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Abstract Background Hepatocellular carcinoma (HCC) is the sixth most commonly diagnosed cancer and third leading cause of cancer-related death worldwide in 2020. Exosomes derived from cancer-associated fibroblasts (CAFs-exo) can promote tumor progression in various human cancers. However, the underlying regulatory mechanism controlling how CAFs-exo can promote HCC progression remains poorly understood. Methods CAFs and para-cancer fibroblasts (PAFs) were isolated from HCC tissues and corresponding para-cancer tissues, then were cultured in vitro. CAFs and PAFs were characterized by immunofluorescence and western blot (WB) assays. Exosomes were isolated by ultracentrifugation, and characterized by transmission electron microscopy, nanoflow cytometry, and WB assay. The internalization of exosomes by HCC cells was observed under a fluorescence microscope. Cell Counting Kit-8 (CCK-8) assay was used to evaluate cell proliferation. Wound healing and transwell assays were used for migration and invasion experiments. RT-PCR assay was used to examine differentially expressed microRNAs (miRNAs) in exosomes and HCC cells. The TargetScan database was used to predict miRNA target genes. Hedgehog interacting protein (HHIP) expression analysis, prognostic analysis, and enrichment analysis of HHIP-related co-expressed genes were performed using the TIMER, UALCAN, Kaplan–Meier plotter, and LinkedOmics databases. Results CAFs-exo were internalized by HCC cells. CAFs-exo contributed to the aggressive phenotype of HCC cells, while inhibiting exosome secretion reversed these effects. Mechanistically, miRNAs in the DLK1-DIO3 imprinted region (miR-329-3p, miR-380-3p, miR-410-5p, miR-431-5p) were increased in HCC cells co-cultured with CAFs-exo compared with PAFs-exo. Expression of HHIP, a possible miR-431-5p target gene, was significantly downregulated in HCC cells. Low HHIP expression level in tumor tissues could predict poor prognosis in HCC patients. HHIP-related co-expressed genes were mainly associated with cell adhesion molecules. Conclusions CAFs-exo can promote HCC progression by delivering miRNAs in the DLK1-DIO3 imprinted region to HCC cells, subsequently inhibiting HHIP expression. HHIP is a potential prognostic biomarker in HCC.

Abstract Background Hepatocellular carcinoma (HCC) is a prevalent malignancy with poor prognosis. As a cell adhesion molecule, poliovirus receptor (PVR/CD155) is abnormally overexpressed in tumour cells, and related to tumour proliferation and invasion. However, the potential role and mechanism of CD155 have not yet been elucidated in HCC. Methods Immunohistochemistry, RT‐PCR and Western blot assays were used to determine CD155 expression in HCC cell lines and tissues. Cell Counting Kit‐8 and colony formation assays were used to examine cell proliferation. Transwell and wound healing assays were used to evaluate cell migration and invasion. Cell apoptosis and cycle distribution were assessed by flow cytometry. Cox regression and Kaplan–Meier analyses were performed to explore the clinical significance of CD155. The role of CD155 in vivo was evaluated by establishing liver orthotropic xenograft mice model. RNA sequencing, bioinformatics analysis and co‐immunoprecipitation assay were used to explore the downstream signalling pathway of CD155. Results CD155 was upregulated in HCC tissues and represented a promising prognostic indicator for HCC patients (n = 189) undergoing curative resection. High CD155 expression enhanced cell proliferation, migration and invasion, and contributed to cell survival in HCC. CD155 overexpression also induced epithelial–mesenchymal transition in HCC cells. CD155 function in HCC involved SRC/p38 MAPK signalling pathway. CD155 interacted with SRC homology‐2 domain of SRC and promoted SRC activation, further inhibiting the downstream p38 MAPK signalling pathway in HCC. Conclusions CD155 promotes HCC progression via the SRC/p38 MAPK signalling pathway. CD155 may represent a predictor for poor postsurgery prognosis in HCC patients.

BackgroundInhibitory immune checkpoint proteins promote tumor immune escape and are associated with inferior patient outcome. However, the biological functions and regulatory roles of one of its members, HHLA2, in the tumor immune microenvironment have not been explored.MethodsRandomForest analyses (371 cases), qRT-PCR (15 cases), and immunohistochemical staining (189 cases) were used to validate the prognostic value of HHLA2 in hepatocellular carcinoma (HCC) patients. Bioinformatic analyses were further performed to explore the biological functions and potential signaling pathways affected by HHLA2. Moreover, ESTIMATE, single sample gene set enrichment analysis, CIBERSORT, TIMER, and other deconvolution methods were used to analyze the composition and infiltration level of immune cells. Multiplex immunofluorescence assays were employed to validate the fractions of suppressive immune cells, and HHLA2-related molecular alterations were investigated. Finally, the clinical response to chemotherapy and immune checkpoint blockade was predicted by TIDE, Submap, and several other in silico analyses.ResultsRandomForest analysis revealed that HHLA2 was the most important inhibitory immune checkpoint associated with HCC patient prognosis (relative importance = 1). Our HCC cohorts further revealed that high HHLA2 expression was an independent prognostic biomarker of shorter overall survival (P

