Pancreatic Juice-Derived microRNA-4516 and microRNA-4674 as Novel Biomarkers for Pancreatic Ductal Adenocarcinoma.

Background and Aims: Precise diagnostic biomarkers are urgently required for pancreatic ductal adenocarcinoma (PDAC). Therefore, the aim of this study was to identify PDAC-specific exosomal microRNAs (Ex-miRs) from pancreatic juice (PJ) and evaluate their diagnostic potential. Methods: Exosomes in PJ and serum were extracted using ultracentrifugation and confirmed morphologically and biochemically. PDAC-specific Ex-miRs were identified using our original miR arrays, in which "Ex-miRs derived from the PJ of patients with chronic pancreatitis (CP)" were subtracted from Ex-miRs commonly expressed in both "human PDAC cell lines" and "the PJ of patients with PDAC." We verified the expression of these miRs using quantitative real-time reverse transcription polymerase chain reaction. Changes in serum Ex-miR levels were assessed in 2 patients with PDAC who underwent curative resection. In situ hybridization was performed to directly visualize PDAC-specific miR expression in cancer cells. Results: We identified novel Ex-miR-4516 and Ex-miR-4674 from the PJ of patients with PDAC, and they showed 80.0% and 81.8% sensitivity, 80.8% and 73.3% specificity, and 90.9% and 80.8% accuracy, respectively. The sensitivity, specificity, and accuracy of a triple assay of Ex-miR-4516/4674/PJ cytology increased to 93.3%, 81.8%, and 88.5%, respectively. In serum samples (n = 88), the sensitivity, specificity, and accuracy of Ex-miR-4516 were 97.5%, 34.3%, and 68%, respectively. Presurgical levels of serum-derived Ex-miR-4516 in 2 patients with relatively early disease stages declined after curative resection. In situ hybridization demonstrated that Ex-miR-4516 expression exclusively occurred in cancer cells. Conclusion: Liquid assays using the in situ-proven Ex-miR-4516 may have a high potential for detecting relatively early-stage PDAC and monitoring its clinical course.

ISG15 mediates the function of extracellular vesicles in promoting ovarian cancer progression and metastasis.

The interferon stimulated gene 15 (ISG15), a ubiquitin like protein and its conjugates have been implicated in various human malignancies. However, its role in ovarian cancer progression and metastasis is largely unknown. In high grade serous ovarian cancer (HGSOC), ascites is the major contributor to peritoneal metastasis. In this study, we identified significantly elevated ISG15 protein expression in HGSOC patient ascites, ascites derived primary ovarian cancer cells (POCCs), POCC small extracellular vesicles (sEVs) as well as metastatic tissue. Our results demonstrates that ISG15 increases exocytosis in ascites-derived POCCs by decreasing the endosome-lysosomal fusion, indicating a key role in sEV secretion. Further, knockdown (KD) of ISG15 resulted in a significant decrease in vesicles secretion from HGSOC cells and in vivo mouse models, leading to reduced HGSOC cell migration and invasion. Furthermore, our pre-clinical mouse model studies revealed the influence of vesicular ISG15 on disease progression and metastasis. In addition, knockdown of ISG15 or using the ISG15 inhibitor, DAP5, in combination therapy with carboplatin showed to improve the platinum sensitivity in-vitro and reduce tumour burden in-vivo. We also found that ISG15 expression within sEV represents a promising prognostic marker for HGSOC patients. Our findings suggest that ISG15 is a potential therapeutic target for inhibiting progression and metastasis in HGSOC and that vesicular ISG15 expression could be a promising biomarker in the clinical management of ovarian cancer. Significance: High-grade serous ovarian cancer (HGSOC) has high morbidity and mortality rates, but its progression and metastasis are still poorly understood, and there is an urgent need for early detection and targeted therapies. Our study presents novel findings that implicate ISG15-mediated vesicular proteins in the advancement and spread of HGSOC. These results offer pre-clinical evidence of potential new molecular targets, prognostic markers and therapeutic strategies for HGSOC that could ultimately enhance patient survival.

Association between nonalcholic fatty liver disease and pancreatic cancer: Epidemiology, mechanisms, and antidiabetic medication.

