferior vena cava stenosis model, massive accumulation of NETs in tumor mouse thrombi greatly enhanced the ability to form thrombi.

There are currently a series of corresponding treatments for the etiology of abnormal blood coagulation in gastric cancer patients caused by NETs. Of note, the combined use of sivelestat, deoxyribonuclease I, and activated protein C significantly abolished the procoagulant activity (PCA) of NETs [91]. Abnormal blood coagulation in cancer can be treated not only by inhibiting the procoagulant activity of NETs but also by reducing the production of NETs. Salvia miltiorrhiza (Danshen) is a plant used medicinally for the treatment of cancer [92], and its root extract can prevent neutrophils from transferring to metastatic sites by inhibiting the activities of MPO and NADPH oxidase (NOX), thereby obstructing the formation of NETs [93]. This was also validated by the latest research that showed that one of the bioactive polyphenols extracted from Danshen called SAA, can attenuate oxidative stress, inflammation, and neutrophil NETosis to ameliorate acute lung injury [94]. Compared with traditional anticancer drugs, tumor nanotherapeutics have various unique advantages. A recent study used Abraxane/human neutrophil (NE)-cell-based neoadjuvant chemotherapy with radiation therapy for efficacious cancer management. This cellular drug-based approach to adjuvant radiotherapy increased the release of inflammatory factors that led to NE homing to the tumor site while destroying the tumor. Explosive release of Abraxane triggers remarkable cancer suppression after excessive activation of Abraxane/NEs to generate NETs at the neoplastic regions [95].

All in all, NETs can directly contact tumor cells, regulate gene expression to promote tumor development, and may also be involved in abnormal blood coagulation in patients with gastric cancer, leading to aggravation of symptoms. The multiple action pathways of NETs also provide directions and means for corresponding drug development and treatment.

A positive feedback effect between CRC (colorectal cancer) progression and NET formation has been identified and several mechanisms have been proposed to trigger the formation of NETs in the CRC microenvironment. For example, in KRAS-mutated CRC cells, potential prognostic and predictive markers for CRC [96,97] have been shown to accelerate the deterioration of CRC by promoting NET production through exosomal activation of neutrophils [97]. Furthermore, tumor cell-driven IL-8 expression can recruit and activate neutrophils to generate more NETs, thereby aggravating CRC progression [57]. The link between KRAS and IL-8 is that CRC cells may transfer mutated KRAS to neutrophils via exosomes, thereby promoting NETs formation by modifying IL-8 and ultimately leading to CRC exacerbation. Another study suggested that poly P expressed by CD68+ mast cells in CRC was one of the factors that triggered the release of NETs from neutrophils. Therefore, the detection of CD68+ poly P-expressing mast cells may also represent another promising prognostic marker like KRAS-mutated CRC cells in CRC [98].

Advanced CRC is prone to distant metastasis, the most common site of which is the liver [99]. NETs contribute to the metastasis of colorectal cancer. Numerous studies have shown that approximately 25–30% of CRC patients will develop coetaneous liver metastasis, most of which show a marked increase in NET formation [100,101]. In a mouse model of hepatic IR injury, it was found that increased levels of postoperative NETs promote the progression of liver metastasis and were associated with decreased disease-free survival in a cohort of patients with colorectal liver metastases who underwent curative hepatectomy [102]. IL-8 overexpression activated neutrophils to form NETs, forming a positive loop that boosts CRC liver metastasis [103]. NETs induce colorectal cancer liver metastasis by interacting with some molecules that promote colorectal cancer metastasis. Amino acids 21–25 at the extracellular N-terminus of CCDC25 (a transmembrane protein expressed in CRC cells) were found as the binding sites for NET-DNA by Yang et al. in their remarkable study aimed to explain why CRC cells have the propensity to spread to the liver. Liver metastasis of CRC cells can be induced by initiating the β-Parvin–RACI–CDC42 pathway to enhance cancer cell motility [104]. Another newly identified molecule that promotes metastasis in colorectal cancer is carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), a cell adhesion molecule expressed on endothelial cells. Blocking CEACAM1 on NETs, or knocking it out in mouse models, resulting in a more than 50% reduction in colon cancer cell adhesion and migration [105]. In addition, using high-resolution stimulated emission depletion (STED) microscopy, Antonia M et al. found that citrullinated NETs were significantly associated with high histopathological tumor grades and lymph node metastasis. Their findings suggested that NETs activate an epithelial–mesenchymal transition-like process in CRC cells and may play a crucial role in the metastatic progression of CRC [106].