Abstract Backgrounds The role of sphere-forming culture in enriching subpopulations with stem-cell properties in hepatocellular carcinoma (HCC) is unclear. The present study investigates its value in enriching cancer stem cells (CSCs) subpopulations and the mechanism by which HCC CSCs are maintained. Methods HCC cell lines and fresh primary tumor cells were cultured in serum-free and ultra-low attachment conditions to allow formation of HCC spheres. In vitro and in vivo experiments were performed to evaluate CSC characteristics. Expression levels of CSC-related genes were assessed by qRT-PCR and the correlation between sphere formation and clinical characteristics was investigated. Finally, gene expression profiling was performed to explore the molecular mechanism underlying HCC CSC maintenance. Results We found that both cell lines and primary tumor cells formed spheres. HCC spheres possessed the capacity for self-renewal, proliferation, drug resistance, and contained different subpopulations of CSCs. Of interest, 500 sphere-forming Huh7 cells or 200 primary tumor cells could generate tumors in immunodeficient animals. Sphere formation correlated with size, multiple tumors, satellite lesions, and advanced stage. Further investigation identified that the PPARα-SCD1 axis plays an important role in maintenance of the CSC properties of HCC sphere cells by promoting nuclear accumulation of β-Catenin. Inhibition of SCD1 interfered with sphere formation, down-regulated expression of CSC-related markers, and reduced β-Catenin nuclear accumulation. Conclusions Sphere-forming culture can effectively enrich subpopulations with stem-cell properties, which are maintained through activation of the PPARα-SCD1 axis. Therefore, we suggest that targeting the SCD1-related CSC machinery might provide a novel insight into HCC treatment.

Abstract Background Hepatocellular carcinoma (HCC) is one of the most prevalent malignancies worldwide because of rapid progression and high incidence of metastasis or recurrence. Accumulating evidence shows that CD73-expressing tumor cell is implicated in development of several types of cancer. However, the role of CD73 in HCC cell has not been systematically investigated and its underlying mechanism remains elusive. Methods CD73 expression in HCC cell was determined by RT-PCR, Western blot, and immunohistochemistry staining. Clinical significance of CD73 was evaluated by Cox regression analysis. Cell counting kit-8 and colony formation assays were used for proliferation evaluation. Transwell assays were used for motility evaluations. Co-immunoprecipitation, cytosolic and plasma membrane fractionation separation, and ELISA were applied for evaluating membrane localization of P110β and its catalytic activity. NOD/SCID/γc(null) (NOG) mice model was used to investigate the in vivo functions of CD73. Results In the present study, we demonstrate that CD73 was crucial for epithelial-mesenchymal transition (EMT), progression and metastasis in HCC. CD73 expression is increased in HCC cells and correlated with aggressive clinicopathological characteristics. Clinically, CD73 is identified as an independent poor prognostic indicator for both time to recurrence and overall survival. CD73 knockdown dramatically inhibits HCC cells proliferation, migration, invasion, and EMT in vitro and hinders tumor growth and metastasis in vivo. Opposite results could be observed when CD73 is overexpressed. Mechanistically, adenosine produced by CD73 binds to adenosine A2A receptor (A2AR) and activates Rap1, which recruits P110β to the plasma membrane and triggers PIP3 production, thereby promoting AKT phosphorylation in HCC cells. Notably, a combination of anti-CD73 and anti-A2AR achieves synergistic depression effects on HCC growth and metastasis than single agent alone. Conclusions CD73 promotes progression and metastasis through activating PI3K/AKT signaling, indicating a novel prognostic biomarker for HCC. Our data demonstrate the importance of CD73 in HCC in addition to its immunosuppressive functions and revealed that co-targeting CD73 and A2AR strategy may be a promising novel therapeutic strategy for future HCC management.

Background: The aim of the study was to determine the utility of the dynamic change and serial monitoring of the systemic immune inflammation index (SII), which was based on the numbers of patients' lymphocytes (L), platelets (P), neutrophils (N) and defined as P*N/L, for predicting prognosis of patients with hepatocellular carcinoma (HCC) after curative resection. Methods: We conducted a prospective study of 163 patients with HCC who underwent curative resection at Zhongshan Hospital from January 2012 to May 2013. SII was calculated using data acquired before and approximately 1 month after surgery. An optimal cutoff value stratified patients into groups with high or low SII. Patients were classified into unfavorable and favorable groups using the dynamic change of the SII. Two groups that were further divided into four categories within the entire cohort and the lowrisk subgroups were serially monitored for = 6 months. Prognostic values of the SII and other factors were determined using the Kaplan-Meier method, the Cox proportional hazards model, and the receiver operating characteristics (ROC) curve. Results: The favorable group was likely to have cirrhosis, and the unfavorable group was likely to have larger tumors and a higher recurrence rate. Multivariate analysis revealed that tumor size and dynamic change of the SII were independent risk factors for early recurrence. Moreover, the predictive value of the SII was retained in a-fetoprotein (AFP)-negative and HBeAg-negative-HBVDNA < 2000 IU/mL subgroups. Further, the serial changes of the SII for recurrence and no recurrence groups were statistically significant. Conclusions: The dynamic change and serial monitoring of the SII represent new indicators for predicting the early recurrence of HCC determining advance optimal therapy in advance. [ABSTRACT FROM AUTHOR]