Single-cell landscape of the ecosystem in early-relapse hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) has high relapse and low 5-year survival rates. Single-cell profiling in relapsed HCC may aid in the design of effective anticancer therapies, including immunotherapies. We profiled the transcriptomes of ∼17,000 cells from 18 primary or early-relapse HCC cases. Early-relapse tumors have reduced levels of regulatory T cells, increased dendritic cells (DCs), and increased infiltrated CD8+ T cells, compared with primary tumors, in two independent cohorts. Remarkably, CD8+ T cells in recurrent tumors overexpressed KLRB1 (CD161) and displayed an innate-like low cytotoxic state, with low clonal expansion, unlike the classical exhausted state observed in primary HCC. The enrichment of these cells was associated with a worse prognosis. Differential gene expression and interaction analyses revealed potential immune evasion mechanisms in recurrent tumor cells that dampen DC antigen presentation and recruit innate-like CD8+ T cells. Our comprehensive picture of the HCC ecosystem provides deeper insights into immune evasion mechanisms associated with tumor relapse.

Detection of circulating tumour cells enables early recurrence prediction in hepatocellular carcinoma patients undergoing liver transplantation.

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 < .05). Post-operative CTC count ≥ 1 per 5 mL peripheral blood was identified as a potential biomarker for predicting tumour recurrence after LTx, especially in patients with no detectable CTCs prior to LTx and negative tumour serological biomarkers. The predictive value of post-operative CTC count ≥ 1 per 5 mL blood was retained in patients who did not meet the Milan criteria, University of California San Francisco (UCSF) criteria, or Fudan criteria (all P < .05). Furthermore, post-operative serial CTC detection may be useful in post-surgical surveillance for HCC recurrence. CONCLUSIONS: CTCs may be a useful biomarker to evaluate recurrence risk following LTx in patients with HCC. Evaluation based on CTC detection may enhance the post-transplant management of HCC, and improve the therapeutic efficacy of LTx.

Deep learning-based automated detection of glaucomatous optic neuropathy on color fundus photographs.

PURPOSE: To develop a deep learning approach based on deep residual neural network (ResNet101) for the automated detection of glaucomatous optic neuropathy (GON) using color fundus images, understand the process by which the model makes predictions, and explore the effect of the integration of fundus images and the medical history data from patients. METHODS: A total of 34,279 fundus images and the corresponding medical history data were retrospectively collected from cohorts of 2371 adult patients, and these images were labeled by 8 glaucoma experts, in which 26,585 fundus images (12,618 images with GON-confirmed eyes, 1114 images with GON-suspected eyes, and 12,853 NORMAL eye images) were included. We adopted 10-fold cross-validation strategy to train and optimize our model. This model was tested in an independent testing dataset consisting of 3481 images (1524 images from NORMAL eyes, 1442 images from GON-confirmed eyes, and 515 images from GON-suspected eyes) from 249 patients. Moreover, the performance of the best model was compared with results obtained by two experts. Accuracy, sensitivity, specificity, kappa value, and area under receiver operating characteristic (AUC) were calculated. Further, we performed qualitative evaluation of model predictions and occlusion testing. Finally, we assessed the effect of integrating medical history data in the final classification. RESULTS: In a multiclass comparison between GON-confirmed eyes, GON-suspected eyes and NORMAL eyes, our model achieved 0.941 (95% confidence interval [CI], 0.936-0.946) accuracy, 0.957 (95% CI, 0.953-0.961) sensitivity, and 0.929 (95% CI, 0.923-0.935) specificity. The AUC distinguishing referrals (GON-confirmed and GON-suspected eyes) from observation was 0.992 (95% CI, 0.991-0.993). Our best model had a kappa value of 0.927, while the two experts' kappa values were 0.928 and 0.925 independently. The best 2 binary classifiers distinguishing GON-confirmed/GON-suspected eyes from NORMAL eyes obtained 0.955, 0.965 accuracy, 0.977, 0.998 sensitivity, and 0.929, 0.954 specificity, while the AUC was 0.992, 0.999 respectively. Additionally, the occlusion testing showed that our model identified the neuroretinal rim region, retinal nerve fiber layer (RNFL) defect areas (superior or inferior) as the most important parts for the discrimination of GON, which evaluated fundus images in a way similar to clinicians. Finally, the results of integration of fundus images with medical history data showed a slight improvement in sensitivity and specificity with similar AUCs. CONCLUSIONS: This approach could discriminate GON with high accuracy, sensitivity, specificity, and AUC using color fundus photographs. It may provide a second opinion on the diagnosis of glaucoma to the specialist quickly, efficiently and at low cost, and assist doctors and the public in large-scale screening for glaucoma.

