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Although the treatment of hepatocellular carcinoma (HCC) has been revolutionized by the advent of effective systemic therapies, the prognosis of patients with HCC remains dismal. Herein, we examined the pathophysiological role of PARG and assessed the utility of targeting dePARylation for HCC therapy.The oncogenic function of PARG was evaluated in 2 orthotopic xenograft models and a PargHigh PARG expression was strongly associated with poor HCC prognosis. Hepatocyte-specific PARG deletion significantly impaired liver tumorigenesis. PARG promoted HCC growth and metastasis through DDB1-dependent modulation of c-Myc. Specifically, PARG dePARylated DDB1 and consequently promoted DDB1 autoubiquitination, thus stabilizing the c-Myc protein in HCC cells. PARG downregulation attenuated c-Myc-induced MMR expression and PARG deficiency was correlated with a favorable prognosis in patients with HCC treated with anti-PD-1-based immunotherapy. In addition, PARG inhibitors could act in synergy with anti-PD-1 antibodies in orthotopic mouse models.PARG can act as an oncogene in HCC by modulating PARG/DDB1/c-Myc signaling and could be used as a biomarker to identify patients with HCC who may benefit from anti-PD-1 treatment. Our findings suggest that co-inhibition of PARG and PD-1 is an effective novel combination strategy for patients with HCC.The increase in deaths due to hepatocellular carcinoma (HCC) is a growing concern, with the mechanisms responsible for HCC development still incompletely understood. Herein, we identify a novel mechanism by which the protein PARG contributes to HCC development. Inhibition of PARG increased the efficacy of anti-PD-1 therapy (a type of immunotherapy) in HCC. These findings support the future clinical development of PARG inhibitors, potentially in combination with anti-PD-1 inhibitors.

Liver metastasis is a leading indicator of poor prognosis in patients with colorectal cancer (CRC). Exosomal intercellular communication has been reported to play an important role in cancer invasion and metastasis. Here, we characterized exosomal miRNAs underlying liver metastasis in CRC patients (Cohort 1, n = 30) using miRNA arrays. Exosomal miR-150 was found to be downregulated in CRC patients with liver metastases compared to those without (P = 0.025, fold change [FC] = 2.01). These results were then validated using another independent cohort of CRC patients (Cohort 2, n = 64). Patients with low expression of exosomal miR-150 had significantly shorter overall survival (OS) time (33.3 months versus 43.3 months, P = 0.002). In addition, the low expression of exosomal miR-150 was significantly correlated with advanced tumor node metastasis staging (P = 0.013), higher CA199 level (P = 0.018), and the presence of liver metastasis (P = 0.048). Multivariate analysis showed that low expression of exosomal miR-150 (P = 0.035) and liver metastasis (P < 0.001) were independent prognostic factors for overall survival. In vivo and in vitro studies showed that the viability and invasion of CRC cells were both significantly suppressed by ExomiR-150. Target-prediction assessment and dual-luciferase reporter assay indicated that FTO (the fat mass and obesity-associated gene) was a direct target for miR-150. This study first demonstrated that exosomal miR-150 may be a potential prognostic factor and treatment target for CRC.

Background Brahma-related gene 1 (BRG1) is essential for embryogenesis and cellular metabolism. A deficiency of BRG1 in vivo decreases lipid droplets, but the molecular mechanism underlying its role in lipid metabolism associated with hepatocellular carcinoma (HCC) remains unknown. Aims We aimed to determine the role of BRG1 in lipid metabolism in HCC. Methods We assessed the differential expression of BRG1 in HCC and adjacent non-tumorous tissues using tissue microarrays. We stained lipid droplets in HCC cells with Bodipy fluorescence and Oil Red O, and verified BRG1 binding to the promoter region of glycosylated lysosomal membrane protein (GLMP) using chromatin immunoprecipitation. Results The expression of GLMP, a potential lipid metabolism regulator, was suppressed by BRG1 via transcriptional activity. Knockdown of BRG1 decreased lipid droplets, increased GLMP expression and altered the phosphoinositide-3-kinase adaptor protein 1 (PIK3AP1)/phosphatidylinositol-3 kinase (PI3K)/protein kinase B (AKT) pathway in HCC, which further GLMP knockdown partially restored. Thus, GLMP knockdown increased lipid droplets and differentially altered the PI3K/AKT pathway. Conclusions Downregulating BRG1 decreased lipid droplet deposition in HCC cells by upregulating GLMP and altering the PI3K/AKT pathway. Both BRG1 and GLMP might serve as therapeutic targets for disorders associated with dysregulated lipid metabolism, such as NAFLD and NAFLD-associated HCC.

There exist no treatment guidelines for spontaneously ruptured hepatocellular carcinoma (srHCC) and its prognosis remains controversial.Patients were retrospectively enrolled and grouped based on hemodynamics and tumor resectability. The 30-day mortality, 5-year overall survival (OS), progression-free survival (PFS), peritoneal metastasis (PM) and intrahepatic metastasis (IM) rates were compared.In general, 239 patients were classified into four groups: patients with stable hemodynamics underwent semi-elective hepatectomy (n = 119), and those with unstable hemodynamics received emergent hepatectomy (n = 17), sequential hemostatic-transcatheter arterial chemoembolization (TACE)/-laparotomy with late hepatectomy (n = 49), or TACE only (n = 54). Hepatectomy was safer and provided better OS and PFS than TACE both before and after propensity score matching. Emergent hepatectomy was associated with higher 30-day mortality (6.2%, P 0.05) and poorer prognosis whereas semi-elective hepatectomy and sequential treatment had comparable mortality (both 0%) and survival (36.3% vs 45.2%, P 0.05). Compared with hemostatic TACE in the sequential treatment group, early surgical intervention (semi-elective hepatectomy, emergent hepatectomy, and sequential laparotomy with late hepatectomy) decreased PM (13.6% vs 34.2%, P = 0.003) whereas had higher IM (68.0% vs 50.0%, P = 0.039), but neither procedure had affected OS. In srHCC patients with high risk of recurrence (multiple tumors, micro- and macro-vascular invasion), postoperative adjuvant TACE improved OS.Hepatectomy could provide better prognosis than TACE for srHCC patients while semi-elective hepatectomy and sequential hemostatic-TACE with staged hepatectomy are viable options for srHCCs with stable and unstable hemodynamics, respectively.