Extrahepatic malignancies are the leading cause of death in patients with nonalcoholic fatty liver disease (NAFLD). Of these cancers, pancreatic cancer is one of the most lethal; however, the link between NAFLD and pancreatic cancer remains unclear. Recently, various research results have been reported on the association between NAFLD and pancreatic cancer, and the results of compiling this information revealed the following. First, the prevalence of pancreatic cancer in patients with NAFLD is at 0.26%. Second, the currently evident pathogenesis includes intrapancreatic risk factors, such as: (1) non-alcoholic fatty pancreas disease, and (2) intraductal papillary mucinous neoplasm; and extrapancreatic risk factors, such as: (1) insulin resistance and adipocytokines, (2) proinflammatory cytokines, and (3) dysbiosis. Finally, metformin and sodium-glucose cotransporter 2 inhibitors may reduce the risk of pancreatic cancer in diabetes patients with NAFLD. In this review, we summarize the recent evidence on the epidemiology and mechanisms for NAFLD-related pancreatic cancer. We further discuss the impact of anti-diabetic medication on pancreatic cancer.

Ex-vivo expanded CD34+ cell transplantation alleviates fibrotic liver injury via innate immune modulation in metabolic dysfunction-associated steatohepatitis mice.

BACKGROUND: In drug-induced liver injury, vascular endothelial progenitor cells, specifically the CD34+ cell fractions, have been found to decrease liver fibrosis and promote regeneration. However, it is unclear whether CD34+ cell transplantation has anti-fibrogenic effects on MASH, which has previously been treated effectively with anti-angiogenic therapy. We investigated the efficacy of ex vivo-expanded CD34+ cells in treating MASH livers. MATERIALS AND METHODS: Diet-induced MASH mice were fed a choline-deficient, L-amino acid-defined, high-fat diet for 12 or 20 weeks, and were designated as a mild and a severe fibrosis model, respectively. Mouse bone marrow CD34+ cells were expanded for 7 days, transplanted into each mouse once or twice 2 weeks later, and sacrificed at 4 weeks after the first transplantation. RESULTS: Expanded CD34+ cell transplantation ameliorated liver fibrosis, regardless of fibrosis degree, as indicated by the decrease in α-smooth muscle actin-positive cells, hydroxyproline concentration, and fibrogenic gene expression of Col1a1 and Timp1. Furthermore, engrafted CD34+ cells reduced alanine transaminase levels, the number of TUNEL+ hepatocytes, and 8-OHdG concentration. RNA-sequencing data showed that "defense response to virus" was the most down-regulated category in the Gene Ontology analysis and subsequent analysis revealed the suppression of RIG-I-like receptors/Irf7/Stat1/Cxcl10 axis in expanded CD34+ cell-transplanted livers. Finally, the downregulation of CXCL10 expression inhibits the mobilization of inflammatory immune cells, macrophages, T cells, and natural killer cells to the MASH liver. CONCLUSIONS: These findings suggest that transplanted expanded CD34+ cells alleviate fibrotic liver injury in MASH mouse models through possible modulation of the innate immune response, which is abnormally activated by hepatocyte lipotoxicity.

A tumor endothelial cell-specific microRNA replacement therapy for hepatocellular carcinoma.

Current approved anti-angiogenic drugs (AAD) for hepatocellular carcinoma (HCC) inhibit tumor angiogenesis, but affect the hepatic vasculature resulting in adverse effects. Tumor endothelial cells (TECs) differ from normal endothelial cells. In this study, we aimed to detect TEC-specific miRNAs and develop an anti-angiogenic treatment specific for TECs. We established HCC orthotopic mouse models. TEC-specific miRNAs were detected using a microRNA array. Finally, we evaluated the therapeutic effects of the TEC-specific miRNA agonist cocktail. In total, 260 TEC-specific genes were detected. Among the top ten downregulated TEC-specific miRNAs, miR-139-3p and 214-3p were important for the TEC phenotype. The TEC-specific microRNA agonist cocktail showed significant anti-tumor effects by inhibiting tumor angiogenesis without affecting hepatic vasculatures in HCC orthotopic mouse models. Moreover, it significantly downregulated tip-cell sprouting-related genes. We identified two downregulated TEC-specific miRNAs; microRNA replacement therapy, which targets the downregulated TEC-specific miRNAs, is an effective and promising treatment for HCC.

Fibroblast growth factor inhibition by molecular-targeted agents mitigates immunosuppressive tissue microenvironment in hepatocellular carcinoma.

