Hepatocellular Carcinoma In Recipients Research Articles

Abstract 3985: S100A10 promotes HCC development and progression via transfer in extracellular vesicles and regulating their protein cargos

Abstract Growing evidence indicates that tumor cells exhibit characteristics similar to their lineage progenitor cells. We found that S100 calcium binding protein A10 (S100A10) exhibited an expression pattern similar to that of liver progenitor genes. However, the role of S100A10 in hepatocellular carcinoma (HCC) progression is unclear. Furthermore, extracellular vesicles (EVs) or exosomes are critical mediator of tumorigenesis and metastasis, but the extracellular functions of S100A10, particularly those related to EVs (EV-S100A10), are unknown. In this study, we observed that S100A10 was upregulated at mRNA level in patients’ HCC tumors as compared to the corresponding non-tumorous livers, associated with a copy-number gain. Overexpression of S100A10 was correlated with more aggressive tumor features and poor prognosis. Furthermore, functional assays demonstrated it promoted HCC initiation and progression, including increased sphere forming ability in vitro, enhanced chemoresistance, upregulated liver cancer stemness markers and activated AKT and ERK pathways. In addition, in orthotopic injection mouse model, S100A10 enhanced both the intrahepatic and pulmonary metastasis of HCC xenografts, with upregulated epithelial-mesenchymal transition (EMT) changes. Of significance, we found that S100A10 was present in EVs secreted by HCC cells. Functionally, the EVs secreted by HCC cells promoted the migratory and invasive abilities of recipient HCC cells. Consistent with the oncogenic role of S100A10 in HCC, EVs from S100A10-overexpressing HCC cells enhanced the tumor promoting effects, while the S100A10-depleted HCC cells abrogated the effects, as compared to the vector control group. To further consolidate the tumor promoting role of S100A10-EV, we blocked the effects using neutralizing antibody (NA) by pretreating HCC cells with S100A10 NA together with EV. Indeed, the NA significantly abrogated the stemness and metastatic properties induced by S100A10-EVs both in vitro and in vivo. In addition, the EMT changes and activation of AKT and ERK were also validated in the recipient HCC cells with the S100A10-EV treatment, while they were abolished with the NA treatment. Moreover, we found that S100A10 governed the protein cargos in EVs and mediated the binding of MMP2, fibronectin and EGF to EV membranes through physical binding with integrin αV to promote the motility of recipient cells. Of note, we observed a simultaneous change of p-EGFR, indicating potential activation of AKT and ERK induced by EGFR. On the other hand, we found the plasma EV-S100A10 level was relatively higher in HCC patients than healthy donors. Collectively, the data showed that S100A10 could be transferred among cells through EVs and activated signaling pathways to facilitate metastasis. Targeting S100A10 may be a potential therapeutic strategy for HCC. Citation Format: Lu TIAN, Xia WANG, Jingyi LU, HongYang Huang, Goofy Yu-Man Tsui, Karen Man-Fong Sze, Tan-To Cheung, Irene Oi-Lin NG. S100A10 promotes HCC development and progression via transfer in extracellular vesicles and regulating their protein cargos. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3985.

Exosome-transmitted circ_002136 promotes hepatocellular carcinoma progression by miR-19a-3p/RAB1A pathway