Iron dyshomeostasis is associated with hepatocellular carcinoma (HCC) development. However, the role of iron in HCC metastasis is unknown. This study aimed to elucidate the underlying mechanisms of iron’s enhancement activity on HCC metastasis. In addition to the HCC cell lines and clinical samples in vitro, iron deficient (ID) mouse models were generated using iron-free diet and transferrin receptor protein knock out, followed by administration of HCC tumors through either orthotopic or ectopic route. Clinical metastatic HCC samples showed significant ID status, accompanied by overexpression of sphingosine-1-phosphate transporter spinster homologue 2 (SPNS2). Mechanistically, ID increased SPNS2 expression, leading to HCC metastasis in both cell cultures and mouse models. ID not only altered the anti-tumor immunity, which was indicated by phenotypes of lymphatic subsets in the liver and lung of tumor-bearing mice, but also promoted HCC metastasis in a cancer cell autonomous manner through the SPNS2. Since germline knockout of globe SPNS2 showed significantly reduced HCC metastasis, we further developed hepatic-targeting recombinant adeno-associated virus vectors to knockdown SPNS2 expression and to inhibit iron-regulated HCC metastasis. Our observation indicates the role of iron in HCC pulmonary metastasis and suggests SPNS2 as a potential therapeutic target for the prevention of HCC pulmonary metastasis.

Liver metastasis, the leading cause of colorectal cancer mortality, exhibits a highly heterogeneous and suppressive immune microenvironment. Here, we sequenced 97 matched samples by using single-cell RNA sequencing and spatial transcriptomics. Strikingly, the metastatic microenvironment underwent remarkable spatial reprogramming of immunosuppressive cells such as MRC1+ CCL18+ M2-like macrophages. We further developed scMetabolism, a computational pipeline for quantifying single-cell metabolism, and observed that those macrophages harbored enhanced metabolic activity. Interestingly, neoadjuvant chemotherapy could block this status and restore the antitumor immune balance in responsive patients, whereas the nonresponsive patients deteriorated into a more suppressive one. Our work described the immune evolution of metastasis and uncovered the black box of how tumors respond to neoadjuvant chemotherapy. Significance: We present a single-cell and spatial atlas of colorectal liver metastasis and found the highly metabolically activated MRC1+ CCL18+ M2-like macrophages in metastatic sites. Efficient neoadjuvant chemotherapy can slow down such metabolic activation, raising the possibility to target metabolism pathways in metastasis. This article is highlighted in the In This Issue feature, p. 1

Hepatocellular carcinoma (HCC) is a major cause of cancer-related death worldwide. Although it has been known that hepatic stellate cells (HSCs) play critical roles in the development and progression of HCC, the molecular mechanism underlying crosstalk between HSCs and cancer cells still remains unclear. Here, we investigated the interactions between HSCs and cancer cells through an indirect co-culture system. The expressions of cellular and exosomal miR-148a-3p were evaluated by quantitative real-time PCR. Cell counting kit-8 was used for evaluating cell growth

Patients with hepatocellular carcinoma (HCC) have poor long-term survival following curative resection because of the high rate of tumor early recurrence. Little is known about the trajectory of genomic evolution from primary to early-recurrent HCC. In this study, we performed whole-genome sequencing (WGS) on 40 pairs of primary and early-recurrent hepatitis B virus (HBV)-related HCC tumors from patients who received curative resection, and from four patients whose primary and recurrent tumor were extensively sampled. We identified two recurrence patterns: de novo recurrence (18/40), which developed genetically independently of the primary tumor and carried different HCC drivers, and ancestral recurrence (22/40), which was clonally related to the primary tumor and progressed more rapidly than de novo recurrence. We found that the recurrence location was predictive of the recurrence pattern: distant recurrence tended to display the de novo pattern, whereas local recurrence tended to display the ancestral pattern. We then uncovered the evolutionary trajectories based on the subclonal architecture, driver-gene mutations, and mutational processes observed in the primary and recurrent tumors. Multi-region WGS demonstrated spatiotemporal heterogeneity and polyclonal, monophyletic dissemination in HCC ancestral recurrence. In addition, we identified recurrence-specific mutations and copy-number gains in BCL9, leading to WNT/β-catenin signaling activation and an immune-excluded tumor microenvironment, which suggests that BCL9 might serve as a new therapeutic target for recurrent HCC. Collectively, our results allow us to view with unprecedented clarity the genomic evolution during HBV-related HCC early recurrence, providing an important molecular foundation for enhanced understanding of HCC with implications for personalized therapy to improve patient survival.

Cancer stemness and immune evasion are closely associated and play critical roles in tumor development and resistance to immunotherapy. However, little is known about the underlying molecular mechanisms that coordinate this association.The expressions of heterogeneous nuclear ribonucleoprotein M (HNRNPM) in 240 hepatocellular carcinoma (HCC) samples, public databases, and liver development databases were analyzed. Chromatin immunoprecipitation assays were performed to explore the associations between stem-cell transcription factors and HNRNPM. HNRNPM-regulated alternative splicing (AS) and its binding motif were identified by RNA-seq and RIP-seq. HNRNPM-specific antisense oligonucleotides were developed to explore potential therapeutic targets in HCC. CD8+ T cells that were co-cultured with tumor cells were sorted by flow cytometry assays.We identified an elevated oncofetal splicing factor in HCC, HNRNPM, that unifies and regulates the positive association between cancer stemness and immune evasion. HNRNPM knockdown abolished HCC tumorigenesis and diminished cancer stem cell properties in vitro and in vivo. Mechanistically, HNRNPM regulated the AS of MBD2 by binding its flanking introns, whose isoforms played opposing roles. Although MBD2a and MBD2c competitively bound to CpG islands in the FZD3 promoter, MBD2a preferentially increased FZD3 expression and then activated the WNT/β-catenin pathway. Interestingly, FZD3 and β-catenin further provided additional regulation by targeting OCT4 and SOX2. We found that HNRNPM inhibition significantly promoted CD8+ T cell activation and that HNRNPM- antisense oligonucleotides effectively inhibited WNT/β-catenin to enhance anti-programmed cell death protein-1 immunotherapy by promoting CD8+ T cell infiltration.HNRNPM has a tumor-intrinsic function in generating an immunosuppressive HCC environment through an AS-dependent mechanism and demonstrates proof of the concept of targeting HNRNPM in tailoring HCC immunotherapeutic approaches.