After realizing the possible role of NETs in colorectal cancer metastasis, some studies further verified this relationship and explored new directions for future treatment by inhibiting the formation of NETs. Using preclinical murine models of lung and colon cancer combined with in vivo video microscopy, Rayes et al. found that NETs functionally contributed to metastatic progression and that blocking NETosis through multiple measures markedly inhibited spontaneous metastasis to the lung and liver [100]. DNase I can disrupt NET formation by cleaving DNA strands. Xia et al. used an adeno-associated virus (AAV) gene therapy vector which specifically expressed DNase I in the liver. NET formation was significantly inhibited in tumor tissues with AAV-DNase I treatment. This result demonstrated that AAV-mediated DNase I liver gene transfer can be a potential therapeutic target for preventing CRC metastasis [101].

It is well known that cancer-related thrombosis is strongly associated with poor prognosis, and patients with CRC are generally at higher risk for venous thrombosis. NETs, as intermediate substances, also mediate the mutual promotion of cancer and blood coagulation. A study revealed a novel link between coagulation, neutrophilia, and complement activation, in which NETs are also important factors. It found that elevated circulating lipopolysaccharide (LPS) induced the upregulation of complement C3a receptor on neutrophils and activation of the complement cascade, which led to NETosis, induction of N2 polarization, and coagulation, thus accelerating tumorigenesis. This provides a favorable explanation for the promotion of tumor development through coagulation [107]. On the other hand, the occurrence of cancer cells can also further promote blood coagulation. Cancer cells may promote NET formation through TLR9 and mitochondria-activated protein kinase signaling in LPS-injected CRC mice [108]. The generation of NETs in cancer patients resulted in a marked increase in thrombin–antithrombin (TAT) complexes and fibrin fibers [109]. This eventually led to a shortened neutrophil clotting time in colorectal cancer patients compared with healthy controls. Knowing the relationship between NETs and thrombus in cancer patients, we can use NETs and substances related to them as biomarkers. For instance, Brice et al. first showed that circulating DNA (cirDNA) might appear as a marker of NETs and speculated that a significant portion of the cirDNA amount is derived from NETs in metastatic colorectal cancer (mCRC) patients. High levels of antiphospholipid antibodies (aPL) in the plasma may trigger thrombosis in cancer patients [110]. Furthermore, a study showed for the first time that aPL was closely associated with NET markers and cirDNA in cancer patients, suggesting that examining these markers may help prevent blood clots in cancer patients [111].

The two key immune cells of the liver that contribute to the capture of pathogens are neutrophils and Kupffer cells [112]. Although Kupffer cells play a key role in antibacterial defense, in some cases Kupffer cells after capturing bacteria can also become potential pathogens [113]. However, NETs released by neutrophils can prevent the escape of infected Kupffer cells. Since neutrophils lack CRIg receptors, they cannot directly capture bacteria in the blood, but they can directly capture and kill bacteria by releasing NETs. While NETs can kill bacteria, excessive formation of NETs can lead to worsening of inflammation. Several factors are associated with the development of HCC, including hepatitis B or C virus, metabolic disorders, nonalcoholic steatohepatitis, or alcohol intoxication, with some of these etiologies are potentially associated with NETs [114]. NETs have been found to contribute to the pathogenesis of autoimmune liver disease, non-alcoholic steatohepatitis (NASH), and other chronic liver diseases [115], which are closely related to the occurrence of HCC. It suggests that NETs may play a significant role in HCC development.