BackgroundCircular RNAs (circRNAs) are enriched in exosomes and are extremely stable. Exosome-mediated intercellular transfer of specific biologically active circRNA molecules can drive the transformation of the tumor microenvironment and accelerate or inhibit the local spread and multifocal growth of hepatocellular carcinoma (HCC). In this study, we explored in depth about the biological roles of HCC cell-derived exosomes and exosome-transported circRNAs on HCC in vivo and in vitro.MethodsExosomes extracted from HCC cells (Huh7 and HA22T) were characterized using transmission electron microscopy, nanoparticle size tracer analysis, and western blotting. Exosomes were observed for endocytosis using fluorescent labeling. The effects of HCC cell-derived exosomes and the circ_002136 they carried on cell growth, metastasis and apoptosis were determined by CCK-8 assay, transwell assay, flow cytometry analysis and TUNEL staining, respectively. The expressions of circ_002136, miR-19a-3p and RAB1A were detected by quantitative RT-PCR (qRT-PCR). Targeted binding between miR-19a-3p and circ_002136 or RAB1A was predicted and verified by bioinformatics analysis, dual-luciferase reporter and RNA pull-down experiments. The in vivo effect of circ_002136 was determined by constructing a xenograft tumor model.ResultsThe findings revealed that Huh7 and HA22T exosomes conferred enhanced viability as well as invasive ability to recipient HCC cells. Circ_002136 was shown for the first time to be differentially upregulated in HCC tissues and cells and transferred by HCC cell-derived exosomes. More importantly, selective silencing of circ_002136 depleted the malignant biological behaviors of HCC exosome-activated Huh7 and HA22T cells. Depletion of circ_002136 in vivo effectively retarded the growth of HCC xenograft tumors. Furthermore, a well-established circ_002136 ceRNA regulatory network was constructed, namely circ_002136 blocked miR-19a-3p expression, elevated RAB1A expression activity and stimulated HCC development. Finally, high levels of circ_002136 or RAB1A, as well as low levels of miR-19a-3p, negatively affected HCC patient survival.ConclusionThe study on circ_002136 provides good data to support our insight into the mechanism of to-be-silenced circRNA as a therapeutic agent in the progression of HCC.

α2,6-Sialylation promotes hepatocellular carcinoma cells migration and invasion via enhancement of nSmase2-mediated exosomal miRNA sorting.

Exosomes have a critical role in the intercellular communication and metastatic progression of hepatocellular carcinoma (HCC). Recently, our group showed that α2, 6-sialylation played an important role in the proliferation- and migration-promoting effects of cancer-derived exosomes. However, the molecular basis remains elusive. In this study, the mechanism of α2, 6-sialylation-mediated specific microRNAs (miRNA) sorting into exosomes was illustrated. We performed miRNA profiling analysis to compare exosomes from HCC cell lines that differ only in α2, 6-sialylation status. A total of 388 differentially distributed miRNAs were identified in wild-type and β-galactoside α2, 6-sialyltransferase I (ST6Gal-I) knockdown MHCC-97H cells-derived exosomes. Neutral sphingomyelinase-2 (nSmase2), an important regulator mediating the sorting of exosomal miRNAs, was found to be a target of ST6Gal-I. The reduction of α2, 6-sialylation could impair the activity of nSmase2, as well as the nSmase2-dependent exosomal miRNAs sorting. This α2,6-sialylation-dependent sorting exerted an augmentation of motility on recipient HCC cells. Our data further demonstrated that α2,6-sialylation-mediated sorting of exosomal miR-100-5p promoted the migration and invasion of recipient HepG2 cells via the PI3K/AKT signaling pathway. The cellular metastasis-related gene CLDN11 was confirmed as a direct target of exosomal miR-100-5p, which elevated the mobility of recipient HCC cells. In conclusion, our results showed that α2,6-sialylation modulates nSmase2-dependent exosomal miRNAs sorting and promotes HCC progression.

M2 Macrophage-Derived Exosomes Facilitate HCC Metastasis by Transferring αM β2 Integrin to Tumor Cells.

Background and AimsThe development and progression of hepatocellular carcinoma (HCC) is dependent on its local microenvironment. Tumor‐associated macrophages (TAMs) are deemed a key factor for the tumor microenvironment and attribute to contribute to tumor aggressiveness. However, the detailed mechanism underlying the pro‐metastatic effect of TAMs on HCC remains undefined.Approach and ResultsThe present study proved that TAMs were enriched in HCC. TAMs were characterized by an M2‐polarized phenotype and accelerated the migratory potential of HCC cells in vitro and in vivo. Furthermore, we found that M2‐derived exosomes induced TAM‐mediated pro‐migratory activity. With the use of mass spectrometry, we identified that integrin, αMβ2 (CD11b/CD18), was notably specific and efficient in M2 macrophage–derived exosomes (M2 exos). Blocking either CD11b and/or CD18 elicited a significant decrease in M2 exos–mediated HCC cell metastasis. Mechanistically, M2 exos mediated an intercellular transfer of the CD11b/CD18, activating the matrix metalloproteinase‐9 signaling pathway in recipient HCC cells to support tumor migration.ConclusionsCollectively, the exosome‐mediated transfer of functional CD11b/CD18 protein from TAMs to tumor cells may have the potency to boost the migratory potential of HCC cells, thus providing insights into the mechanism of tumor metastasis.

