Human umbilical cord mesenchymal stem cells reverse depression in rats induced by chronic unpredictable mild stress combined with lipopolysaccharide.

BACKGROUND: Inflammation and oxidative stress are considered crucial to the pathogenesis of depression. Rat models of depression can be created by combined treatments of chronic unpredictable mild stress (CUMS) and lipopolysaccharide (LPS). Behaviors associated with depression could be improved by treatment with mesenchymal stem cells (MSCs) owing to immunomodulatory functions of the cells. Therapeutic potentials of the MSCs to reverse pro-inflammatory cytokines, proteins, and metabolites were identified by transcriptomic, proteomic, and metabolomic analysis, respectively. METHODS: A depression model was established in male SD rats by 2 weeks of CUMS combined with LPS. The models were verified by behavioral tests, namely SPT, OFT, EPM, and qRT-PCR for pro-inflammatory cytokines. Such depressed rats were administered human umbilical cord MSCs (hUC-MSCs) via the tail vein once a week for 2 and 4 weeks. The homing capacity was confirmed by detection of the fluorescent dye on day 7 after the hUC-MSCs were labeled with CM-Dil and administered. The expression of GFAP in astrocytes serves as a biomarker of CNS disorders and IBA1 in microglia serves as a marker of microglia activation were detected by immunohistochemistry at 2 and 4 weeks after final administration of hUC-MSCs. At the same time, transcriptomics of rat hippocampal tissue, proteomic and metabolomic analysis of the serum from the normal, depressed, and treated rats were also compared. RESULTS: Reliable models of rat depression were successfully induced by treatments of CUMS combined with LPS. Rat depression behaviors, pro-inflammatory cytokines, and morphological disorders of the hippocampus associated with depression were reversed in 4 weeks by hUC-MSC treatment. hUC-MSCs could reach the hippocampus CA1 region through the blood circulation on day 7 after administration owing to the disruption of blood brain barrier (BBB) by microglial activation from depression. Differentiations of whole-genome expression, protein, and metabolite profiles between the normal and depression-modeled rats, which were analyzed by transcriptomic, proteomics, and metabolomics, further verified the high association with depression behaviors. CONCLUSIONS: Rat depression can be reversed or recovered by treatment with hUC-MSCs.

Molecular sieving of iso-butene from C4 olefins with simultaneous high 1,3-butadiene and n-butene uptakes.

Iso-butene (iso-C4H8) is an important raw material in chemical industry, whereas its efficient separation remains challenging due to similar molecular properties of C4 olefins. The ideal adsorbent should possess simultaneous high uptakes for 1,3-butadiene (C4H6) and n-butene (n-C4H8) counterparts, endowing high efficiency for iso-C4H8 separation in adsorption columns. Herein, a sulfate-pillared adsorbent, SOFOUR-DPDS-Ni (DPDS = 4,4'-dipyridyldisulfide), is reported for the efficient iso-C4H8 separation from binary and ternary C4 olefin mixtures. The rigidity in pore sizes and shapes of SOFOUR-DPDS-Ni exerts the molecular sieving of iso-C4H8, while exhibiting high C4H6 and n-C4H8 uptakes. The benchmark Henry's selectivity for C4H6/iso-C4H8 (2321.8) and n-C4H8/iso-C4H8 (233.5) outperforms most reported adsorbents. Computational simulations reveal the strong interactions for C4H6 and n-C4H8. Furthermore, dynamic breakthrough experiments demonstrate the direct production of high-purity iso-C4H8 (>99.9%) from C4H6/iso-C4H8 (50/50, v/v), n-C4H8/iso-C4H8 (50/50, v/v), and C4H6/n-C4H8/iso-C4H8 (50/15/35, v/v/v) gas-mixtures.

Association of Oxidative Stress and Proinflammation with Insomnia in Perimenopause.