NASH is a progressive, inflammatory fatty liver disorder and one of the risk factors for HCC. The chronic inflammation caused by steatosis through production of NETs plays an important role in its pathogenesis. Studies have found that elevated free fatty acids such as linoleic acid and palmitic acid in NASH can stimulate liver-infiltrating neutrophils to form NETs, which then cause monocyte-macrophage infiltration and increases in inflammatory cytokines such as IL-6 and TNF-α. Increased inflammatory cytokines affect the tumorigenic, inflammatory microenvironment of the liver and participate in the occurrence and development of HCC. Furthermore, a study has shown that the production of inflammatory cytokines is caused by an increase in NET-activated TLR4 signaling. The active TLR4 signaling induced naive T cells to differentiate into Tregs. The differentiation of naive T cells into Tregs led to the malignant transformation of epithelial cells and the occurrence of liver cancer [116]. That is to say, fatty liver can lead to the formation of NETs, affect the inflammatory microenvironment, and promote the occurrence of liver cancer. Consistent with the findings of this study, inhibition of NETs by DNase treatment or PAD4 gene knockout did not affect the progression of fatty liver but may slow the growth of HCC [117]. Along the same line, NETs enriched with IL-1β and IL-17A were found to participate in the hepatic inflammatory process of patients with NASH [118]. Patients with NASH are at high risk for venous thromboembolism and high morbidity and mortality. Therefore, a deeper study of the neutrophil/NETs/IL-1β/IL-17α axis in both upstream and downstream of polarized Th17 cell-driven adaptive immune responses is warranted.

NETs can not only affect the local inflammatory microenvironment in NASH patients, but may also lead to the hypercoagulable state of blood in patients. A recent study found that plasma levels of NET markers were considerably higher in NASH patients than in healthy controls. NETs exert cytotoxic effects on endothelial cells, rendering them with a pro-coagulant and pro-inflammatory phenotype [119]. Subsequently, Armando also found that the release of NETs in non-alcoholic fatty liver disease (NAFLD) patients may enhance the hypercoagulant state of NASH patients [120]. These discoveries led to the recognition that NETs may have a critical role in the procoagulant events of NASH. According to the above studies, we realized the influence of NETs on the inflammation and blood coagulation of NASH patients and that NETs may induce hepatocellular carcinoma, which provides an optimal treatment strategy to reduce inflammation and prevent hepatocellular carcinoma in NASH patients or reduce the pathological hypercoagulation state of NASH patients.

Many studies have illustrated that neutrophils and the NETs they produce play complex roles in the pathophysiology of alcoholic liver disease [121,122]. Excessive alcohol consumption can stimulate neutrophils and promote the production of NETs leading to pathological damage and even liver cancer. Recently, Yang Liu analyzed potential molecular mechanisms that might engage the migration of bacterial metabolites from the intestine to the liver and the activation of NETs [123]. They found that NETs formed after LPS activation of TLR4 following chronic alcohol consumption caused elevated alcoholic steatosis, which consequently led to HCC. Hepatitis virus infection is widely known as a common cause of liver cancer. The pathogenic mechanism of hepatitis virus infection is related to NETs. A study revealed that the hepatitis B virus (HBV) can suppress NETs release by modulating the production of ROS and autophagy to evade the immune system and promote the establishment of chronic infections [124]. In addition to causing chronic infection, HBV may also promote the growth and metastasis of HCC cells. According to recent findings published in Cancer Letters, HBV used NETs to exacerbate the progression of hepatocellular carcinoma. Specifically, HBV induced S100A9 to activate the RAGE/TLR4–ROS signaling pathway to allow massive NET formation [125]. In addition, based on the mechanism of HBV aggravating hepatocellular carcinoma metastasis, the prediction of HBV-associated extrahepatic metastasis of HCC can use NETs as a biomarker in the future. In chronic viral hepatitis, the infection is promoted by inhibiting the function of NETs, while in fulminant viral hepatitis (FVH), NETs may aggravate liver damage. FVH is a life-threatening disease, but its pathogenesis is not fully understood. The study by Li et al. proposed a mechanism whereby in FVH, NETs exacerbate FVH liver injury by promoting fibrin deposition and inflammation. They illustrated that the formation of NETs was regulated by the fibrinogen-like protein 2 (Fgl2)–mucolipin 3–autophagy axis [126]. Due to the remarkable heterogeneity of human viral hepatitis syndromes and pathogens, the applicability of their study to human disease is unclear. We look forward to more nuanced studies to clarify the emerging role of NETs in FVH.

