Title : Chronic stress promotes colorectal cancer liver metastasis via gut barrier disruption–activated intratumoral microbiota–NETs axis
Abstract:
Objective: Chronic stress represents a significant modern health threat, yet its mechanisms for promoting colorectal cancer liver metastasis (CRLM) remain unclear. This study aims to elucidate the complete mechanism by which stress modulates the hepatic pre-metastatic niche via the gut-liver axis, focusing on the core role of gut barrier disruption-induced microbiota-immune interactions, and to reveal the pivotal position of neutrophil extracellular traps (NETs) within this process, thereby providing novel targets for prevention and treatment in high-risk patients.
Methods: Chronic restraint stress (CRS) mouse models were employed, combined with orthotopic colorectal cancer transplantation and spleen injection liver metastasis models. Multi-omics technologies were utilized to systematically analyze the effects of stress on gut barrier function, microbial translocation, and liver immune microenvironment reprogramming. Molecular biology techniques, immunohistochemistry (IHC), and live-cell imaging were used to assess tight junction protein expression, TLR4/MyD88 pathway activation, and NET release dynamics. NETs' causal contribution to metastatic colonization was validated using in vitro tumor-immune cell co-culture systems and in vivo targeted interventions. A clinical study enrolled a CRLM patient cohort to analyze pathological associations between stress biomarkers and spatial NETs distribution in liver tissues.
Results: Chronic stress significantly disrupted the intestinal mucosal barrier, manifested by elevated serum stress hormones, increased intestinal permeability, and widespread downregulation of tight junction proteins. Concomitant with barrier integrity loss, substantial enrichment of gut-derived bacteria and their metabolites was detected in the liver microenvironment, a phenomenon also confirmed in metastatic lesions of high-stress patients. Single-cell transcriptome analysis revealed that translocated microbial metabolites activated the TLR4/ROS signaling axis in liver sinusoidal endothelial cells (LSECs), driving robust expression of chemokines CXCL1/CXCL2, which recruited large numbers of neutrophils with an immunosuppressive phenotype. Mechanistically, bacterial LPS triggered a dramatic accumulation of reactive oxygen species (ROS) within neutrophils via the TLR4/MyD88 pathway, potently inducing NETosis (blocking this pathway significantly suppressed NET formation). NETs shaped the pro-metastatic niche through a tripartite mechanism: ① Remodeling the extracellular matrix to form fibronectin-rich adhesion scaffolds for tumor cells; ② Releasing proteases to directly activate tumor cell invasion programs; ③ Profoundly suppressing cytotoxic T lymphocyte (CTL) function and inducing their apoptosis. In vivo intervention confirmed that clearing NETs or depleting neutrophils markedly reduced stress-induced hepatic metastatic burden. Clinical specimens further demonstrated that NETs density in patient liver tissues positively correlated with psychological stress indicators, and high NETs levels predicted worse metastatic progression.
Conclusion: This study is the first to delineate the complete mechanistic chain of chronic stress driving CRLM: Stress dismantles the gut barrier, leading to pathological translocation of gut microbiota and metabolites to the liver; Gut-derived signals activate the neutrophil NETosis program via the LSEC TLR4/ROS axis; NETs collaboratively establish the hepatic pre-metastatic microenvironment through a tripartite synergistic mechanism—matrix remodeling, tumor cell empowerment, and immune suppression. This discovery not only provides a theoretical breakthrough for stress-associated metastasis but also establishes NETs as a key therapeutic target: NETs levels effectively predict high-risk patients, and strategies targeting NET formation or degradation demonstrated significant efficacy in experimental models. This research lays the molecular foundation for developing precision prevention and treatment strategies for stress-related CRLM and provides a scientific basis for integrating psychological interventions into comprehensive cancer management.