Recipient Hepatic Tumor-Associated Immunologic Infiltrates Predict Outcomes After Liver Transplantation for Hepatocellular Carcinoma.

BackgroundTransplantation of the liver entails a state of altered recipient immunologic competence. There are only scarce data concerning the impact of host immunologic factors on the outcome of liver transplant recipients in the context of hepatocellular carcinoma (HCC).Material/MethodsOur study focused on evaluating the presence of tumor necrosis and frequency levels of angiopoietins and monocytes/macrophages subtypes in the host liver prior to liver transplantation (LTX) and their association with recurrence, graft rejection, survival, and clinical prognosis after LTX. Formation of tumor necrosis and tissue densities of angiopoietins and cellular immunologic infiltrates – CD68+ and CD163+ macrophages (TAMs) and TIE2-expressing monocytes (TEMs) – were quantified in recipient HCC specimens. The densities were then matched with clinicopathologic variables and patient survival after LTX (n=88). Some patients were treated prior to LTX by neoadjuvant transarterial chemoembolization (TACE, n=55).ResultsRecipient hepatic infiltration with TEMs and CD68+ TAMs was associated with decreased 1-, 3-, and 5-year survival, as well as metastatic and recurrent HCC after LTX (all p<0.05). TEMs and infiltrating monocytes/macrophages were associated with angiopoietin expression, metastatic, and recurrent HCC (all p<0.05). Furthermore, hepatic angiopoietin-2 expression was associated with graft rejection after LTX (p<0.05). After TACE and LTX, formation of tumor necrosis was associated with an increased presence of monocytes/macrophages and a reduced incidence of recurrent HCC in the graft (all p<0.05).ConclusionsInfiltrating monocytes/macrophages subsets and related angiopoietin axis are associated with worse survival, tumor recurrence, and clinical outcome after LTX for HCC.

Exosomes derived from HCC cells with different invasion characteristics mediated EMT through TGF-β/Smad signaling pathway.

BackgroundExosomes are nano-sized biological vesicles released by many kinds of cells, which play an important role in tumor metastasis through transporting cytokines, RNAs and proteins. However, the molecular mechanisms of exosomes in hepatocellular carcinoma (HCC) metastasis are not completely understood.Materials and methodsExosomes derived from hepatoma cell lines with different invasion characteristics. Exosome characteristics, cell migration and invasion, and effects on major signal transduction pathways deregulated in cancer cells were analyzed by transmission electron microscopy (TEM), wound-healing assay, Trans-well invasion assay and Western blot-based assays, respectively. Moreover, exosomes effects on tumor metastasis in vivo were investigated by subcutaneous transplantation tumor model of athymic nude mice.ResultsExosomes derived from hepatoma cells can promote the migration and invasion of recipient cells, induce the decrease of E-cadherin expression, increase the expression of Vimentin and promote epithelial-mesenchymal transition (EMT) in cells. Moreover, highly invasive hepatoma-cells-derived exosomes effects are stronger than low-invasive hepatoma cells and normal liver cells exosomes. Mechanistic studies showed that the biological alterations in recipient HCC cells treated with MHCC97H and MHCC97L-derived exosomes were caused by inducing EMT via TGF-β/Smad signaling pathway. In vivo experiments also suggest that highly invasive hepatoma cells derived exosomes are more likely to promote lung metastasis of HCC in nude mice.ConclusionOur results reveal the important role of tumor-derived exosomes in the migration and invasion of recipient cells and exosomes may be the novel therapeutic and prognostic targets for HCC patients.