BACKGROUND & AIMS: Combination immunotherapy refers to the use of immune checkpoint inhibitors (ICI) and molecular-targeted agents (MTA), which have recently been approved for the treatment of advanced hepatocellular carcinoma (HCC). Owing to its relatively low antitumor effect (up to 30%), sequential therapy following ICIs treatment is required in patients with HCC. This study aimed to determine the impact of MTAs on the tumor immune microenvironment (TIME). METHODS: We established immune syngeneic orthotopic HCC mouse models using Hep-55.1C and Hep-53.4, and treated them with MTAs (lenvatinib, sorafenib, regorafenib, cabozantinib, and DC101 as anti-vascular endothelial growth factor receptor-2 antibodies, and AZD4547 as a fibroblast growth factor receptor (FGFR)-1/2/3/4 inhibitor) for 2 weeks. Subsequently, alterations in the TIME caused by MTAs were evaluated using immunohistochemistry (antibodies for CD3, CD8, Foxp3, Granzyme B, Arginase-1, NK1.1, F4/80, CD11c, PD-1, and PD-L1). We conducted RNA-seq analysis using lenvatinib- and AZD4547-treated tumors. To confirm the clinical relevance of these findings, we analyzed the transcriptome data of human HCC cells (MHCC-97H) treated with various concentrations of lenvatinib for 24 h using RNA-seq data from the Gene Expression Omnibus database. RESULTS: The number of Foxp3- and F4/80-positive cells in the TIME was decreased in many MTAs. Cabozantinib increased the numbers in NK1.1-, Granzyme B, and CD11c-positive cells. Lenvatinib and AZD4547 increased the number of CD8, Granzyme B, and PD-L1-positive cells. Gene ontology enrichment analysis revealed that lipid metabolism-related genes were downregulated by lenvatinib and AZD4547. In total, 161 genes downregulated by FGFR inhibition in rodent models overlapped with those downregulated by lenvatinib in human HCC cells. CONCLUSIONS: In this study, we showed that cabozantinib activated the innate immune system, and lenvatinib and AZD4547, which commonly inhibit FGFR signaling, altered TIME to a hot immune state by downregulating lipid metabolism-related genes. These findings support the therapeutic use of combination immunotherapies.

Anti-PD-L1 antibodies promote cellular proliferation by activating the PD-L1-AXL signal relay in liver cancer cells.

BACKGROUND: Immune checkpoint inhibitors (ICIs) are emerging treatments for advanced hepatocellular carcinoma (HCC); however, evidence has shown they may induce hyperprogressive disease via unexplained mechanisms. METHODS: In this study, we investigated the possible stimulative effect of ICIs on programmed cell death-ligand 1 (PD-L1)-harboring liver cancer cells under immunocompetent cell-free conditions. RESULTS: The sarcomatous HAK-5 cell line displayed the highest expression of PD-L1 among 11 human liver cancer cell lines used in this study. HLF showed moderate expression, while HepG2, Hep3B, and HuH-7 did not show any. Moreover, sarcomatous HCC tissues expressed high levels of PD-L1. We observed approximately 20% increase in cell proliferation in HAK-5 cells treated with anti-PD-L1 antibodies, such as durvalumab and atezolizumab, for 48 h compared with that of those treated with the control IgG and the anti-PD-1 antibody pembrolizumab. No response to durvalumab or atezolizumab was shown in PD-L1-nonexpressing cells. Loss-of-function and gain-of-function experiments for PD-L1 in HAK-5 and HepG2 cells resulted in a significant decrease and increase in cell proliferation, respectively. Phosphorylated receptor tyrosine kinase array and immunoprecipitation revealed direct interactions between PD-L1 and AXL in tumor cells. This was stabilized by extrinsic anti-PD-L1 antibodies in a glycosylated PD-L1-dependent manner. Activation of AXL, triggering signal relay to the Akt and Erk pathways, boosted tumor cell proliferation both in vitro and in xenografted tumors in NOD/SCID mice. CONCLUSION: Collectively, this suggests that anti-PD-L1 antibodies stimulate cell proliferation via stabilization of the PD-L1-AXL complex in specific types of liver cancer, including in HCC with mesenchymal components. SIGNIFICANCE: Therapeutic anti-PD-L1 antibodies promote cell proliferation by stabilizing the PD-L1-AXL complex in PD-L1-abundant neoplasms, including in HCC with mesenchymal components. Such a mechanism may contribute to the development of hyperprogressive disease.

Prognostic Factors for Distal Bile Duct Carcinoma After Surgery.