Clinical Characteristics and Prognostic Factors of Patients with Intrahepatic Cholangiocarcinoma with Fever: A Propensity Score Matching Analysis.

BACKGROUND: Patients with intrahepatic cholangiocarcinoma (ICC) rarely present fever as the initial symptom. We aimed to identify clinical characteristics and prognostic factors for these feverish patients. SUBJECTS, MATERIALS, AND METHODS: This study retrospectively reviewed 31 patients with ICC with fever (≥38.0°C) treated at our hospital between January 2002 and December 2014. A propensity score was used to match patients with and without fever at a ratio of 1:2. RESULTS: Patients with ICC with fever had higher serum γ-glutamyl transferase and carcinoembryonic antigen levels, larger tumors, poorer tumor differentiation, and worse prognosis (all p < .05) than those without fever. This was supported by propensity score matching (PSM) analysis. Univariate and multivariate analyses indicated that microvascular invasion, hilar lymph node metastasis, and temperature ≥ 38.6°C were related to prognosis. Patients with ICC with fever had higher levels of leucocytes, neutrophils, neutrophil-to-lymphocyte ratio (NLR), and platelet-to-lymphocyte ratio (PLR) in peripheral blood before and after PSM analysis. Body temperature positively correlated with leucocytes (r = 0.599, p < .001), neutrophils (r = 0.644, p < .001), NLR (r = 0.681, p < .001), and PLR (r = 0.457, p = .010). CONCLUSION: Patients with ICC with fever ≥38.0°C and ≥38.6°C had poor and extremely poor prognosis, respectively. Radical surgical treatment may improve the prognosis of patients with ICC with fever <38.6°C. However, systemic therapy (e.g., anti-inflammatory and immune therapy) may be preferable to surgery for these patients with fever ≥38.6°C. IMPLICATIONS FOR PRACTICE: Patients with intrahepatic cholangiocarcinoma (ICC) with fever (≥38.0°C) as the initial symptom are extremely rare. Because their symptoms are similar to those of liver abscess, diagnosis is challenging, and most of these patients are already at an advanced stage at the time of diagnosis. Patients with ICC with fever had different clinical characteristics and worse prognosis than those without fever. The prognosis of those with temperature <38.6°C would be improved by timely surgical intervention. Those with fever ≥38.6°C had an extremely dismal outcome, although they all received radical surgical treatment. New therapeutic strategies are needed to improve survival for patients with ICC with temperature ≥38.6°C.

Tiliroside attenuates acute kidney injury by inhibiting ferroptosis through the disruption of NRF2-KEAP1 interaction.

