[Effect and mechanism of taurocholic acid in promoting colorectal cancer liver metastasis by regulating neutrophil extracellular trap formation].

To investigate the effect and underlying mechanism by which taurocholic acid (TCA) promotes colorectal cancer liver metastasis (CRLM) through regulation of neutrophil extracellular trap (NET) formation. A total of 20 CRLM patients and 20 non-metastatic colorectal cancer (non-mCRC) patients admitted to Qilu Hospital of Shandong University from 2021 to 2022 were retrospectively included. The age of the CRLM patients was (57±12) years, including 11 males and 9 females; the age of the non-mCRC patients was (60±9) years, including 15 males and 5 females. Non-targeted metabolomics was employed to identify differential serum metabolites associated with liver metastasis, and the distinguish efficacy of TCA was evaluated by receiver operating characteristic (ROC) curve analysis. Neutrophils from healthy donors were isolated by density gradient centrifugation. The differentiated HL-60 (dHL-60) model was established by inducing the acute promyelocytic leukemia cell line HL-60 to differentiate into neutrophil-like cells with all-trans retinoic acid (ATRA). The expression characteristics of carcinoembryonic antigen-related cell adhesion molecule 8 (CD66b) and integrin subunit alpha M (CD11b) were detected by flow cytometry. The cell morphology and the proportion of live cells were assessed respectively by wright-giemsa staining and trypan blue staining. In neutrophils and dHL-60 models, cells were divided into a negative control group, a phorbol 12-myristate 13-acetate (PMA, 0.5 μmol/L) positive control group and different concentrations of TCA treatment groups (0.01, 0.1, 1, 10 and 100 μmol/L). The formation of the NET reticular structure after stimulation was observed by Sytox Green staining, and the content of double-stranded deoxyribonucleic acid (ds-DNA) released after stimulation was quantitatively evaluated by PicoGreen. Western blotting was used to detect the expression levels of p44/42 mitogen-activated protein kinase (p44/42 MAPK) and its phosphorylated form (p-p44/42 MAPK), mammalian target of rapamycin (mTOR) and its phosphorylated form (p-mTOR), and peptidylarginine deiminase 4 (PAD4) in dHL-60 following stimulation. NET induced by TCA were divided into a control group, a low-dose NET group (0.1 μg/ml) and a high-dose NET group (0.3 μg/ml) according to dose, and co-cultured respectively with colorectal cancer (CRC) cell lines DLD1 and HCT116. Transwell assay was used to analyze the effect of TCA-induced NET on the migration behavior of CRC cells. The expression levels of epithelial cadherin (E-cadherin), neural cadherin (N-cadherin), and vascular endothelial growth factor A (VEGFA) were detected by Western blotting in CRC cells after co-culture. Comparisons of measurement data between groups were performed using the independent samples -test, Mann-Whitney test, and one-way ANOVA. Comparisons of enumeration data between groups were performed using the χ test or Fisher's exact test. The relative abundance of 64 metabolites, including TCA [ (, )] [4.445 (1.669, 9.579) vs 0.956 (0.649, 1.372), <0.01], in the serum metabolic profile of CRLM patients was higher than that of non-mCRC patients (all <0.05), while the relative abundance of 27 metabolites was lower than that of non-mCRC patients (all <0.05). In the ROC analysis distinguishing the 2 sample groups, TCA achieved an area under the curve (AUC) of 0.873 (95%: 0.741-0.984). In cell identification, both peripheral blood neutrophils and dHL-60 models displayed typical neutrophil morphology: nuclear lobulation and cytoplasm rich in fine neutral granules; the positive rates of CD66b/CD11b were>90%, and the cell viability was>90%. In stimulation of peripheral blood neutrophils for 1 h or 3 h, when the TCA stimulation concentration was greater than 0.01 μmol/L, the formation of NET was more than that of the negative control group (all <0.05). After intervention of dHL-60 for 4 h and 6 h, when the TCA stimulation concentration was greater than 0.1 μmol/L, the NET reticular structure was more than that of the negative control (all <0.05). When the TCA stimulation concentration was greater than 10 μmol/L, the ds-DNA release was higher than that of the negative control group (<0.001). The expression levels of p44/42 MAPK, p-p44/42 MAPK, mTOR, p-mTOR, and PAD4 in the TCA treatment group were higher than those in the negative control group (<0.001). In co-culture experiments of NET and CRC cells, after high-dose NET treatment of DLD1 cells, the expression of E-cadherin was lower than that of the control group (<0.001). After high and low-dose NET treatment of HCT116 cells, the expression of E-cadherin was lower than that of the control group (both <0.001). In both CRC cell lines, after high and low-doses of NET intervention, the expression of N-cadherin and VEGFA was higher than that of the control group (all <0.001), and their migration and invasion abilities were higher than those of the control group (all <0.001). CRLM patients exhibit distinct serum metabolic profiles, among which high-abundance TCA can induce NET release and thereby promote CRC cell metastasis. This process is associated with the activation of the p44/42 MAPK/mTOR signaling pathways in neutrophils and the epithelial-mesenchymal transition in CRC cells.