Liver metastasis is the leading cause of mortality in patients with colorectal cancer. Given the significance of both epithelial–mesenchymal transition (EMT) of tumor cells and the immune microenvironment in colorectal cancer liver metastasis (CRLM), the interplay between them could hold the key for developing improved treatment options. We employed multiomics analysis of 130 samples from 18 patients with synchronous CRLM integrated with external datasets to comprehensively evaluate the interaction between immune cells and EMT of tumor cells in liver metastasis. Single-cell RNA sequencing analysis revealed distinct distributions of nonmalignant cells between primary tumors from patients with metastatic colorectal cancer (mCRC) and non-metastatic colorectal cancer, showing that Th17 cells were predominantly enriched in the primary lesion of mCRC. TWEAK, a cytokine secreted by Th17 cells, promoted EMT by binding to receptor Fn14 on tumor cells, and the TWEAK–Fn14 interaction enhanced tumor migration and invasion. In mouse models, targeting Fn14 using CRISPR-induced knockout or lipid nanoparticle–encapsulated siRNA alleviated metastasis and prolonged survival. Mice lacking Il17a or Tnfsf12 (encoding TWEAK) exhibited fewer metastases compared with wild-type mice, while cotransfer of Th17 with tumor cells promoted liver metastasis. Higher TWEAK expression was associated with a worse prognosis in patients with colorectal cancer. In addition, CD163L1+ macrophages interacted with Th17 cells, recruiting Th17 via the CCL4-CCR5 axis. Collectively, this study unveils the role of immune cells in the EMT process and identifies TWEAK secreted by Th17 as a driver of CRLM. Significance: TWEAK secreted by Th17 cells promotes EMT by binding to Fn14 on colorectal cancer cells, suggesting that blocking the TWEAK–Fn14 interaction may be a promising therapeutic approach to inhibit liver metastasis.

Gut microbiota and the tumor microenvironment are thought to be critical factors that modulate the processes of liver diseases, including hepatocellular carcinoma (HCC). Interleukin-25 (IL-25) promotes type 2 immunity via alternative activation of macrophages, and is closely associated with inflammation-related diseases, even malignancies. However, it is not clear which role IL-25 plays in the development of HCC, and whether gut microbiota are involved. IL-25 was detected by ELISA, Western blotting (WB), and immunohistochemistry. Chemokines were measured by RT-qPCR and WB. After co-culture with IL-25-stimulated macrophages, the cell growth, migration, invasion and EMT marker of HCC cell lines (MHCC97L and HepG2) were evaluated by Brdu proliferation, Transwell assays and WB. An antibody neutralization assay of chemokine CXCL10 was performed to confirm its role in HCC development. Furthermore, the effects of IL-25 in HCC were investigated in vivo. Dysbiosis of gut microflora was induced by antibiotics (vancomycin, cefoperazone or combination of ampicillin, neomycin, metronidazole, and vancomycin). We used feces suspension to treat colonic epithelial NCM460 cells, and detected IL-25 and tuft cell marker DCLK1 using WB and immunofluorescence staining. We found that the level of IL-25 was significantly elevated in HCC patients, and was negatively correlated with survival rate after hepatectomy. However, IL-25 did not directly promote the development of HCC cells. Then, we observed the significant positive correlation between IL-25 level and M2 percentage (CD206/CD68) in HCC tumors. In vitro and in vivo, IL-25 induced alternative activation of macrophages promoted HCC cell migration, invasion and tumorigenesis, increased the expression of vimentin, Snail and phospho-ERK, and decreased the expression of E-cadherin in HCC cells. After IL-25 treatment, chemokine CXCL10 was increased in macrophages. Neutralizing CXCL10 in macrophage-conditioned medium reversed the IL-25-mediated effect on HCC cells. Vancomycin-induced dysbiosis promoted the growth of orthotopic HCC homograft. Surprisedly, we found the hyperplasia of colonic epithelial tuft cells, from which more IL-25 was secreted . IL-25 promotes the progression of HCC through inducing alternative activation and CXCL10 secretion of macrophages in tumor microenvironment, and IL-25 secretion may partly result from hyperplastic epithelial tuft cells in colon, induced by gut microbiota dysbiosis.

Objective: Intrahepatic cholangiocarcinoma (ICC) exhibits very low response rate to immune checkpoint inhibitors (ICIs) and the underlying mechanism is largely unknown. We investigate the tumour immune microenvironment (TIME) of ICCs and the underlying regulatory mechanisms with the aim of developing new target to inhibit tumour growth and improve anti-programmed cell death protein-1 (PD-1) efficacy. Design: Tumour tissues from patients with ICC together with hydrodynamic ICC mouse models were employed to identify the key cell population in TIME of ICCs. Functional analysis and mechanism studies were performed using cell culture, conditional knockout mouse model and hydrodynamic transfection ICC model. The efficacy of single or combined therapy with anti-PD-1 antibody, gene knockout and chemical inhibitor were evaluated in vivo. Results: Polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) are enriched in advanced ICCs and significantly correlated with N ⁷ -methylguanosine tRNA methyltransferase METTL1. Using diverse in vivo cancer models, we demonstrate the crucial immunomodulator function of METTL1 in regulation of PMN-MDSC accumulation in TIME and ICC progression. Mechanistically, CXCL8 in human and Cxcl5 in mouse are key translational targets of METTL1 that facilitate its function in promoting PMN-MDSC accumulation in TIME and ICC progression in vivo. Co-blockade of METTL1 and its downstream chemokine pathway enhances the anti-PD-1 efficacy in ICC preclinical mouse models. Conclusions: Our data uncover novel mechanisms underlying chemokine regulation and TIME shaping at the layer of messenger RNA translation level and provide new insights for development of efficient cancer immunotherapeutic strategies.