NETs are closely related to the development of HCC. Van der Windt et al. used neonatal streptozotocin and a high-fat diet to induce a NASH–HCC mouse model [117]. At 20 weeks, a large number of cancer cells accumulated on the liver surface of all male mice. After inhibiting the formation of NETs with drugs or knocking out the PAD4 gene, the size and number of cancer cells on the liver surface were significantly reduced. This preliminarily suggested that the expression of NETs played a role in promoting the development of HCC. Furthermore, NETs were found to promote the development of HCC by affecting the inflammation and invasion ability of liver cancer, and the pathways affected by NETs were further studied. Yang et al. found that, compared with healthy people, serum NET marker MPO-DNA levels were increased in HCC patients, and it was more significant in patients with metastatic liver cancer. Through in vitro research, they confirmed that the increased expression level of NETs can enhance the cytotoxicity and invasiveness of liver cancer cells. NETs induced the inflammatory response of HCC cells by up-regulating COX2, thereby activating the TLR4/9 signaling pathway, and enhancing the metastasis ability of liver cancer cells. This result suggests that NETs can promote tumor inflammation and liver cancer metastasis [127]. In addition, subsequent research illustrated that NETs could also downregulate tight junction molecules on adjacent endothelial cells, thereby facilitating tumor infiltration and metastasis [128]. In addition, Xiangqian found that NETs-associated cathepsin G (cG) facilitated HCC metastasis in vitro as well as in vivo. Clinically, the expression of cG protein in tumor tissues was closely related to the prognosis of HCC patients [129]. From the above three molecular signals and signaling pathways related to NETs, we can see that the generation of NETs is accompanied by the regulation of various signaling molecules and signaling pathways, which jointly affect the occurrence and development of HCC. Furthermore, a clinical retrospective investigation found that higher NET levels in preoperative sera were associated with shorter relapse-free survival/overall survival in HCC patients [130]. These results suggest a role for NETs as a prognostic factor in patients with liver malignancies.

The fundamental impact of mitochondrial metabolism on all steps of tumorigenesis (i.e., malignant transformation, tumor progression, and response to therapy) is widely recognized [131]. The role of NETs in the relationship between mitochondria and tumors has also been preliminarily recognized. NETs can enhance the function of mitochondria, which can then promote cancer cell growth. CRC research has found that NETs can directly stimulate Hepa1-6 and Huh 7 cell proliferation by enhancing mitochondrial function and biogenesis in vitro [132]. Neutrophil elastase (NE) released by NETs activated TLR-4 on cancer cells, inducing PGC-1α upregulation, increased mitochondrial biogenesis, and accelerated growth. The interaction between mitochondria and NETs is bidirectional, and mitochondria can also affect the state of cancer cells by affecting the formation of NETs. A recent study found that neutrophils in HCC patients contained high levels of mitochondrial ROS and formed NETs enriched with oxidized mtDNA in a mitochondrion-adherent manner. Hepatocellular carcinoma can stimulate NETs rich in oxidized mtDNA, which play a crucial role in promoting inflammation and metastasis [133]. Related to this mechanism, a new finding demonstrated that mesenchymal stromal cell-derived extracellular vesicles (MSC-EVs) have a nanotherapeutic effect [134]. They can inhibit the formation of local NETs by transferring functional mitochondria to intrahepatic neutrophils and repairing their mitochondrial function. The restricted synthesis of NETs can further prevent and limit the inflammation and metastasis of HCC. That is to say, after recognizing the relationship between NETs and mitochondria in promoting hepatocellular carcinogenesis, we can use NETs and overexpressed mitochondria or even the mediators of their interaction as therapeutic targets.

Notably, the NP-neutrophil targeting approach can be used to prevent NET formation in GI cancers [135]. Some recent studies, which revealed the relationship between nanoparticles (NPs) and liver injury [136], used nanoparticles to adjust the level of NETs to prevent and treat liver inflammation. A study demonstrated that zinc oxide nanoparticles (ZnO-NPs) could elevate the levels of malondialdehyde (MDA), decrease superoxide dismutase (SOD) levels, and increase the levels of NETs in the liver of crucian carp [137]. In addition, DNase I can prevent ZnO–NPs-induced liver injury, which provides new insights into the immunotoxicity of ZnO–NPs in fish. DNase I is a key enzyme in the degradation of NETs induced by NPs and the alleviation of liver inflammation also was found in the human liver. Some studies also used nanoparticles (NPs) to further reveal the relationship between NETs and liver inflammation. Qianru Chi et al. have revealed the mechanism underlying NETs formation in polystyrene nanoparticle (PSNP) exposure-induced liver inflammation [138] providing an applicable and efficient target for anti-cancer therapies.