BACKGROUND: Ferroptosis, an iron-dependent process that regulates cell death. Emerging evidences suggest that ferroptosis induces acute kidney injury (AKI) progression, and inhibiting ferroptosis provides an effect strategy for AKI treatment. The disruption of the NRF2-KEAP1 protein to protein interaction (PPI) induces NRF2 activation, which provides a promising strategy that can identify new ferroptosis inhibitors. A previous study revealed that tiliroside, a glycosidic flavonoid extracted from Edgeworthia chrysantha Lindl (buds), has anti-neuroinflammatory and neuroprotective effects via NRF2 activation. However, the mechanism through which tiliroside activates NRF2 is unknown, and it remains unclear whether it has protective effects against AKI. PURPOSE: To investigate whether tiliroside has protective effects against AKI in mice and the associated mechanisms. METHODS: Possible tiliroside substrates were analyzed using molecular docking. Cisplatin- and ischemia-reperfusion injury (IRI)-induced AKI mouse models and HK2 cells model were constructed to evaluate the protective effects of tiliroside. CRISPR/Cas9 mediated NRF2 knockout HK2 cells were used to verify whether NRF2 mediates tiliroside protective effects. RESULTS: In vivo, our results showed that tiliroside treatment preserved kidney functions in AKI mice models, as showed by lower levels of serum creatinine (SCr), blood urea nitrogen (BUN), and renal injury markers, including neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule 1 (KIM1), compared with the mice in control groups. In vitro, tiliroside treatment greatly ameliorated cisplatin-induced ferroptosis through NRF2 activation in cultured HK2 cells, as evidenced by the protective effects of tiliroside being greatly blunted after the knockout of NRF2 in HK2 cells. Mechanistic studies indicated that tiliroside promoted NRF2/GPX4 pathway activation and ferroptosis inhibition, perhaps via the disruption of the NRF2-KEAP1 PPI. CONCLUSION: Together, our results demonstrate that tiliroside may serve as a NRF2-KEAP1 PPI inhibitor and prevents ferroptosis-induced AKI, indicating its potential for clinical AKI treatment.

Olesoxime protects against cisplatin-induced acute kidney injury by attenuating mitochondrial dysfunction.

BACKGROUND: Mitochondrial dysfunction is a critical factor in the pathogenesis of acute kidney injury (AKI). Agents that ameliorate mitochondrial dysfunction hold potential for AKI treatment. The objective of this study was to investigate the impact of olesoxime, a novel mitochondrial-targeted agent, on cisplatin-induced AKI. METHODS: In vivo, a cisplatin-induced AKI mouse model was established by administering a single intraperitoneal dose of cisplatin (25 mg/kg) to male C57BL/6 mice for 72 hours, followed by gavage of either olesoxime or a control solution. In vitro, human proximal tubular HK2 cells were cultured and subjected to treatments with cisplatin, either in the presence or absence of olesoxime. RESULTS: In vivo, our findings demonstrated that olesoxime administration significantly mitigated the nephrotoxic effects of cisplatin in mice, as evidenced by reduced blood urea nitrogen (BUN) and serum creatinine (SCr) levels, improved renal histopathology, and decreased expression of renal tubular injury markers such as kidney injury molecule 1 (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL). Furthermore, olesoxime administration markedly reduced cisplatin-induced apoptosis, inflammation, and oxidative stress in the kidneys of AKI mice. Additionally, olesoxime treatment effectively restored mitochondrial function in the kidneys of AKI mice. In vitro, our results indicated that olesoxime treatment protected against cisplatin-induced apoptosis and mitochondrial dysfunction in cultured HK2 cells. Notably, cisplatin's anticancer effects were unaffected by olesoxime treatment in human cancer cells. CONCLUSION: The results of this study suggest that olesoxime is a viable and efficient therapeutic agent in the treatment of cisplatin-induced acute kidney injury presumably by alleviating mitochondrial dysfunction.

Integrated multi-omics profiling to dissect the spatiotemporal evolution of metastatic hepatocellular carcinoma.

Comprehensive molecular analyses of metastatic hepatocellular carcinoma (HCC) are lacking. Here, we generate multi-omic profiling of 257 primary and 176 metastatic regions from 182 HCC patients. Primary tumors rich in hypoxia signatures facilitated polyclonal dissemination. Genomic divergence between primary and metastatic HCC is high, and early dissemination is prevalent. The remarkable neoantigen intratumor heterogeneity observed in metastases is associated with decreased T cell reactivity, resulting from disruptions to neoantigen presentation. We identify somatic copy number alterations as highly selected events driving metastasis. Subclones without Wnt mutations show a stronger selective advantage for metastasis than those with Wnt mutations and are characterized by a microenvironment rich in activated fibroblasts favoring a pro-metastatic phenotype. Finally, metastases without Wnt mutations exhibit higher enrichment of immunosuppressive B cells that mediate terminal exhaustion of CD8+ T cells via HLA-E:CD94-NKG2A checkpoint axis. Collectively, our results provide a multi-dimensional dissection of the complex evolutionary process of metastasis.