Background: The levels of oxidative stress and proinflammatory factors in perimenopausal females increased, and they were also deeply troubled by insomnia. The occurrence of insomnia is related to the changes of oxidative stress and inflammation levels in the body. Perimenopausal insomnia may be related to mild systemic inflammation, and oxidative stress can promote chronic inflammation. However, the underlying mechanism behind the phenomenon is still unclear. Objective: The aim was to investigate whether the occurrence of perimenopausal insomnia disorder is related to higher levels of oxidative stress and inflammation in the body, and to explore the role of inducible nitric oxide synthase (iNOS) in perimenopausal insomnia. Methods: A total of 127 perimenopausal participants were recruited in this study. Participants with global scores of the Pittsburgh sleep quality index (PSQI) >7 were diagnosed with insomnia (n = 54). The patient health questionnaire-9 (PHQ-9) and generalized anxiety disorder-7 (GAD-7) were evaluated, and sociodemographic data were obtained. The serum concentrations of iNOS, interleukin 6 (IL6), and tumor necrosis factor α (TNFα) were measured using commercial assays. Results: In the insomnia group, IL6 levels were positively correlated with scores of component 5 and component 7 of PSQI, respectively. PHQ-9 and GAD-7 were positively correlated with the global score of PSQI component 7 and PSQI, respectively; PHQ-9 was positively correlated with the global score of PSQI component 1. Finally, PHQ-9, iNOS, and IL6 were found to be independent predictors of perimenopausal insomnia using logistic regression. Conclusions: Moderate oxidative stress caused by a certain concentration of iNOS plays a protective role in perimenopausal insomnia, while proinflammation and depression are potential risk factors.

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.

Dissecting the tumor ecosystem of liver cancers in the single-cell era.

Primary liver cancers (PLCs) are a broad class of malignancies that include HCC, intrahepatic cholangiocarcinoma, and combined hepatocellular and intrahepatic cholangiocarcinoma. PLCs are often associated with a poor prognosis due to their high relapse and low therapeutic response rates. Importantly, PLCs exist within a dynamic and complex tumor ecosystem, which includes malignant, immune, and stromal cells. It is critical to dissect the PLC tumor ecosystem to uncover the underlying mechanisms associated with tumorigenesis, relapse, and treatment resistance to facilitate the discovery of novel therapeutic targets. Single-cell and spatial multi-omics sequencing techniques offer an unprecedented opportunity to elucidate spatiotemporal interactions among heterogeneous cell types within the complex tumor ecosystem. In this review, we describe the latest advances in single-cell and spatial technologies and review their applications with respect to dissecting liver cancer tumor ecosystems.

An invasive zone in human liver cancer identified by Stereo-seq promotes hepatocyte-tumor cell crosstalk, local immunosuppression and tumor progression.

Dissecting and understanding the cancer ecosystem, especially that around the tumor margins, which have strong implications for tumor cell infiltration and invasion, are essential for exploring the mechanisms of tumor metastasis and developing effective new treatments. Using a novel tumor border scanning and digitization model enabled by nanoscale resolution-SpaTial Enhanced REsolution Omics-sequencing (Stereo-seq), we identified a 500 µm-wide zone centered around the tumor border in patients with liver cancer, referred to as "the invasive zone". We detected strong immunosuppression, metabolic reprogramming, and severely damaged hepatocytes in this zone. We also identified a subpopulation of damaged hepatocytes with increased expression of serum amyloid A1 and A2 (referred to collectively as SAAs) located close to the border on the paratumor side. Overexpression of CXCL6 in adjacent malignant cells could induce activation of the JAK-STAT3 pathway in nearby hepatocytes, which subsequently caused SAAs' overexpression in these hepatocytes. Furthermore, overexpression and secretion of SAAs by hepatocytes in the invasive zone could lead to the recruitment of macrophages and M2 polarization, further promoting local immunosuppression, potentially resulting in tumor progression. Clinical association analysis in additional five independent cohorts of patients with primary and secondary liver cancer (n = 423) showed that patients with overexpression of SAAs in the invasive zone had a worse prognosis. Further in vivo experiments using mouse liver tumor models in situ confirmed that the knockdown of genes encoding SAAs in hepatocytes decreased macrophage accumulation around the tumor border and delayed tumor growth. The identification and characterization of a novel invasive zone in human cancer patients not only add an important layer of understanding regarding the mechanisms of tumor invasion and metastasis, but may also pave the way for developing novel therapeutic strategies for advanced liver cancer and other solid tumors.

Dihydromyricetin Alleviates H9C2 Cell Apoptosis and Autophagy by Regulating CircHIPK3 Expression and PI3K/AKT/mTOR Pathway.