Although the use of nanoparticles and other strategies combined with NETs provides a useful direction for clinical anticancer treatment, it has not yet been put into clinical practice, and cancer surgery or liver transplantation is still relatively common. In these surgical operations for liver cancer, hepatic ischemia-reperfusion injury (IRI) often occurs [139]. Recent studies have implicated NETs in the pathogenesis of I/R. Injury-associated molecular patterns such as histones and HMGB1 protein are released from damaged hepatocytes and induce NETs formation by activating neutrophil TLR4 and TLR9 [140]. Using a mouse model of hepatic IRI, it was found that induction of NETs can further activate platelets, leading to systemic immune thrombosis and distal organ damage [141,142]. There are corresponding strategies for this. Researchers found that the level of NETs in mice was reduced and liver damage was improved by pretreating mice undergoing I/R with allopurinol and N-acetylcysteine, which aimed to reduce circulating superoxide [143]. These results suggest that antioxidant therapy may prevent liver I/R by attenuating NETs formation. In addition, there are some biomolecules and related signaling pathways that can be used to inhibit the formation of NETs to suppress the degree of IRI damage. A recent study found that human thrombomodulin (rTM) significantly inhibited the formation of NETs by neutrophils through blocking Toll-like receptor 4 and the downstream extracellular signal-regulated kinase/c-Jun NH2 terminal kinase and nicotinamide adenine dinucleotide phosphate (NADPH)/reactive oxygen species/peptidyl arginine deiminase 4 signaling pathways. This effect contributed to the reduction in hepatocyte apoptosis, alleviation of rat liver IRI, and improvement of liver function [144].

Pancreatic cancer is often associated with poor prognosis, in which neutrophils are abnormally recruited into the tumor microenvironment, leading to tumorigenesis [145]. Significantly increased NET formation and decreased NETs degradation were observed in the serum of PDAC patients [146]. The etiology of PDAC patients may be related to abnormally high NETs in the body. More specifically, the amount of NETs is negatively associated with recurrence-free survival and OS rate and could serve as a separate factor to evaluate the prognosis of PDAC patients [147]. A study found that the mechanism of the role of NETs in pancreatic cancer is related to the overexpression of the tissue inhibitor metalloproteinase-1 (TIMP1), whereas abrogation of NETs formation or TIMP1 expression was associated with prolonged survival [148]. This effect depended on the interaction of TIMP-1 with its receptor CD63 and subsequent induction of MEK/ERK signaling. Notably, we can use signaling molecules in this pathway leading to pancreatic cancer as prognostic targets. Plasma levels of TIMP-1 and NETs combined with the clinically established marker CA19-9 have a better prognostic value than CA19–9 alone as TIMP1 overexpression and NET formation are inseparably associated with PDAC progression [149]. This pioneering study is very important because TIMP1 has been proposed as a potential serum marker to detect early stages of familial PDAC [150], which allows for more precise prognoses. In addition, lysine(K)-specific demethylase 6A (KDM6A) is a frequently mutated tumor suppressor gene in PDAC. A recent study analyzed the effects of KDM6A loss on the immune microenvironment of PDAC tumors. They demonstrated that KDM6A-deficient PDAC cells alter the immune microenvironment by increasing CXCL1 secretion and neutrophil recruitment. Loss of KDM6A was associated with increased tumor-associated neutrophils and NET formation. Their study provides evidence for targeting the CXCL1–CXCR2 signal axis in low-KDM6A tumors [151].