BACKGROUND: Distal bile duct carcinoma continues to be one of the most difficult cancers to manage in terms of staging and radical resection. Pancreaticoduodenectomy (PD) with regional lymph node dissection has become the standard treatment of distal bile duct carcinoma. We evaluated treatment outcomes and histological factors in patients with distal bile duct carcinoma. METHODS: Seventy-four cases of resection of carcinoma of the distal bile ducts treated at our department during the period from January 2002 and December 2016 using PD and regional lymph node dissection as the standard surgical procedure were investigated. Survival rates of factors were analyzed using uni- and multivariate analyses. RESULTS: The median survival time was 47.8 months. On univariate analysis, age of 70 years or older, histologically pap, pPanc2,3, pN1, pEM0, v2,3, ly2,3, ne2,3 and postoperative adjuvant chemotherapy were statistically significant factors. On multivariate analysis, histologically pap was identified as a significant independent prognostic factor. The multivariate analysis identified age of 70 years or older, pEM0, ne2,3 and postoperative adjuvant chemotherapy as showing a significant trend towards independent prognostic relevance. CONCLUSION: The good news about resected distal bile duct carcinoma is that the percentage of those who achieved R0 resection has risen to 89.1%. Our multivariate analysis identified age of 70 years or older, pEM0, ne2,3 and postoperative adjuvant chemotherapy as prognostic factors. In order to improve the outcome of treatment, it is necessary to improve preoperative diagnostic imaging of pancreatic invasion and lymph node metastasis, establish the optimal operation range and clarify whether aortic lymph node dissection is needed to control lymph node metastasis, and establish effective regimens of chemotherapy.

Tumor-derived insulin-like growth factor-binding protein-1 contributes to resistance of hepatocellular carcinoma to tyrosine kinase inhibitors.

BACKGROUND: Antiangiogenic tyrosine kinase inhibitors (TKIs) provide one of the few therapeutic options for effective treatment of hepatocellular carcinoma (HCC). However, patients with HCC often develop resistance toward antiangiogenic TKIs, and the underlying mechanisms are not understood. The aim of this study was to determine the mechanisms underlying antiangiogenic TKI resistance in HCC. METHODS: We used an unbiased proteomic approach to define proteins that were responsible for the resistance to antiangiogenic TKIs in HCC patients. We evaluated the prognosis, therapeutic response, and serum insulin-like growth factor-binding protein-1 (IGFBP-1) levels of 31 lenvatinib-treated HCC patients. Based on the array of results, a retrospective clinical study and preclinical experiments using mouse and human hepatoma cells were conducted. Additionally, in vivo genetic and pharmacological gain- and loss-of-function experiments were performed. RESULTS: In the patient cohort, IGFBP-1 was identified as the signaling molecule with the highest expression that was inversely associated with overall survival. Mechanistically, antiangiogenic TKI treatment markedly elevated tumor IGFBP-1 levels via the hypoxia-hypoxia inducible factor signaling. IGFBP-1 stimulated angiogenesis through activation of the integrin α5ß1-focal adhesion kinase pathway. Consequently, loss of IGFBP-1 and integrin α5ß1 by genetic and pharmacological approaches re-sensitized HCC to lenvatinib treatment. CONCLUSIONS: Together, our data shed light on mechanisms underlying acquired resistance of HCC to antiangiogenic TKIs. Antiangiogenic TKIs induced an increase of tumor IGFBP-1, which promoted angiogenesis through activating the IGFBP-1-integrin α5ß1 pathway. These data bolster the application of a new therapeutic concept by combining antiangiogenic TKIs with IGFBP-1 inhibitors.

Hyperlipidemia as a risk factor for Trousseau syndrome-related cerebral infarction in patients with advanced gastrointestinal cancer.

Trousseau syndrome-related cerebral infarction rarely occurs during chemotherapy in patients with gastrointestinal (GI) cancer, and its clinical features remain unclear. The present study aimed to examine the clinical features of Trousseau syndrome-related cerebral infarction developed during chemotherapy for GI cancer. The present retrospective cohort study consecutively enrolled 878 patients with unresectable GI cancer who received chemotherapy at the Multidisciplinary Treatment Cancer Center, Kurume University Hospital (Kurume, Japan) between April 2014 and March 2020. Patients with colorectal cancer (n=308) were the most common, followed by those with pancreatic (n=242), gastric (n=222) and biliary tract (n=59) cancer, neuroendocrine tumors (n=34) and duodenal cancer (n=11). Among the 878 patients, Trousseau syndrome-related cerebral infarction occurred in 8 (0.9%) patients with a median age of 70.5 years (range, 58-75 years), and 50% of the patients were male (4/8). In total, 3 patients had gastric cancer, 3 had pancreatic cancer and 2 had biliary tract cancer. A greater percentage of patients with Trousseau syndrome-related cerebral infarction had hyperlipidemia (38.0%) than those without (8.2%; P=0.005). Hyperlipidemia was a risk factor for occurrence of Trousseau syndrome-related cerebral infarction with an odds ratio of 7.009 (95% confidence interval, 1.785-27.513). Trousseau syndrome-related cerebral infarction developed during GI chemotherapy was rare and hyperlipidemia may predict its onset.