OBJECTIVE: To investigate the effect and potential mechanism of dihydromyricetin (Dmy) on H9C2 cell proliferation, apoptosis, and autophagy. METHODS: H9C2 cells were randomly divided into 7 groups, namely control, model, EV (empty pCDH-CMV-MCS-EF1-CopGFP-T2A-Puro vector), IV (circHIPK3 interference), Dmy (50 µ mol/L), Dmy+IV, and Dmy+EV groups. Cell proliferation and apoptosis were detected by cell counting kit-8 assay and flow cytometry, respectivley. Western blot was used to evaluate the levels of light chain 3 II/I (LC3II/I), phospho-phosphoinositide 3-kinase (p-PI3K), protein kinase B (p-AKT), and phospho-mammalian target of rapamycin (p-mTOR). The level of circHIPK3 was determined using reverse transcriptase polymerase chain reaction. Electron microscopy was used to observe autophagosomes in H9C2 cells. RESULTS: Compared to H9C2 cells, the expression of circHIPK in H9C2 hypoxia model cells increased significantly (P<0.05). Compared to the control group, the cell apoptosis and autophagosomes increased, cell proliferation rate decreased significantly, and the expression of LC3 II/I significantly increased (all P<0.05). Compared to the model group, the rate of apoptosis and autophagosomes in IV, Dmy, and Dmy+IV group decreased, the cell proliferation rate increased, and the expression of LC3 II/I decreased significantly (all P<0.05). Compared to the control group, the expressions of p-PI3K, p-AKT, and p-mTOR in the model group significantly reduced (P<0.05), whereas after treatment with Dmy and sh-circHIPK3, the above situation was reversed (P<0.05). CONCLUSION: Dmy plays a protective role in H9C2 cells by inhibiting circHIPK expression and cell apoptosis and autophagy, and the mechanism may be related to PI3K/AKT/mTOR pathway.

Gastrodin Regulates the Notch-1 Signal Pathway via Renin-Angiotensin System in Activated Microglia.

Notch-1 and renin angiotensin system (RAS) are involved in microglia activation. It has been reported that gastrodin inhibited inflammatory responses mediated by activated microglia. This study explored the possible interaction between this two pathways, and to determine whether gastrodin would exert its effects on both of them. Expression of RAS, Notch-1 signaling and proinflammatory mediators in lipopolysaccharide (LPS) activated BV-2 microglia subjected to various treatments was determined by Western blot and immunofluorescence. The protein expression of RAS, Notch-1 pathway and TNF-α and IL-1ß was significantly increased in activated microglia. Exogenous Ang II markedly enhanced the expression of these biomarkers. Meanwhile, Azilsartan [a specific inhibitor of AT1 (AT1I)] inhibited the expression of Notch-1 pathway and proinflammatory cytokines. When Notch-1 signaling was inhibited with DAPT, ACE and AT1 expression remained unaffected, indicating that RAS can regulate the Notch-1 pathway in activated microglia but not reciprocally. Additionally, we showed here that gastrodin inhibited the RAS, Notch-1 pathway and inflammatory response. Remarkably, gastrodin did not exert any effect on expression of Notch-1 signaling when RAS was blocked by AT1I, suggesting that gastrodin acts on the RAS directly, not through the Notch-1 pathway. Furthermore, TNF-α and IL-1ß expression was significantly increased in activated microglia treated with exogenous Ang II; the expression, however, was suppressed by gastrodin. Of note, expression of proinflammatory cytokines was further decreased in gastrodin and AT1I combination treatment. The results suggest that gastrodin acts via the RAS which regulates the Notch-1 signaling and inflammation in LPS-induced microglia.

Uncovering the Heterogeneity and Clinical Relevance of Circulating Tumor-Initiating Cells in Hepatocellular Carcinoma Using an Integrated Immunomagnetic-Microfluidic Platform.

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+ CTICs in the blood samples from 33 HCC patients than the FDA-approved CellSearch system (0.92 ± 0.94 vs 0.23 ± 0.36, P < 0.001). The number of EpCAM+ CTICs (≥0.75/mL) detected by iMAC was a predictor of early recurrence (P = 0.007). The distinct stem-related markers' expression of CTICs could distinguish primary HCC, recurrent HCC, and TACE-resistant HCC. Single-cell transcriptional profiling proved the heterogeneity among individual CTICs and separated the four CTIC subsets into distinct phenotypes. Dissecting the heterogeneity of CTICs using the iMAC represents a novel and informative method for accurate CTIC detection and characterization. This innovative technology will enable more indepth cancer biology research and clinical cancer management than is currently available.