Endoscopic management of colorectal polyps.

Colorectal polyps are premalignant lesions in the lower gastrointestinal tract. Endoscopic polypectomy is an effective strategy to prevent colorectal cancer morbidity and more invasive procedures. Techniques for the endoscopic resection of polyps keep evolving, and endoscopists are required to perform the most appropriate technique for each polyp. In this review, we outline the evaluation and classification of polyps, update the recommendations for optimal treatment, describe the polypectomy procedures and their strengths/weaknesses, and discuss the promising innovative methods or concepts.

Synthetic miR-26a mimics delivered by tumor exosomes repress hepatocellular carcinoma through downregulating lymphoid enhancer factor 1.

BACKGROUND: The dysregulation of exosomal microRNAs plays an important role in the progression of hepatocarcinogenesis. In this study, we investigated the therapeutic potential of synthetic exosomal miR-26a against HCC cells and explored the feasibility of tumor-derived exosomes as drug delivery vehicles. METHODS: Proliferation and migration assays were performed to examine the effects of miR-26a on HCC in vitro. The direct target gene of miR-26a was identified through miRecords analysis and target validation. The transferring efficiency and anti-HCC effect of exosomes with different origin were studied and the optimal miR-26a delivery method was established and verified in vitro and in vivo. In addition, the relationships between prognosis of HCC patients and miR-26a expression in HCC serum and exosomes were retrospectively analyzed. RESULTS: Here, we found that tumor cell-derived exosomes were taken in preferentially by HCC cells and promoted HCC progression through Wnt pathway by low-density lipoprotein receptor-related protein 6 (LRP6). HCC cells with vacuolar protein sorting-associated protein 35 knocked down were adopted to generate engineered LRP6-exosomes. The engineered HCC-derived exosomes loading miR-26a inhibited HCC progression in vitro and in vivo effectively. Overexpression of miR-26a impaired the growth and migration of HCC by targeting lymphoid enhancer factor 1 (LEF1). Moreover, low expression of exosomal miR-26a was an independent prognostic factor for recurrence and survival in HCC patients. CONCLUSIONS: Our findings suggested the exosomal miR-26a could serve as a non-invasive prognostic marker for HCC patients. Genetically modified tumor-derived exosomes showed preferable transfection efficiency but reduced Wnt activity, which provides a novel therapeutic strategy for HCC.

Long-term outcomes and prognosis for patients with sarcomatoid hepatocellular carcinoma.

Background: Characterized by spindle cell composition in hepatocellular carcinoma tumor, sarcomatoid hepatocellular carcinoma (SHC) is a rare malignant with poor prognosis. In this study, we aimed to evaluate the clinical and pathological features of SHC and establish a nomogram that can predict long-term outcomes of the disease. Methods: We retrospectively analyzed 63 patients who were diagnosed with SHC between October 2007 and November 2016 and used immunohistochemistry (IHC) to assessed various markers in liver samples. The clinical data and the histological and pathological findings were collected and used to build a nomogram to predict survival. Results: The median overall survival (OS) and the recurrence-free survival (RFS) in SHC were 23.2 and 8.4 months, respectively. High expression levels of tyrosine-protein kinase Met (17/63, 27.0%) were associated with poorer RFS (P=0.040). A panel of markers, consisting heat-shock protein 70 (HSP70), glutamine synthetase (GS), and glypican-3 (GPC3), merged as an independent risk factor for treatment outcomes. The nomogram, which including this panel of markers, predicted OS times with a concordance-index (C-index) score of 0.758 (95% CI: 0.672-0.843) in the training set and 0.832 (95% CI: 0.712-0.952) in the validation set. The use of the nomogram showed marked improvements in the prediction of patient outcomes compared with conventional staging systems (P<0.05). Conclusions: Diagnosis of SHC is rare and has a relatively poor prognosis. A panel of markers HSP70, GS and GPC3 served as an independent prognostic factor for SHC.