Background Immune checkpoint blockade resistance narrows the efficacy of cancer immunotherapies, but the underlying mechanism remains elusive. Delineating the inherent mechanisms of anti-PD1 resistance is important to improve outcome of patients with advanced HCC. Method The level of cricTMEM181 was measured in HCC patients with anti-PD1 therapy by RNA sequencing and then confirmed by qPCR and Sanger sequencing. Immune status in tumor microenvironment of HCC patients or mice models was evaluated by flow cytometry and IHC. Exosomes from HCC cell lines were isolated by ultracentrifugation, and their internalization by macrophage was confirmed by immunofluorescence. The underlying mechanism of HCC-derived exosomal circTMEM181 to macrophage was confirmed by SILAC, RNA FISH and RNA immunoprecipitation. The ATP–ADO pathway amplified by HCC–macrophage interaction was evaluated through ATP, AMP and ADO measurement and macrophage-specific CD39 knockout mice. The role of circTMEM181 in anti-PD1 therapy and its clinical significance were also determined in our retrospective HCC cohorts. Results Here, we found that circTMEM181 was elevated in hepatocellular carcinoma (HCC) patients responding poorly to anti-PD1 therapy and in HCC patients with a poor prognosis after operation. Moreover, we also found that high exosomal circTMEM181 favored the immunosuppressive microenvironment and endowed anti-PD1 resistance in HCC. Mechanistically, exosomal circTMEM181 sponged miR-488-3p and upregulated CD39 expression in macrophages. Using macrophage-specific CD39 knockout mice and pharmacologic approaches, we revealed a novel mode of anti-PD1 resistance in HCC. We discovered that cell-specific CD39 expression in macrophages and CD73 expression in HCC cells synergistically activated the eATP–adenosine pathway and produced more adenosine, thereby impairing CD8+ T cell function and driving anti-PD1 resistance. Conclusion In summary, HCC-derived exosomal circTMEM181 contributes to immunosuppression and anti-PD1 resistance by elevating CD39 expression, and inhibiting the ATP–adenosine pathway by targeting CD39 on macrophages can rescue anti-PD1 therapy resistance in HCC. Graphical Abstract

We studied the value of circulating tumor DNA (ctDNA) in predicting early postoperative tumor recurrence and monitoring tumor burden in patients with hepatocellular carcinoma (HCC). Plasma‐free DNA, germline DNA, and tissue DNA were isolated from 41 patients with HCC. Serial ctDNAs were analyzed by next‐generation sequencing before and after operation. Whole‐exome sequencing was used to detect the DNA of HCC and adjacent tissues. In total, 47 gene mutations were identified in the ctDNA of the 41 patients analyzed before surgery. ctDNA was detected in 63.4% and 46% of the patient plasma pre‐ and postoperation, respectively. The preoperative ctDNA positivity rate was significantly lower in the nonrecurrence group than in the recurrence group. With a median follow‐up of 17.7 months, nine patients (22%) experienced tumor recurrence. ctDNA positivity at two time‐points was associated with significantly shorter recurrence‐free survival (RFS). Tumors with NRAS, NEF2L2, and MET mutations had significantly shorter times to recurrence than those without mutations and showed high recurrence prediction performance by machine learning. Multivariate analyses showed that the median variant allele frequency (VAF) of mutations in preoperative ctDNA was a strong independent predictor of RFS. ctDNA is a real‐time monitoring indicator that can accurately reflect tumor burden. The median VAF of baseline ctDNA is a strong independent predictor of RFS in individuals with HCC., Here, we studied the value of circulating tumor DNA (ctDNA), in combination with germline and tissue DNA by whole‐exome sequencing in patients withhepatocellular carcinoma (HCC). Our analysis demonstrated that ctDNA may serve as a real‐time monitoring indicator by accurately reflecting tumor burden, and as a strong independent predictor of recurrence‐free survival in HCC.

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.

BackgroundConversion therapy is feasible in patients with oncologically unresectable hepatocellular carcinoma (HCC). However, it is challenging to prospectively identify patients who are more likely to achieve successful conversion before initiating systemic therapy, either alone or combined with locoregional therapy.MethodsCriteria for identifying potentially resectable patients with initially oncologically unresectable HCC before treatment with lenvatinib plus an anti-PD-1 antibody were proposed based on real-world evidence. Multivariate Firth logistic regression was used to validate the proposed criteria in a retrospective cohort of consecutive patients with advanced HCC, who received combination therapy with lenvatinib plus an anti-PD-1 antibody between September 2018 and September 2021.ResultsThe proposed criteria were as follows: (1) Eastern Cooperative Oncology Group performance status of 0 or 1; (2) Child-Pugh class A; (3) intrahepatic tumors confined to one lobe (left, right, or middle lobe), or present in one lobe alongside a single tumor with diameter ≤5 cm or up to three tumors each with diameter ≤3 cm in the remaining lobes, with R0 resection achievable by hemihepatectomy, alone or combined with locoregional therapy to the remaining lobes during surgery; and (4) no portal vein tumor thrombus involving the contralateral liver lobe or reaching the superior mesenteric vein, no hepatic vein tumor thrombus involving more than two major hepatic vein branches on the tumor side, and no tumor thrombus of the inferior vena cava reaching the atrium. Firth logistic regression confirmed the criteria were an independent predictor of surgery following conversion therapy with lenvatinib plus an anti-PD-1 antibody.ConclusionsThis study proposed and validated criteria for identifying patients with initially oncologically unresectable HCC who are potentially resectable when treated with combination therapy with lenvatinib plus an anti-PD-1 antibody. The proposed criteria could help standardize conversion therapy studies in advanced HCC.