Primary tumor-derived exosomes facilitate metastasis by regulating adhesion of circulating tumor cells via SMAD3 in liver cancer

Hepatocellular carcinoma (HCC) is a fatal disease and patients with HCC frequently die from metastasis. The mechanisms of HCC metastasis are not completely understood. In the present study, in vitro and in vivo data showed that HCC cells promoted cancer cell proliferation and lung metastases formation in a paracrinal/endocrinal way. We found that HCC-derived exosomes mediated this phenomenon and observed enhanced cell adhesion in the presence of these malignant exosomes. We further identified that reactive oxygen species (ROS) regulated the adhesive molecules. Intriguingly, attached HCC cells released exosomes containing both SMAD Family Member 3 (SMAD3) protein and mRNA, which were delivered to detached HCC cells and facilitated their adhesion. These exosomes induced enhanced SMAD3 signaling in the recipient HCC cells and increased their adhesive ability. In addition, we showed that SMAD3-abundant exosomes existed in the peripheral blood of patients with HCC, and their levels correlated with disease stage and the SMAD3 expression of primary tumors. Our study suggested a possible mechanism by which primary HCC supported metastases formation and revealed the role of SMAD3 in the exosomes-mediated crosstalk between primary and circulating HCC cells.

Tumor-derived exosomes promote tumor self-seeding in hepatocellular carcinoma by transferring miRNA-25-5p to enhance cell motility.

Tumor self-seeding occurs when circulating malignant cells reinfiltrate the original tumor. The process may breed more aggressive tumor cells, which may contribute to cancer progression. In this study, we observed tumor self-seeding in mouse xenograft models of hepatocellular carcinoma (HCC) for the first time. We confirmed that circulating tumor cell uptake of tumor-derived exosomes, which are increasingly recognized as key instigators of cancer progression by facilitating cell-cell communication, promoted tumor self-seeding by enhancing the invasive and migration capability of recipient HCC cells. Horizontal transfer of exosomal microRNA-25-5p to anoikis-resistant HCC cells significantly enhanced their migratory and invasive abilities, whereas inhibiting microRNA-25-5p alleviated these effects. Our experiments delineate an exosome-based novel pathway employed by functional microRNA from the original tumor cells that can influence the biological fate of circulating tumor cells.

Exosome miR-335 as a novel therapeutic strategy in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a common and deadly cancer. Most cases of HCC arise in a cirrhotic/fibrotic liver, indicating that environment may play a paramount role in cancer genesis. Previous studies from our group and others have shown that, in desmoplastic cancers, there is a rich intercellular communication between activated, cancer-associated fibroblasts and cancer cells. Moreover, extracellular vesicles (EVs), or exosomes, have been identified as an important arm of this intercellular communication platform. Finally, these studies have shown that EVs can carry microRNA (miR) species in vivo and deliver them to desmoplastic cancers. The precise role played by activated liver fibroblasts/stellate cells in HCC development is insufficiently known. Based on previous studies, it appears plausible that activated fibroblasts produce signals carried by EVs that promote HCC genesis. In the current study, we first hypothesized and then demonstrated that stellate cell-derived EVs 1) can be loaded with an miR species of choice (miR-335-5p); 2) are taken up by HCC cells in vitro and more importantly in vivo; 3) can supply the miR-335-5p cargo to recipient HCC cells in vitro as well as in vivo; and 4) inhibit HCC cell proliferation and invasion in vitro as well as induce HCC tumor shrinkage in vivo. Finally, we identified messenger RNA targets for miR-335 that are down-regulated after treatment with EV-miR-335-5p. This study informs potential therapeutic strategies in HCC, whereby stellate cell-derived EVs are loaded with therapeutic nucleic acids and delivered in vivo. (Hepatology 2018;67:940-954).

Characterizing the Risk of False-Positive Hepatocellular Carcinoma in Recipients Transplanted With T2 MELD Exceptions.