Characteristics and Clinical Significance of T-Cell Receptor Repertoire in Hepatocellular Carcinoma.

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 Vß, Jß genes, and TCRß 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 TCRß 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.

Exosome-depleted MiR-148a-3p derived from Hepatic Stellate Cells Promotes Tumor Progression via ITGA5/PI3K/Akt Axis in Hepatocellular Carcinoma.

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 in vitro. Cell migration and invasion ability were evaluated by wound-healing and Transwell assays. Western blot, quantitative real-time PCR and Luciferase reporter assay were performed to determine the target gene of miR-148a-3p. A xenograft liver cancer model was established to study the function of exosomal miR-148a-3p in vivo. We found that miR-148a-3p was downregulated in co-cultured HSCs and overexpression of miR-148a-3p in HSCs impaired the proliferation and invasiveness of HCC both in vitro and in vivo. Moreover, further study showed that the miR-148a-3p was also downexpressed in HSCs-derived exosomes, and increased HSCs-derived exosomal miR-148a-3p suppressed HCC tumorigenesis through ITGA5/PI3K/Akt pathway. In conclusion, our study demonstrated that exosome-depleted miR-148a-3p derived from activated HSCs accelerates HCC progression through ITGA5/PI3K/Akt axis.

Development and validation of a non-invasive model for diagnosing HBV-related liver cirrhosis.

BACKGROUND: Liver cirrhosis is closely related to the abnormal liver function and occurrence of liver cancer. Accurate non-invasive assessment of liver cirrhosis is of great significance for preventing disease progression and treatment decision-making. We aim to develop and validate a non-invasive diagnostic model for liver cirrhosis in patients with chronic hepatitis B (CHB). METHODS: From July 2015 to April 2017, seven-hundred fifty-four patients with primary HBV-related liver cancer who underwent hepatectomy were reprospectively recruited. All patients were examined with 2D-SWE and serologic testing preoperatively, which were utilized for measurement of liver stiffness and serum fibrosis models. The stage of liver fibrosis was evaluated using a resected liver specimen. Least absolute shrinkage and selection operator (Lasso) regression was used for feature selection and binary logistic regression analysis was chosen to build a diagnostic model, which was presented as a nomogram and evaluated for calibration, discrimination and clinical usefulness. The performance of noninvasive model was then prospectively validated in an independent cohort (361 patients) by the ROC curve analysis. RESULTS: The diagnostic model, which consists of 5 selected clinical characteristics (PIII-NP, IV-C, Hyaluronan, Platelet and Liver stiffness), showed the strongest correlation with liver fibrosis stage (ρ = 0.702, P < 0.05). Compared with APRI, FIB-4, King's Score, and Forns Index, the model presented the optimal discrimination and the best predictive performance with the highest AUC in the training cohort (0.866, 95%CI 0.840-0.892, P < 0.05) and validation cohorts (0.852, 95%CI 0.813-0.890, P < 0.05). Decision curve analysis demonstrated that nomogram based on the model was extremely useful for diagnosing cirrhosis in patients with chronic hepatitis B. CONCLUSION: This study proposes a non-invasive diagnostic model that incorporates the clinical predictors which can be conveniently used in the individualized diagnosis of HBV-related liver cirrhosis.

scDPN for High-throughput Single-cell CNV Detection to Uncover Clonal Evolution During HCC Recurrence.