Background: Circular RNAs (circRNAs) are a class of regulatory RNAs with complex roles in healthy and diseased tissues. However, the oncogenic role of circRNAs in hepatocellular carcinoma (HCC) remains poorly understood, including the mechanisms by which the circRNA UBAP2 contributes to tumorigenesis. Methods: We analyzed the expression of circUBAP2 in 20 paired samples of HCC and healthy tissue as well as in seven HCC cell lines via quantitative real-time polymerase chain reaction (qRT-PCR). Functional experiments, such as CCK8 viability assays, colony formation assays, wound healing, transwell assays, and flow cytometry, were conducted to assess the effects of circUBAP2 in vitro. To further elucidate the mechanisms by which circUBAP2 acts, we conducted dual-luciferase assays, western blots, RNA pull-down assays, and rescue experiments. Results: circUBAP2 was highly upregulated in most HCC tissues and was associated with poor prognosis. HCC patients with high circUBAP2 expression had greater vascular invasion and worse differentiation. Functionally, circUBAP2 overexpression enhanced HCC cell proliferation, migration, and invasion and inhibited apoptosis. Furthermore, we found that circUBAP2 upregulated c-Myc expression by sponging miR-1294, thus contributing to hepatocarcinogenesis. Inhibiting circUBAP2 expression in HCC attenuated the oncogenic effects of c-Myc. Conclusions: These findings suggest that circUBAP2 promotes HCC growth and metastasis. circUBAP2 may have value as an independent prognostic biomarker or as a new target for the treatment of HCC.

Little is known about the transcriptomic plasticity and adaptive mechanisms of circulating tumor cells (CTCs) during hematogeneous dissemination. Here we interrogate the transcriptome of 113 single CTCs from 4 different vascular sites, including hepatic vein (HV), peripheral artery (PA), peripheral vein (PV) and portal vein (PoV) using single-cell full-length RNA sequencing in hepatocellular carcinoma (HCC) patients. We reveal that the transcriptional dynamics of CTCs were associated with stress response, cell cycle and immune-evasion signaling during hematogeneous transportation. Besides, we identify chemokine CCL5 as an important mediator for CTC immune evasion. Mechanistically, overexpression of CCL5 in CTCs is transcriptionally regulated by p38-MAX signaling, which recruites regulatory T cells (Tregs) to facilitate immune escape and metastatic seeding of CTCs. Collectively, our results reveal a previously unappreciated spatial heterogeneity and an immune-escape mechanism of CTC, which may aid in designing new anti-metastasis therapeutic strategies in HCC., Circulating tumour cells can be useful for monitoring disease progression but how they survive in the circulatory system is unclear. Here, the authors use single-cell sequencing of circulating tumour cells from multiple vascular sites in liver cancer patients and identify genes that may help the cells survive.

Gut microbiome is an important regulator of hepatic inflammation, detoxification, and immunity, and contributes to the carcinogenesis of liver cancer. Intratumoral bacteria are supposed to be closer to the tumor cells, forming a microenvironment that may be relevant to the pathological process of hepatocellular carcinoma (HCC).

Studies of the gut-liver axis have enhanced our understanding of the pathophysiology of various liver diseases and the mechanisms underlying the regulation of the effectiveness of therapies. Radiotherapy (RT) is an important therapeutic option for patients with unresectable hepatocellular carcinoma (HCC). However, the role of the microbiome in regulating the response to RT remains unclear. The present study characterizes the gut microbiome of patients responsive or non-responsive to RT and investigates the molecular mechanisms underlying the differences in patient response. We collected fecal samples for 16S rRNA sequencing from a prospective longitudinal trial of 24 HCC patients receiving RT. We used fecal microbiota transplantation (FMT), flow cytometry, and transcriptome sequencing to explore the effects of dysbiosis on RT. We also examined the role of stimulator of interferon genes (STING) in RT-associated antitumor immune responses mediated by gut microbiota in STING- (Tmem173sup-/-/sup) and cGAS-knockout (Mb21d1sup-/-/sup) mouse models. We propose that primary resistance to RT could be attributed to the disruption of the gut microbiome. Mechanistically, gut microbiome dysbiosis impairs antitumor immune responses by suppressing antigen presentation and inhibiting effector T cell functions through the cGAS-STING-IFN-I pathway. Cyclic-di-AMP - an emerging second messenger of bacteria - may act as a STING agonist and is thus a potential target for the prediction and modulation of responses to RT in HCC patients. Our study highlights the therapeutic potential of modulating the gut microbiome in HCC patients receiving RT and provides a new strategy for the radiosensitization of liver cancer.

Background Combination conversion therapies afforded curative surgery chance for initially unresectable hepatocellular carcinoma (uHCC). This study aimed to evaluate the conversion rate and clinical outcomes of a first-line conversion regimen of lenvatinib combined with transarterial chemoembolization (TACE) plus immunotherapy for initial uHCC by interpreting real-world data. Methods Conversion therapy data of patients with uHCC from November 2018 to January 2021 were analysed. The regimens included triple combination therapy (t-CT: lenvatinib, TACE, plus toripalimab) and dual combination therapy (d-CT: lenvatinib plus TACE). Another study population diagnosed with hepatocellular carcinoma of macrovascular invasion disease were included as the upfront surgery cohort. Treatment responses and conversion rate were primary outcomes. Survival and adverse events were analysed. Results Fifty-one patients receiving t-CT (n = 30) and d-CT (n = 21) were enrolled. Higher overall response rates (76.7 per cent versus 47.6 per cent, P = 0.042) and disease control rates (90.0 per cent versus 57.1 per cent, P = 0.042) were observed via t-CT than d-CT. Both median overall survival and event-free survival were not reached in the t-CT cohort. A higher rate of curative conversion resection was achieved through t-CT than d-CT (50.0 per cent versus 19.0 per cent, P = 0.039). The disease-free survival of patients undergoing conversion resection in the t-CT cohort (n = 15) was higher than that in the upfront surgery cohort (n = 68, P = 0.039). Both t-CT and d-CT regimens were tolerable. Conclusions Better treatment responses and conversion rate for patients with uHCC were obtained with first-line t-CT. Neoadjuvant t-CT before surgery should be recommended for patients with macrovascular invasion.