Patients with hepatocellular carcinoma (HCC) can receive Model for End-Stage Liver Disease (MELD) exception points to increase waitlist priority for liver transplantation. This process does not require a biopsy and is based on radiologic criteria. However, imaging modalities are imperfect, and some will ultimately have no HCC on explant. This was a retrospective cohort study using national explant pathology data from 2012 to 2015. False-positive HCC was defined as answering "no" to the question: "was evidence of HCC (viable or nonviable) found in the explant?" in patients with T2 MELD exceptions. Four thousand one hundred seventeen patients received T2 MELD exceptions, of which 245 (6%) had false-positive HCC. Maximal tumor diameter of 3 to 5 cm and serum α fetoprotein (AFP) greater than 100 ng/mL at transplant yielded a 50% lower risk of false-positive HCC (odds ratio [OR], 0.45; 95% confidence interval [CI], 0.27-0.73 and OR, 0.57; 95% CI, 0.37-0.88, respectively). Recipients with immune-mediated liver disease were twice as likely to have no HCC on explant (OR, 2.12; 95% CI, 1.18-3.83) and had a predicted probability of false positive HCC greater than 10% regardless of largest tumor size or AFP. Significant among-center variability in the rate of false-positive HCC was seen. The risk of false-positive HCC is markedly higher in certain groups, such that biopsy may be warranted before T2 MELD exception point approval. Transplant centers with high false-positive HCC rates may benefit from greater oversight.

The impact of the recipient and donor interferon lambda-3 polymorphism on the course of HCV infection following liver transplantation.

Aim of the studyAim of the study was to assess the impact of the recipient and donor interferon lambda-3 (IFNL3) single-nucleotide polymorphisms (SNPs) rs12979860 and rs8099917 on the course of hepatitis C virus (HCV) reinfection following liver transplantation.Material and methodsThe study involved 141 subjects after liver transplantation for HCV-induced cirrhosis, performed between 2000 and 2015. It assessed the impact of both SNPs on the outcomes of interferon/ribavirin (IFN/RBV) treatment following transplantation, HCV viral load, laboratory test results, histological lesions in the liver graft, the risk of acute rejection, and the development of hepatocellular carcinoma (HCC) in patient’s own liver.ResultsIn the case of rs12979860, SVR was achieved in 58.8% of recipients with the CC genotype, and only 12% of recipients with the TT genotype (p = 0.016). Recipients with the rs12979860 CC variant had lower viral load and lower alanine transaminase (ALT) activity than recipients with a non-CC variant. Opposite effects were demonstrated in the analysis of the donors’ genotype. Recipients with the unfavorable variants (rs12979860 TT and rs8099917 GG) had a lower risk of graft rejection and tended to have a higher risk of developing HCC in their own liver.ConclusionsThe IFNL3 rs12979860 polymorphism may be considered a predictor for IFN/RBV effectiveness following liver transplantation. The course of HCV reinfection following liver transplantation may be more aggressive if an unfavorable variant in the recipient coexists with a promising variant in the donor. Particularly careful monitoring for HCC in recipients with unfavorable IFNL3 variants is warranted.

Exosomes derived from miR-122-modified adipose tissue-derived MSCs increase chemosensitivity of hepatocellular carcinoma.