CD155/SRC complex promotes hepatocellular carcinoma progression via inhibiting the p38 MAPK signalling pathway and correlates with poor prognosis.

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

Development of a Modified QuEChERS Method Based on Magnetic Multi-Walled Carbon Nanotubes as a Clean-Up Adsorbent for the Analysis of Heterocyclic Aromatic Amines in Braised Sauce Beef.

Heterocyclic aromatic amines (HAAs) generated during the cooking of meats cause adverse effects on human health. The purpose of the current research was to develop a modified QuEChERS (Quick, Easy, Cheap, Effective, Rugged, Safe) method using magnetic multi-walled carbon nanotubes (Fe3O4-MWCNTs) as clean-up adsorbents for the rapid determination of HAAs in braised sauce beef. The significant parameters in extraction and clean-up processes were screened and optimized. Under optimal conditions, the LODs ranged from 3.0 ng/g to 4.2 ng/g. The recoveries (78.5−103.2%) and relative standard deviations RSDs (<4.6%) of five HAAs were obtained. These are in accordance with the validation criteria (recovery in the range of 70−120% with RSD less than 20%). Compared with conventional clean-up adsorbents (PSA or C18), Fe3O4-MWCNTs displayed equivalent or better matrix removal efficiency, while making the pretreatment process easier and more time-saving through magnetic separation. Less usage of adsorbent makes the method possess another advantage of being lower in cost per sample. The method developed was successfully applied to analyze real samples collected from local deli counters, demonstrating Fe3O4-MWCNTs could be considered as an effective alternative adsorbent with great potential in the QuEChERS process.

Mucin 1 promotes tumor progression through activating WNT/ß-catenin signaling pathway in intrahepatic cholangiocarcinoma.

Background: Current treatment options for intrahepatic cholangiocarcinoma (ICC) are limited by the lack of understanding of the disease pathogenesis. It has been known that mucin 1 (MUC1) is a cell surface mucin that highly expressed in various cancer tissues. However, its role in ICC has not been well studied. The purpose of this study was to investigate the clinical significance and biological function of MUC1 in ICC. Methods: qRT-PCR and western blot assays were performed to examine MUC1 expression. RNA-Seq (RNA Sequencing) s conducted to explore the RNA expression. A tissue microarray study including 214 ICC cases was also conducted to evaluate the clinical relevance and prognostic significance of MUC1. The role and underlying mechanisms of MUC1 in regulating cell growth and invasion were further explored both in vitro and in vivo models. Results: The mRNA and protein levels of MUC1 were significantly up-regulated in ICC compared to paired non-tumor tissues. Depletion of MUC1 in HCCC9810 cells significantly inhibited cell proliferation, migration and invasion in vitro and overexpression of MUC1 in RBE cells resulted in increased cell proliferation, migration and invasion. Both univariate and multivariate analysis revealed that the protein expression of MUC1 was associated with overall survival and relapse-free survival after tumor resection. Clinically, high MUC1 expression was more commonly observed in aggressive tumors. Further studies indicated that MUC1 exerted its function through activating Wnt/ ß-catenin pathway. Conclusions: Our data suggests that MUC1 promoted ICC progression via activating Wnt / ß-catenin pathway. This study not only deciphered the role of MUC in ICC pathogenesis, but also shed light upon identifying novel potential therapeutic targets.

Emergency use of COVID-19 vaccines recommended by the World Health Organization (WHO) as of June 2021.

In December 2019, the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused the outbreak of coronavirus disease 2019 (COVID-19), and the resulting pandemic has caused widespread health problems and social and economic disruption. Thus far in 2021, more than 4 million people worldwide have died from COVID-19, so safe and efficacious vaccines are urgently needed to restore normal economic and social activities. According to the official guidance documents of the World Health Organization (WHO), vaccines based on four major strategies including mRNA, adenoviral vectors, inactivated viruses, and recombinant proteins have entered the stage of emergency use authorization and pre-certification evaluation. The current review summarizes these vaccines and it looks ahead to the development of additional COVID-19 vaccines in the future.