The complement cascade plays a "complementing" role in human immunity. However, the potential roles of complement system in impacting molecular and clinical features of hepatocellular carcinoma (HCC) remain unclear. In this study, eleven public datasets are analyzed to compare the complement status between normal and cancerous samples based on 18 classical complement-associated genes. The complement scores are constructed to quantify complement signatures of individual tumors. HCC patients in the The Cancer Genome Atlas (TCGA) cohort are focused to perform systematical analyses between complement status and immune infiltration, miRNA expression, DNA methylation, clinicopathological features, and drug response. The results show that the complement scores in normal tissues are dramatically higher than those of tumor tissues. Tumor samples in the TCGA cohort are classified into complement score-low and score-high groups. Pathway analysis reveals that tumor-promoting pathways are typically inhibited in complement score-high group. This study also shows that tumor-killing immune cells, such as CD8

Circulating tumor-initiating cells (CTICs) with stem cell-like properties play pivotal roles in tumor metastasis and recurrence. However, little is known about the biology and clinical relevance of CTICs in hepatocellular carcinoma (HCC). Here, we investigated the molecular heterogeneity and clinical relevance of CTICs in HCC using a novel integrated immunomagnetic-microfluidic platform (iMAC). We constructed the iMAC and evaluated its ability to detect CTICs using a series of spiked cell experiments. A four-channel microfluidic chip was applied to investigate the composition of CTICs in patients with primary and recurrent HCC utilizing microbeads labeled with one of four stem-related markers: epithelial cell adhesion molecule (EpCAM), CD133, CD90, and CD24. The dynamic changes of these four CTIC subsets were serially monitored during treatment courses. Finally, single-cell RNA profiling was used to reveal the molecular characteristics of the four CTIC subsets. The iMAC platform detected significantly more EpCAM

Sarcomatoid hepatocellular carcinoma (sHCC) is a rare type of liver malignancy. Currently, the tumor immune features of sHCC are poorly understood. We recruited 31 patients with resected sHCC for whom tissue samples and complete clinicopathologic and follow-up data were available. To understand the immune infiltration of sHCC, immunohistochemical staining was performed on the resected sHCC samples to compare the expressions of programmed death-1 (PD-1), programmed death-ligand 1 (PD-L1), B7-H3, indoleamine 2,3-dioxygenase (IDO), lymphocyte-activation gene 3 (LAG-3), CD8, FOXP3, and CD68 in tumor and peritumoral tissues. Kaplan-Meier and Cox regression analyses were used to assess the predictive value of immune markers. Sarcomatoid components were characterized with significantly higher expression of PD-L1 and B7-H3 in tumor cells than in conventional HCC components, as well as in peritumoral tissue. Additionally, sarcomatoid components had a higher density of FOXP3+ and LAG-3+ cells and a lower density of CD8+ cells than conventional HCC components or peritumoral tissue. Higher expression of PD-L1 in tumor cells significantly correlated with higher densities of CD8+, PD-1+, and LAG-3+ cells. Increased tumor PD-L1 expression and decreased CD8+ T-cell density were associated with poor overall survival (OS) and disease-free survival (DFS) in patients of sHCC. These findings suggest further characterization on relative mechanism of sHCC immune infiltration may identify therapeutic targets for immunotherapy.

Background We evaluated organ-specific response rates (OSRRs) to first-line lenvatinib plus anti-PD-1 antibodies in patients with advanced hepatocellular carcinoma (HCC). Methods This retrospective analysis included Chinese patients with unresectable/advanced HCC who received first-line lenvatinib (8 mg/day) plus ≥3 infusions of anti-PD-1 antibodies between October 2018 and May 2020. Tumor and macrovascular tumor thrombi (MVTT) treatment responses were evaluated every 2 months using RECIST v1.1. The overall response rate (ORR)/OSRR was defined as the percentage of patients with a best overall response of complete or partial response (CR or PR). Results In total, 60 patients were included in the analysis; 96.7% had measurable intrahepatic lesions, 55% had MVTT and 26.7% had extrahepatic disease. In all 60 patients, the ORR was 33.3%, median progression-free survival was 7.0 months (95% CI, 1.7–12.3) and median overall survival was not reached. The OSRR for MVTT (54.5%) was higher versus intrahepatic tumors (32.8%), extrahepatic lung metastases (37.5%) and lymph node metastases (33.3%). Among 33 patients with intrahepatic tumors and MVTT, 18 had differential responses in each site, including 13 with a better response in MVTT versus intrahepatic lesions. Among 18 patients whose MVTT achieved a radiographic CR or PR, six underwent surgical resection: 4/6 achieved a pathological CR in MVTT and 2/6 in the intrahepatic tumor. Conclusions First-line lenvatinib plus anti-PD-1 antibodies resulted in better tumor responses in MVTT versus intrahepatic lesions. Complete MVTT necrosis may allow downstaging and subsequent eligibility for surgical resection in a proportion of patients with advanced HCC.

Cancer-associated fibroblasts (CAFs) are commonly acquired activated extracellular matrix (ECM)-producing myofibroblasts, a phenotypes with multiple roles in hepatic fibrogenesis and carcinogenesis via crosstalk with cohabitating stromal/cancer cells. Here, we discovered a mechanism whereby CAF-derived cytokines enhance hepatocellular carcinoma (HCC) progression and metastasis by activating the circRNA-miRNA-mRNA axis in tumor cells. CAFs secreted significantly higher levels of CXCL11 than normal fibroblasts (NFs), and CXCL11 also had comparatively higher expressions in HCC tissues, particularly in metastatic tissues, than para-carcinoma tissues. Both CAF-derived and experimentally introduced CXCL11 promoted HCC cell migration. Likewise, CAFs promoted tumor migration in orthotopic models, as shown by an increased number of tumor nodules, whereas CXCL11 silencing triggered a decrease of it. CXCL11 stimulation upregulated circUBAP2 expression, which was significantly higher in HCC tissues than para-carcinoma tissues. Silencing circUBAP2 reversed the effects of CXCL11 on the expression of IL-1β/IL-17 and HCC cell migration. Further downstream, the IFIT1 and IFIT3 levels were significantly upregulated in HCC cells upon CXCL11 stimulation, but downregulated upon circUBAP2 silencing. IFIT1 or IFIT3 silencing reduced the expression of IL-17 and IL-1β, and attenuated the migration capability of HCC cells. Herein, circUBAP2 counteracted miR-4756-mediated inhibition on IFIT1/3 via sponging miR-4756. miR-4756 inhibition reversed the effects induced by circUBAP2 silencing on the IL-17 and IL-1β levels and HCC cell migration. In orthotopic models, miR-4756 inhibition also reversed the effects on metastatic progression induced by silencing circUBAP2.