The occurrence and development of hepatocellular carcinoma (HCC) depends largely on such non-tumor factors as inflammatory condition, immune state, viral infection and liver fibrosis. Various inflammation-based prognostic scores have been associated with survival in patients with HCC, such as the neutrophil/lymphocyte ratio (NLR), the platelet/lymphocyte ratio (PLR) and the prognostic nutritional index (PNI). The aspartate aminotransferase/platelet count ratio index (APRI) is thought to be a biomarker of liver fibrosis and cirrhosis. This study aims to evaluate the ability of these indices to predict survival in HCC patients after curative hepatectomy, and probe the increased prognostic accuracy of APRI combined with established inflammation-based prognostic scores. Data were collected retrospectively from 321 patients who underwent curative resection for HCC. Preoperative NLR, PLR, PNI, APRI and clinico-pathological variables were analyzed. Univariate and multivariate analyses were performed to identify the predictive value of the above factors for disease-free survival (DFS) and overall survival (OS). Univariate analysis showed that NLR, PLR, PNI and APRI were significantly associated with DFS and OS in HCC patients with curative resection. Multivariate analysis showed that NLR and APRI were superior to PLR and PNI, and both were independently correlated with DFS and OS. Preoperative NLR >2 or APRI >1.68 predicted poor prognosis of patients with HCC after hepatectomy. Furthermore, the predictive range of NLR combined with APRI was more sensitive than that of either measure alone. Preoperative NLR and APRI are independent predictors of DFS and OS in patients with HCC after surgical resection. Higher levels of NLR or APRI predict poorer outcomes in HCC patients. Intriguingly, combining NLR and APRI increases the prognostic accuracy of testing.

A high rate of recurrence after curative therapy is a major challenge for the management of hepatocellular carcinoma (HCC). Currently, no effective adjuvant therapy is available to prevent HCC recurrence. We designed a personalized neoantigen-loaded dendritic cell vaccine and neoantigen-activated T-cell therapy, and used it as adjuvant therapy to treat 10 patients with HCC who had undergone curative resection or radiofrequency ablation in the first stage of a phase II trial (NCT03067493). The primary outcomes were safety and neoantigen-specific immune response. Disease-free survival (DFS) was also evaluated. The immunotherapy was successfully administered to all the patients without unexpected delay and demonstrated a reasonable safety profile with no grade ≥3 treatment-related side effects reported. Seventy percent of patients generated de novo circulating multiclonal neoantigen-specific T-cell responses. Induced neoantigen-specific immunity was maintained over time, and epitope spreading was observed. Patients who generated immune responses to treatment exhibited prolonged DFS compared with nonresponders (P = 0.012), with 71.4% experiencing no relapse for 2 years after curative treatment. High expression of an immune stimulatory signature, enhanced immune-cell infiltration (i.e., CD8+ T cells), and upregulated expression of T-cell inflammatory gene profiles were found in the primary tumors of the responders. In addition, neoantigen depletion (immunoediting) was present in the recurrent tumors compared with the primary tumors (7/9 vs. 1/17, P = 0.014), suggesting that immune evasion occurred under the pressure of immunotherapy. Our study indicates that neoantigen-based combination immunotherapy is feasible, safe, and has the potential to reduce HCC recurrence after curative treatment.

Pancreatic fistula (PF) remains the most challenging complication after pancreaticoduodenectomy (PD). The purpose of this study was to identify the risk factors of PF and delineate its impact on patient outcomes. We retrospectively reviewed clinical data of 532 patients who underwent PD and divided them into PF group and no PF group. Risk factors and outcomes of PF following PD were examined. PF was found in 65 (12.2%) cases, of whom 11 were classified into ISGPF grade A, 42 grade B, and 12 grade C. Clinically serious postoperative complications in the PF versus no PF group were mortality, abdominal bleeding, bile leak, intra-abdominal abscess and pneumonia. Univariate and multivariate analysis showed that blood loss ≥ 500 ml, pancreatic duct diameter ≤ 3 mm and pancreaticojejunostomy type were independent risk factors of PF after PD. Blood loss ≥ 500 ml, pancreatic duct diameter ≤ 3 mm and pancreatico-jejunostomy type were independent risk factors of PF after PD. PF was related with higher mortality rate, longer hospital stay, and other complications.