Single-cell genomics provides substantial resources for dissecting cellular heterogeneity and cancer evolution. Unfortunately, classical DNA amplification-based methods have low throughput and introduce coverage bias during sample preamplification. We developed a single-cell DNA library preparation method without preamplification in nanolitre scale (scDPN) to address these issues. The method achieved a throughput of up to 1800 cells per run for copy number variation (CNV) detection. Also, our approach demonstrated a lower level of amplification bias and noise than the multiple displacement amplification (MDA) method and showed high sensitivity and accuracy for cell line and tumor tissue evaluation. We used this approach to profile the tumor clones in paired primary and relapsed tumor samples of hepatocellular carcinoma (HCC). We identified three clonal subpopulations with a multitude of aneuploid alterations across the genome. Furthermore, we observed that a minor clone of the primary tumor containing additional alterations in chromosomes 1q, 10q, and 14q developed into the dominant clone in the recurrent tumor, indicating clonal selection during recurrence in HCC. Overall, this approach provides a comprehensive and scalable solution to understand genome heterogeneity and evolution.

Circulating tumor cell detection and single-cell analysis using an integrated workflow based on ChimeraX® -i120 Platform: A prospective study.

Circulating tumor cell (CTC) analysis holds great potential to be a noninvasive solution for clinical cancer management. A complete workflow that combined CTC detection and single-cell molecular analysis is required. We developed the ChimeraX® -i120 platform to facilitate negative enrichment, immunofluorescent labeling, and machine learning-based identification of CTCs. Analytical performances were evaluated, and a total of 477 participants were enrolled to validate the clinical feasibility of ChimeraX® -i120 CTC detection. We analyzed copy number alteration profiles of isolated single cells. The ChimeraX® -i120 platform had high sensitivity, accuracy, and reproducibility for CTC detection. In clinical samples, an average value of > 60% CTC-positive rate was found for five cancer types (i.e., liver, biliary duct, breast, colorectal, and lung), while CTCs were rarely identified in blood from healthy donors. In hepatocellular carcinoma patients treated with curative resection, CTC status was significantly associated with tumor characteristics, prognosis, and treatment response (all P < 0.05). Single-cell sequencing analysis revealed that heterogeneous genomic alteration patterns resided in different cells, patients, and cancers. Our results suggest that the use of this ChimeraX® -i120 platform and the integrated workflow has validity as a tool for CTC detection and downstream genomic profiling in the clinical setting.

Dissecting spatial heterogeneity and the immune-evasion mechanism of CTCs by single-cell RNA-seq in hepatocellular carcinoma.

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.

Rapid detection of hepatocellular carcinoma metastasis using reverse transcription loop-mediated isothermal amplification.

The main therapies of hepatocellular carcinoma (HCC) are hepatectomy and liver transplantation, but the recurrence rate of HCC after radical resection remains high. We established a novel method based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) for the rapid identification of four cancer stem cell-specific biomarkers to estimate the potential risk of HCC metastasis. After optimizing the final concentrations of Mg2+ and betaine, the sensitivity limits for detection of CK19 and EpCAM could reach 10 to 20 copies, while the sensitivity limits for the detection of CD133 and CD90 could reach 10 copies. We detected clinical specimens from 10 HCC patients and performed analysis before and after receiving hepatectomy using RT-LAMP and quantitative reverse transcription polymerase chain reaction (qRT-PCR). The results of both were consistent, but RT-LAMP was proved to be a more rapid, more sensitive, and more economic approach. This novel method is expected to estimate the recurrence and metastasis of HCC for clinical application by combining various low-cost circulating tumor cell sorting and detection tools.

Integrated Bioinformatics analysis and clinical validation reveals that high expression of mucin 1 in intrahepatic cholangiocarcinoma predicts recurrence after curative resection.