Objective Post-hepatectomy liver failure (PHLF) is a severe complication in patients with hepatocellular carcinoma (HCC) who underwent hepatectomy. This study aims to develop a nomogram of PHLF grade B–C in patients with huge HCC (diameter ≥ 10 cm). Methods We retrospectively collected clinical information of 514 and 97 patients who underwent hepatectomy for huge HCC at two medical centers between 2016 and 2021. Univariate and multivariate analysis were carried out to screen the independent risk factors of PHLF grade B–C, which were visualized as a nomogram. Results Three Hundred Forty Three Thousand One Hundred Seventy One and 97 HCC patients were included in the training cohort, internal validation cohort, and external validation cohort, with probabilities of PHLF grade B–C of 15.1%, 12.9%, and 22.7%, respectively. Pre-operative modified albumin-bilirubin (mALBI) grade (p p = 0.044), international normalized ratio (INR) (p = 0.005), cirrhosis (p = 0.019), and intraoperative blood loss (p = 0.004) were found to be independently associated with PHLF grade B–C in the training cohort. All the five independent factors were considered in the establishment of the nomogram model. In the internal validation cohort and external validation cohort, the area under receiver operating characteristic curve for the nomogram in PHLF grade B–C prediction reached 0.823 and 0.740, respectively. Divided into different risk groups according to the optimal cut-off value, patients in the high-risk group reported significantly higher frequency of PHLF grade B–C than those in the low-risk group, both in the training cohort and the validation cohort (p Conclusions The proposed noninvasive nomogram based on mALBI-Child–Pugh and three other indicators achieved optimal prediction performance of PHLF grade B–C in patients with huge HCC.

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.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.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.CD155 promotes HCC progression via the SRC/p38 MAPK signalling pathway. CD155 may represent a predictor for poor postsurgery prognosis in HCC patients.

ObjectiveDysfunction of endoplasmic reticulum (ER) proteins is closely related to homeostasis disturbance and malignant transformation of hepatocellular carcinoma (HCC). Reticulons (RTN) are a family of ER-resident proteins critical for maintaining ER function. Nevertheless, the precise roles of RTN in HCC remain largely unclear. The aim of the study is to examine the effect of reticulon family member RTN3 on HCC development and explore the underlying mechanisms.DesignClinical HCC samples were collected to assess the relationship between RTN3 expression and patients’ outcome. HCC cell lines were employed to examine the effects of RTN3 on cellular proliferation, apoptosis and signal transduction in vitro. Nude mice model was used to detect the role of RTN3 in modulating tumour growth in vivo.ResultsWe found that RTN3 was highly expressed in normal hepatocytes but frequently downregulated in HCC. Low RTN3 expression predicted poor outcome in patients with HCC in TP53 gene mutation and HBV infection status-dependent manner. RTN3 restrained HCC growth and induced apoptosis by activating p53. Mechanism studies indicated that RTN3 facilitated p53 Ser392 phosphorylation via Chk2 and enhanced subsequent p53 nuclear localisation. RTN3 interacted with Chk2, recruited it to ER and promoted its activation in an ER calcium-dependent manner. Nevertheless, the tumour suppressive effects of RTN3 were abrogated in HBV-positive cells. HBV surface antigen competed with Chk2 for RTN3 binding and blocked RTN3-mediated Chk2/p53 activation.ConclusionThe findings suggest that RTN3 functions as a novel suppressor of HCC by activating Chk2/p53 pathway and provide more clues to better understand the oncogenic effects of HBV.

Background T follicular helper (Tfh) cells and T follicular regulatory (Tfr) cells were newly identified as the subsets of cluster of CD4+ T cells. As major components of human immune system, they were found in tumor microenvironment and reported to play vital roles in the progression of cancer. But their clinical significance in Hepatocellular carcinoma (HCC) was not elucidated. Thus, this research aimed to investigate their prognostic value in HCC. Materials and methods A total of 210 subjects (including 110 HCC patients, 50 chronic hepatitis patients and 50 healthy individuals) were enrolled in the research. Tfh, Tfr cells and Treg cells from peripheral blood were measured by flow cytometry. Receiver operating characteristic (ROC) curves were used to evaluate the diagnostic performance of Tfr-Tfh Index (TTI) in early HCC and relapse status. Its further prognostic valve was assessed by Kaplan-Meier survival estimate and log rank tests. Results Tfh cells, Tfr cells, Treg cells and TTI were all higher in HCC patients than in chronic hepatitis patients and healthy control. TTI was found to have positive correlation with the load of HBV. The AUC of TTI for early HCC and relapse status was better than other clinical indices in HBV positive patients. An optimal cutoff point for the TTI stratified the HCC patients into high (>21.96) and low index (≤21.96) groups. High TTI was significantly correlated with recurrence. Univariate and multivariate analyses revealed TTI could be a predictor for recurrence. Moreover, it retained prognostic performance for patients with lower recurrence risk. Conclusion Our research showed that TTI could be a promising indicator for early recurrence in HCC patients with HBV infection.