最近的研究表明，NET在PDAC进展和炎症相关转移中起着新兴的作用。正如NETs促进胃癌和结直肠癌等细胞的增殖一样，NETs也会促进胰腺癌细胞的增殖。最近，发现NET相关的IL-1β通过EGFR-ERK途径参与胰腺癌的EMT过程[152]。NETs还可以通过激活胰腺星状细胞来促进肿瘤增殖，胰腺星状细胞通过与RAGE受体相互作用形成致密的纤维基质[153]。此外，NET的形成有利于肿瘤细胞的转移。一项研究发现在转移性肿瘤周围会产生NETs。同一研究进一步表明，NET诱导肿瘤细胞的EMT，从而促进其迁移和侵袭。血栓调节素可通过降解NETs来源的HMGB1来减轻癌细胞的恶性肿瘤，并防止胰腺癌向肝脏转移[154]。这进一步证实了NETs在肿瘤细胞转移中的作用。NET还可以作为作用于PDAC的其他生物分子信号通路中的中间物质。大量研究表明，DDR1在PDAC中的表达与临床结局呈负相关[155]，可能在PDAC中胶原蛋白驱动的肿瘤发生中起关键作用[156，157]。Jenying Deng等人确定胶原蛋白诱导的CXCL5产生是通过DDR1 / PKCθ / SYK / NF-κB信号通路介导的。CXCL5通过诱导TAN形成NETs来促进癌细胞侵袭和转移[158]。瓜氨酸组蛋白精氨酸脱亚胺酶4（PAD4）在NET释放中至关重要[159]。在异种移植小鼠模型中，PAD4抑制剂GSK484治疗可减少NET形成并完全抑制胰腺肿瘤生长[73]。

尽管胃肠道癌症的治疗策略取得了进展，但恶性肿瘤的手术切除仍然是主要的治疗方法。随后的手术应激是抑制免疫力和影响胃肠道患者预后的关键因素之一[166，167]。尽管几十年来已经知道增加肿瘤复发风险的手术损伤，但治疗失败的潜在机制仍然知之甚少。中性粒细胞作为手术应激后的一线应答者，可能在炎症和癌症进展中起关键作用[168，169]。具体来说，中性粒细胞可能通过NET的形成诱导手术应激。体外研究观察到癌细胞与NET结构的粘附，表明手术应激的部分不良反应可能是由于NET的形成[170]。临床研究的相关统计数据也表明，NETs产生的增加与术后并发症有关，例如住院时间延长和死亡率增加[171]。

至于NETs影响手术压力的机制，NETs可能与凝血系统相互作用，导致手术结果不佳。张宏基致力于手术压力后的NETs研究。他的小组发现，手术后中性粒细胞的涌入可以促进肿瘤捕获和生长[172]。手术应激可能通过免疫血栓形成促进肿瘤转移，免疫血栓形成是由NETs与凝血系统相互作用引起的[173，174]。Seth等人的研究是最早的实验研究之一，表明手术通过凝血依赖性机制促进肿瘤转移[175]。张宏基还证明NETs可以在体内通过血小板TLR4激活血小板[142]。NETs具有激活血小板引起血栓的能力，因此作为中间物质参与导致血栓的各种途径。Zhang随后的研究表明，血小板TLR4-ERK5轴可以促进循环肿瘤细胞的捕获和手术应激后的远处转移，这一过程是由NET介导的[176]。他们最近的研究发现，HMGB1通过TLR4激活的血小板影响NET的形成[103]。HMGB1不仅通过TLR4与血小板结合，还激活肿瘤细胞中TLR9依赖性途径以产生中间NET[177，178]。NETs形成的增加可以加速手术应激后的癌细胞粘附，增殖，侵袭和迁移。

由于手术仍然是转移性疾病患者最合适且唯一有希望的治疗方法，我们可以做的是更好地了解与术后肿瘤复发相关的潜在机制，并防止复发和情况恶化。综上所述，NET是引发手术压力和术后复发的关键因素。因此，利用NETs作为治疗靶点来治疗手术压力可能是一种有效的治疗选择。使用脱氧核糖核酸酶抑制NET是进一步临床应用的潜在方法。在手术应激和肝转移的小鼠模型中，DNase I抑制NET形成已被证明可以减少术后转移的发展[103]。重组人DNA酶（rhDNase）既往曾用于一项安慰剂对照的随机试验，用于治疗自身免疫性系统性红斑狼疮患者[179]。rhDNase给药耐受性良好，无显著不良事件。这一结果表明，DNA酶可能会降低接受转移性癌症切除术的患者复发风险。