Patient-Derived Xenograft Models for Intrahepatic Cholangiocarcinoma and Their Application in Guiding Personalized Medicine.

BACKGROUND: Intrahepatic cholangiocarcinoma (ICC) remains one of the most intractable malignancies. The development of effective drug treatments for ICC is seriously hampered by the lack of reliable tumor models. At present, patient derived xenograft (PDX) models prove to accurately reflect the genetic and biological diversity required to decipher tumor biology and therapeutic vulnerabilities. This study was designed to investigate the establishment and potential application of PDX models for guiding personalized medicine and identifying potential biomarker for lenvatinib resistance. METHODS: We generated PDX models from 89 patients with ICC and compared the morphological and molecular similarities of parental tumors and passaged PDXs. The clinicopathologic features affecting PDX engraftment and the prognostic significance of PDX engraftment were analyzed. Drug treatment responses were analyzed in IMF-138, IMF-114 PDX models and corresponding patients. Finally, lenvatinib treatment response was examined in PDX models and potential drug resistance mechanism was revealed. RESULTS: Forty-nine PDX models were established (take rate: 55.1%). Successful PDX engraftment was associated with negative HbsAg (P = 0.031), presence of mVI (P = 0.001), poorer tumor differentiation (P = 0.023), multiple tumor number (P = 0.003), presence of lymph node metastasis (P = 0.001), and later TNM stage (P = 0.039). Moreover, patients with tumor engraftment had significantly shorter time to recurrence (TTR) (P < 0.001) and worse overall survival (OS) (P < 0.001). Multivariate analysis indicated that PDX engraftment was an independent risk factor for shortened TTR (HR = 1.84; 95% CI, 1.05-3.23; P = 0.034) and OS (HR = 2.13; 95% CI, 1.11-4.11; P = 0.024). PDXs were histologically and genetically similar to their parental tumors. We also applied IMF-138 and IMF-114 PDX drug testing results to guide clinical treatment for patients with ICC and found similar treatment responses. PDX models also facilitated personalized medicine for patients with ICC based on drug screening results using whole exome sequencing data. Additionally, PDX models reflected the heterogeneous sensitivity to lenvatinib treatment and CDH1 might be vital to lenvatinib-resistance. CONCLUSION: PDX models provide a powerful platform for preclinical drug discovery, and potentially facilitate the implementation of personalized medicine and improvement of survival of ICC cancer patient.

Bawei Chenxiang Wan Ameliorates Cardiac Hypertrophy by Activating AMPK/PPAR-α Signaling Pathway Improving Energy Metabolism.

Bawei Chenxiang Wan (BCW), a well-known traditional Chinese Tibetan medicine formula, is effective for the treatment of acute and chronic cardiovascular diseases. In the present study, we investigated the effect of BCW in cardiac hypertrophy and underlying mechanisms. The dose of 0.2, 0.4, and 0.8 g/kg BCW treated cardiac hypertrophy in SD rat model induced by isoprenaline (ISO). Our results showed that BCW (0.4 g/kg) could repress cardiac hypertrophy, indicated by macro morphology, heart weight to body weight ratio (HW/BW), left ventricle heart weight to body weight ratio (LVW/BW), hypertrophy markers, heart function, pathological structure, cross-sectional area (CSA) of myocardial cells, and the myocardial enzymes. Furthermore, we declared the mechanism of BCW anti-hypertrophy effect was associated with activating adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK)/peroxisome proliferator-activated receptor-α (PPAR-α) signals, which regulate carnitine palmitoyltransferase1ß (CPT-1ß) and glucose transport-4 (GLUT-4) to ameliorate glycolipid metabolism. Moreover, BCW also elevated mitochondrial DNA-encoded genes of NADH dehydrogenase subunit 1(ND1), cytochrome b (Cytb), and mitochondrially encoded cytochrome coxidase I (mt-co1) expression, which was associated with mitochondria function and oxidative phosphorylation. Subsequently, knocking down AMPK by siRNA significantly can reverse the anti-hypertrophy effect of BCW indicated by hypertrophy markers and cell surface of cardiomyocytes. In conclusion, BCW prevents ISO-induced cardiomyocyte hypertrophy by activating AMPK/PPAR-α to alleviate the disturbance in energy metabolism. Therefore, BCW can be used as an alternative drug for the treatment of cardiac hypertrophy.