BACKGROUND & AIMS Liver transplantation (LTx) is one of the most effective treatments for hepatocellular carcinoma (HCC); however, tumour recurrence after LTx often leads to poor outcomes. This study investigated the value of circulating tumour cells (CTCs) as a predictor of recurrence following LTx in patients with HCC. METHODS This analysis included 193 patients with HCC who underwent LTx at our institute and accepted pre- and post-operative CTC detection; 38 were selected for serial CTC monitoring. The predictive value of CTCs for tumour recurrence in patients with HCC following LTx was evaluated. Single-cell whole genome sequencing was used to characterize CTCs. RESULTS Overall, the CTC burden decreased after LTx (P

Accumulating evidence indicates that hepatocellular carcinoma (HCC) tumorigenesis, recurrence, metastasis, and therapeutic resistance are strongly associated with liver cancer stem cells (CSCs), a rare subpopulation of highly tumorigenic cells with self-renewal capacity and differentiation potential. Previous studies identified B cell leukemia/lymphoma-11b (BCL11B) as a novel tumor suppressor with impressive capacity to restrain CSC traits. However, the implications of BCL11B in HCC remain unclear. In this study, we found that low BCL11B expression was an independent indicator for shorter overall survival (OS) and time to recurrence (TTR) for HCC patients with surgical resection. In vitro and in vivo experiments confirmed BCL11B as a tumor suppressor in HCC with inhibitory effects on proliferation, cell cycle progression, apoptosis, and mobility. Furthermore, BCL11B could suppress CSC traits, as evidenced by dramatically decreased tumor spheroid formation, self-renewal potential and drug resistance. A Cignal Finder Array and dual-luciferase activity reporter assays revealed that BCL11B could activate the transcription of P73 via an E2F1-dependent manner. Thus, we concluded that BCL11B is a strong suppressor of retaining CSC traits in HCC. Ectopic expression of BCL11B might be a promising strategy for anti-HCC treatment with the potential to cure HBV-related HCC regardless of P53 mutation status.

ObjectiveTumour pathology contains rich information, including tissue structure and cell morphology, that reflects disease progression and patient survival. However, phenotypic information is subtle and complex, making the discovery of prognostic indicators from pathological images challenging.DesignAn interpretable, weakly supervised deep learning framework incorporating prior knowledge was proposed to analyse hepatocellular carcinoma (HCC) and explore new prognostic phenotypes on pathological whole-slide images (WSIs) from the Zhongshan cohort of 1125 HCC patients (2451 WSIs) and TCGA cohort of 320 HCC patients (320 WSIs). A ‘tumour risk score (TRS)’ was established to evaluate patient outcomes, and then risk activation mapping (RAM) was applied to visualise the pathological phenotypes of TRS. The multi-omics data of The Cancer Genome Atlas(TCGA) HCC were used to assess the potential pathogenesis underlying TRS.ResultsSurvival analysis revealed that TRS was an independent prognosticator in both the Zhongshan cohort (ppredictive ability of TRS was superior to and independent of clinical staging systems, and TRS could evenly stratify patients into up to five groups with significantly different prognoses. Notably, sinusoidal capillarisation, prominent nucleoli and karyotheca, the nucleus/cytoplasm ratio and infiltrating inflammatory cells were identified as the main underlying features of TRS. The multi-omics data of TCGA HCC hint at the relevance of TRS to tumour immune infiltration and genetic alterations such as the FAT3 and RYR2 mutations.ConclusionOur deep learning framework is an effective and labour-saving method for decoding pathological images, providing a valuable means for HCC risk stratification and precise patient treatment.

BACKGROUND Postoperative extrahepatic metastases (EHM) contribute to a grim outcome in patients with hepatocellular carcinoma (HCC) who are undergoing curative surgical resection. The current study investigated the clinical value of circulating tumor cells (CTCs) in predicting EHM after curative surgery. METHODS A total of 197 patients with HCC who were undergoing curative surgical resection were assigned to a retrospective training cohort (144 patients) or a prospective validation cohort (53 patients). The CELLSEARCH system was used for the detection of CTCs prior to surgical resection and 1 month thereafter. The cutoff value of CTCs was estimated using receiver operating characteristic analysis. Bonferroni correction was applied for multiple testing in a Cox proportional hazards regression model. RESULTS In the training cohort, EHM was found to be associated with a higher postoperative CTC burden compared with no EHM (mean: 4.33 vs 0.52; P < .001). Receiver operating characteristic analysis demonstrated a postoperative CTC count ≥3 as the optimal cutoff value for the prediction of EHM. Patients with a postoperative CTC count ≥3 experienced a higher EHM risk (56.3% vs 5.5%) and a shorter median overall survival (31.25 months vs not reached) (all P

Background Prognosis of hepatocellular carcinoma (HCC) remains poor despite significant recent improvement in therapy. Recent studies have reported that transglutaminase 3 (TGM3) plays an important role in several human cancer types. However, the role of TGM3 in HCC have not been previously elucidated. Methods We evaluated the role of TGM3 in regulating HCC cell proliferation, migration, and invasion. We also investigated the prognostic significance of TGM3 in an HCC cohort. Finally, we explored the signalling pathways that TGM3 regulates in HCC. Results We identified TGM3 to be overexpressed in HCC compared to normal tissues. Higher expression of TGM3 predicts poor prognosis in HCC patients. TGM3 knockdown led to decreased HCC cell proliferation, invasion, and xenograft tumour growth. TGM3 depletion inhibited AKT, extracellular signal–regulated kinase (ERK), p65, and glycogen synthase kinase 3β (GSK3β)/β-catenin activation, but promoted levels of cleaved caspase 3. Moreover, TGM3 knockdown cells had increased E-cadherin levels and decreased vimentin levels, suggesting that TGM3 contributes to epithelial–mesenchymal transition (EMT) in HCC. Conclusion Our results suggest that TGM3 controls multiple oncogenic pathways in HCC, thereby contributing to increased cell proliferation and EMT, and TGM3 potentially enhances HCC metastasis. TGM3 may serve as a novel therapeutic target in HCC.

Hepatic stellate cells (HSCs) play vital roles in tumorigenesis and progression of hepatocellular carcinoma (HCC). However, there remains a lack of high-throughput studies on gene expression alterations in HCC cells in response to direct interactions with HSCs. In this study, we established a direct co-culture model of HSCs and HCC cells. We found that the expression of a set of miRNAs, most notably miR-1246, was triggered by HSCs. RORα was confirmed as the target gene of miR-1246. Either overexpression of miR-1246 or knockdown of RORα enhanced the proliferation, invasiveness, and metastatic capability of HCC both in vitro and in vivo, through Wnt/β-catenin pathway activation and promotion of epithelial-mesenchymal transition (EMT). Moreover, upregulation of miR-1246 and repression of RORα were prominent features of aggressive clinical HCC. The miR-1246-RORα-Wnt/β-catenin axis is a novel pathway through which HSCs accelerate HCC progression.