BackgroundHepatocellular carcinoma (HCC) displays high resistance to conventional chemotherapy. Considering that microRNA-122 (miR-122) performs an essential function to promote chemosensitivity of HCC cells, an effective vehicle-mediated miR-122 delivery may represent a promising strategy for HCC chemotherapy. An increasing interest is focused on the use of exosomes as biological vehicles for microRNAs (miRNA) transfer. Mesenchymal stem cells (MSCs) are known for their capacity to produce large amounts of exosomes. This study aimed to determine whether adipose tissue-derived MSC (AMSC) exosomes can be used for miR-122 delivery.MethodsAMSCs were transfected with a miR-122 expression plasmid. At 48 h after transfection, AMSC-derived exosomes (122-Exo) were harvested and added to recipient HCC cells. Expression levels of miR-122 in AMSCs, exosomes, and HCC cells were quantified by real-time PCR. The mRNA and protein levels of miR-122-target genes in recipient HCC cells were quantified by real-time PCR and Western blot, respectively. The effects of 122-Exo on cell viability, apoptosis, and cell cycle of HCC cells were evaluated by MTT and flow cytometry analysis. Xenograft models were used to determine whether 122-Exo can sensitize HCC cells to sorafenib in vivo.ResultsData showed that miR-122-transfected AMSC can effectively package miR-122 into secreted exosomes, which can mediate miR-122 communication between AMSCs and HCC cells, thereby rendering cancer cells sensitive to chemotherapeutic agents through alteration of miR-122-target gene expression in HCC cells. Moreover, intra-tumor injection of 122-Exo significantly increased the antitumor efficacy of sorafenib on HCC in vivo.ConclusionsThe findings suggest that the export of miR-122 via AMSC exosomes represents a novel strategy to enhance HCC chemosensitivity.

Performance of gadoxetic acid‐enhanced MRI for detecting hepatocellular carcinoma in recipients of living‐related‐liver‐transplantation: Comparison with dynamic multidetector row computed tomography and angiography‐assisted computed tomography

To clarify the diagnostic performance of gadoxetic acid-enhanced MRI for the detection of hepatocellular carcinoma (HCC) in recipients of living related-liver transplantation (LRLT). This retrospective study group consisted of 15 patients with 61 HCCs who each underwent multidetector row computed tomography (MDCT), gadoxetic acid-enhanced MRI, and angiography-assisted computed tomography (CT) before LRLT. The three modalities were compared for their ability to detect HCC. Two blinded readers independently reviewed the images obtained by each modality for the presence of HCC on a segment-by-segment basis using a 5-point confidence scale. The diagnostic performance of the modalities was evaluated in a receiver operating characteristic (ROC) analysis. The area under the ROC curve (Az), sensitivity, specificity, and accuracy were compared for the three modalities. No significant difference in Az, sensitivity, specificity, or accuracy was obtained among gadoxetic acid-enhanced MRI, MDCT, and angiography-assisted CT for both readers. For reader 1, the sensitivity (55.6%) and the accuracy (84.7%) of angiography-assisted CT were significantly higher than those of MDCT (33.3% and 78.0%) (P < 0.05). Gadoxetic acid-enhanced MRI has a relatively high diagnostic ability to detect HCC even in recipients of LRLT, equivalent to the abilities of MDCT and angiography-assisted CT.

A comprehensive risk assessment of mortality following donation after cardiac death liver transplant – An analysis of the national registry

Organ scarcity has resulted in increased utilization of donation after cardiac death (DCD) donors. Prior analysis of patient survival following DCD liver transplantation has been restricted to single institution cohorts and a limited national experience. We compared the current national experience with DCD and DBD livers to better understand survival after transplantation. We compared 1113 DCD and 42,254 DBD recipients from the Scientific Registry of Transplant Recipients database between 1996 and 2007. Patient survival was analyzed using the Kaplan-Meier methodology and Cox regression. DCD recipients experienced worse patient survival compared to DBD recipients (p<0.001). One and 3 year survival was 82% and 71% for DCD compared to 86% and 77% for DBD recipients. Moreover, DCD recipients required re-transplantation more frequently (DCD 14.7% vs. DBD 6.8%, p<0.001), and re-transplantation survival was markedly inferior to survival after primary transplant irrespective of graft type. Amplification of mortality risk was observed when DCD was combined with cold ischemia time >12h (HR = 1.81), shared organs (HR = 1.69), recipient hepatocellular carcinoma (HR=1.80), recipient age >60 years (HR = 1.92), and recipient renal insufficiency (HR = 1.82). DCD recipients experience significantly worse patient survival after transplantation. This increased risk of mortality is comparable in magnitude to, but often exacerbated by other well-established risk predictors. Utilization decisions should carefully consider DCD graft risks in combination with these other factors.