Intrahepatic cholangiocarcinoma (ICC) is a cancer type with high malignancy and a current lack of biomarkers to predict recurrence. In the present study, to identify potential biomarkers, five ICC datasets from the Gene Expression Omnibus database were analyzed to construct initial datasets by using a robust rank aggregation approach. A total of 19 upregulated genes were identified in the initial datasets. The genes identified were then further analysed using data from The Cancer Genome Atlas. Only mucin 1 (MUC1) exhibited significance regarding differential expression and survival prediction. Finally, the expression levels of MUC1 were assessed using reverse transcription-quantitative PCR in 61 pairs of ICC tumor and matched non-cancerous samples. The expression of MUC1 was significantly elevated in ICC tissues compared with that in matched non-cancerous counterparts (P=0.001). Patients with high MUC1 expression levels had significantly shorter overall survival (OS, P=0.009) and recurrence-free survival (RFS, P=0.012). MUC1 was identified as an independent prognostic factor for OS [hazard ratio (HR)=2.364, 95%CI: 1.214-4.485; P=0.023] and RFS (HR=2.552 95%CI: 1.294-5.032; P=0.007) in the multivariate analysis. Using receiver operating characteristic analysis, a co-index including MUC1 had a high accuracy for predicting survival [MUC1 combined with serum levels of CEA and cancer antigen 19-9, and lymph node metastasis, area under curve (AUC)=0.746, 95%CI: 0.620-0.872] and recurrence (MUC1 combined with bile duct invasion and lymph node metastasis, AUC=0.729, 95%CI: 0.605-854). In conclusion, MUC1 is highly expressed in ICC tissue and is a potential prognostic biomarker and therapeutic target for ICC.

Anlotinib suppresses tumor progression via blocking the VEGFR2/PI3K/AKT cascade in intrahepatic cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (ICC) is a malignant tumor derived from bile duct epithelium. Its characteristics include an insidious onset and frequent recurrence or metastasis after surgery. Current chemotherapies and molecular target therapies provide only modest survival benefits to patients with ICC. Anlotinib is a novel multi-target tyrosine kinase inhibitor that has good antitumor effects in a variety of solid tumors. However, there are few studies of anlotinib-associated mechanisms and use as a treatment in ICC. In this study using in vitro experiments, we found that anlotinib had significant effects on proliferation inhibition, migration and invasion restraint, and cell-cycle arrestment. Anlotinib treatment affected induction of apoptosis and the mesenchymal-epithelial transition. Patient-derived xenograft models generated directly from patients with ICC revealed that anlotinib treatment dramatically hindered in vivo tumor growth. We also examined anlotinib's mechanism of action using transcriptional profiling. We found that anlotinib treatment might mainly inhibit tumor cell proliferation and invasion and promote apoptosis via cell-cycle arrestment by inactivating the VEGF/PI3K/AKT signaling pathway, as evidenced by significantly decreased phosphorylation levels of these kinases. The activation of vascular endothelial growth factor receptor 2 (VEGFR2) can subsequently activate PI3K/AKT signaling. We identified VEGRF2 as the main target of anlotinib. High VEGFR2 expression might serve as a promising indicator when used to predict a favorable therapeutic response. Taken together, these results indicated that anlotinib had excellent antitumor activity in ICC, mainly via inhibiting the phosphorylation level of VEGFR2 and subsequent inactivation of PIK3/AKT signaling. This work provides evidence and a rationale for using anlotinib to treat patients with ICC in the future.

USP1 Maintains the Survival of Liver Circulating Tumor Cells by Deubiquitinating and Stabilizing TBLR1.

The prognosis of hepatocellular carcinoma (HCC) is closely associated with the occurrence of distant metastases, which is likely due to circulating tumor cells (CTCs). However, the low number of CTCs is the main obstacle limiting research of the mechanism of CTC metastasis. Here, We evaluated the role of ubiquitin-specific protease 1 (USP1) in promoting CTC survival during blood-borne metastases. We observed that USP1 was frequently upregulated in CTCs and correlated with metastasis and a reduced overall survival rate of patients. Additionally, genetic knockout of USP1 the survival rate of CTCs. Further analyses showed that USP1 mediates oncogenic activity by deubiquitinating and stabilizing transducin ß-like 1 X-linked receptor 1 (TBLR1), which plays essential roles in regulating Wnt signaling. These results demonstrated that USP1 may act as an essential factor in promoting the survival of CTCs and suggest that inhibition of USP1 is a potential strategy for HCC treatment.