Liver cancer is a lethal disease that is associated with poor prognosis. In order to identify the functionally important genes associated with liver cancer that may reveal novel therapeutic avenues, we performed integrated analysis to profile miRNA and mRNA expression levels for liver tumors compared to normal samples in The Cancer Genome Atlas (TCGA) database. We identified 405 differentially expressed genes and 233 differentially expressed miRNAs in tumor samples compared with controls. In addition, we also performed the pathway analysis and found that mitogen-activated protein kinases (MAPKs) and G-protein coupled receptor (GPCR) pathway were two of the top significant pathway nodes dysregulated in liver cancer. Furthermore, by examining these signaling networks, we discovered that FOS (Fos proto-oncogene, AP-1 transcription factor subunit), LAMC2 (laminin subunit gamma 2), and CALML3 (calmodulin like 3) were the most significant gene nodes with high degrees involved in liver cancer. The expression and disease prediction accuracy of FOS, LAMC2, CALML3, and their interacting miRNAs were further performed using a HCC cohort. Finally, we investigated the prognostic significance of FOS in another HCC cohort. Patients with higher FOS expression displayed significantly shorter time to recurrence (TTR) and overall survival (OS) compared with patients with lower expression. Collectively, our study demonstrates that FOS is a potential prognostic marker for liver cancer that may reveal a novel therapeutic avenue in this lethal disease.

OBJECTIVE The aim of the study is to assess the efficacy and safety of associating liver partition and portal vein ligation for staged hepatectomy (ALPPS) in patients with hepatitis B virus-related hepatocellular carcinoma (HCC). BACKGROUND ALPSS allows curative resection of conventionally-unresectable liver tumors. However, its role in HCC is largely unknown. METHODS Consecutive HCC patients who underwent ALPPS at our center between April 2013 and September 2017 were retrospectively studied. The oncological results were compared with patients receiving transcatheter arterial chemoembolization (TACE), and patients undergoing one-stage resection by using propensity score matching (PSM) analysis. RESULTS The median tumor diameter was 13 cm (range: 6-22 cm) in patients with a single tumor (n = 28), whereas the median total tumor diameter was 12 cm (range: 9-31 cm) in patients with multiple tumors (n = 17). After stage-1 ALPPS, the median future liver remnant (FLR) increased by 56.8%. The stage-2 ALPPS was completed in 41 patients (91.1%) after a median of 12 days. The 90-day mortality rate was 11.1% (5/45). The overall survival (OS) rates at 1- and 3-year were 64.2% and 60.2%, whereas the disease-free survival (DFS) rates at 1 and 3 years were 47.6% and 43.9%, respectively. On PSM analysis, the long-term survival of patients undergoing ALPPS was significantly better than those receiving TACE (OS, P = 0.004; DFS, P < 0.0001) and similar to those subjected to one-stage liver resection (OS, P = 0.514; DFS, P = 0.849). CONCLUSIONS The long-term survival after ALPPS was significantly better than TACE, and similar to those after one-stage liver resection. ALPPS is a viable treatment option for patients with unresectable HCC in selected patients.

Dysregulation of microRNA (miRNA) is a frequent event in hepatocellular carcinoma (HCC), but little is known whether it is a bystander or an actual player on residual HCC metastasis during liver microenvironment remodeling initiated by hepatectomy. Methods: The differently expressed miRNAs and mRNAs were identified from RNA-seq data. Western blot, qRT-PCR, fluorescence in situ hybridization, immunofluorescence and immunohistochemical were used to detect the expression of miRNA and mRNA in cell lines and patient tissues. The biological functions were investigated in vitro and in vivo. Chromatin immunoprecipitation, proximity ligation and luciferase reporter assay were used to explore the specific binding of target genes. The expression of HGF/ERBB3 signaling was detected by Western blot. Results: In this study, HGF induced by hepatectomy was shown to promote metastasis of residual HCC cells. miR-17-5p and miR-20a-5p were confirmed to play inhibitory roles on HCC metastasis. And ERBB3 was found to be the common target of miR-17-5p and miR-20a-5p. HCC cells with lower levels of miR-17-5p and miR-20a-5p or higher level of ERBB3 were often more sensitive to response HGF stimuli and to facilitate metastatic colonization both in vitro and in vivo experimental systems. Furthermore, HGF reinforced ERBB3 expression by NF-κB transcriptional activity in a positive feedback loop. Of particular importance, HCC patients with lower levels of miR-17-5p and miR-20a-5p or higher level of ERBB3 had significantly shorter OS and PFS survivals after surgical resection. Conclusion: miR-17-5p and miR-20a-5p could suppress postoperative metastasis of hepatocellular carcinoma via blocking HGF/ERBB3-NF-κB positive feedback loop and offer a new probable strategy for metastasis prevention after HCC resection.

Several studies have demonstrated that the T-cell receptor (TCR) repertoire is associated with prognosis and immune therapy response in several types of cancer. However, the comprehensive features of TCR repertoire in tumor-infiltrating and circulating T cells, as well as its clinical significance of diagnosis in hepatocellular carcinoma (HCC) patients, are still unknown. In this study, we perform paired tumor/peritumoral tissues and peripheral blood samples from 58 patients with HCC and sequenced them with high-throughput TCR to comprehensively analyze the characteristics of TCR and the clinical significance of peripheral TCR sequence. By exploring the abundance and diversity of TCR repertoires, we observe that there was a significantly higher TCR diversity in peripheral blood than in tumoral and peritumoral tissues, while tumoral and peritumoral tissues showed similar TCR diversity. A substantial difference in the usage frequencies of several Vβ, Jβ genes, and TCRβ VJ pairings was found among three types of tissues. Moreover, we reveal that HCC patients have a unique profile of TCR repertoire in peripheral blood in contrast to healthy individuals. We further establish an HCC diagnostic model based on TCRβ VJ pairing usage in peripheral blood, which yields a best-fit area under the curve (AUC) of 0.9746 ± 0.0481 (sensitivity = 0.9675 ± 0.0603, specificity = 0.9998 ± 0.0007, average of 100 repeats) in the test set. Our study describes the characteristics of tissue infiltration and circulating T-cell bank in patients with HCC and shows the potential of using circulating TCR sequence as a biomarker for the non-invasive diagnosis of patients with HCC.