BRCA1-associated protein 1 serves as a tumor suppressor in hepatocellular carcinoma by deubiquitinating and stabilizing PTEN.

BRCA1-associated protein 1 (BAP1) or its mutants have been known to play critical regulatory roles in tumor biology, yet their role in hepatocellular carcinoma (HCC) remains largely unclear. In this study, we detected the mutations of all the exons of BAP1 in 105 HCC patients using Sanger sequencing, and found eight somatic mutations in 6 (5.71%) patients. We also found that the mRNA and protein levels of BAP1 were markedly downregulated in HCC versus the adjacent non-tumor tissues. Wild-type BAP1 but not mutant BAP1 significantly inhibited HCC cell proliferation, invasion, epithelial-mesenchymal transition (EMT) in vitro, and tumor progression and metastasis in vivo. Mechanistically, BAP1 complexed with PTEN and stabilized PTEN via deubiquitination and, furthermore, negatively regulated HCC cell EMT by deactivating the AKT/GSK-3ß/Snail pathway. However, those tumor-inhibitory effects of BAP1 were abolished by inactivating mutations. Clinically, low BAP1 expression was positively correlated to aggressive tumor phenotypes, which also independently associated with poorer recurrence-free survival and overall survival after curative hepatectomy. Conclusively, our results indicate that BAP1, significantly downregulated, somatically mutated and negatively regulating EMT in HCC, serves as a tumor suppressor of HCC by deubiquitinating and stabilizing PTEN.

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.

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.

CD13 promotes hepatocellular carcinogenesis and sorafenib resistance by activating HDAC5-LSD1-NF-κB oncogenic signaling.

RATIONALE: CD13 is a new marker for liver cancer stem cells (CSCs) that contributes to sorafenib resistance in hepatocellular carcinoma (HCC). However, the underlying mechanism of CD13 in HCC sorafenib resistance remains enigmatic. METHODS: The expression of CD13 in HCC cell lines and tissues was assayed by RT-PCR, western-blot, and immunohistochemistry staining. Athymic BALB/c nu/nu mice model was used to study the in vivo functions of CD13. Clinical significance of CD13 was evaluated by Kaplan-Meier methods. Cellular proliferation rate was evaluated by cell counting kit-8 cell proliferation assay and colony formation assay. Tunel assay was used to detect cell death ratio. Transwell assay was used to evaluate the motility of cells. Immunoprecipitation (IP), liquid chromatography-mass spectrometry (LC-MS)/MS, and co-IP were applied to investigate potential protein interactions of CD13. RESULTS: In this research, we found that CD13 expression was higher in metastatic HCC samples, and its overexpression was predicted worse prognosis for patients after surgical resection. Functionally, CD13 promoted HCC proliferation, invasion, cell cycle progression as well as sorafenib resistance. Mechanistically, CD13 interacted with histone deacetylase5 (HDAC5) to promote its protein stability, thus resulting in HDAC5-mediated lysine-specific demethylase 1 (LSD1) deacetylation and protein stabilization. Consequently, LSD1 decreased the NF-κB catalytic unit p65 methylation that led to p65 protein stability. A CD13 inhibitor ubenimex in combination with sorafenib, suppressed the tumor growth and attenuated the resistance of HCC cells toward sorafenib in patient-derived xenograft models. CONCLUSIONS: CD13 promotes HCC progression and induces sorafenib resistance, mainly via interacting with HDAC5 to prevent the degradation of p65 and activate NF-kB signaling pathway. CD13 is a prognostic indicator for HCC patients underwent curative resection as well as a predictor of response to treatment with sorafenib. Our study establishes the new therapeutic potential of targeting CD13-HDAC5-LSD1-NF-κB in HCC.

Effect of surgical margin on recurrence based on preoperative circulating tumor cell status in hepatocellular carcinoma.

BACKGROUND: High rates of recurrence after resection severely worsen hepatocellular carcinoma (HCC) prognosis. This study aims to explore whether circulating tumor cell (CTC) is helpful in determine the appropriate liver resection margins for HCC patients. METHODS: HCC patients who underwent liver resection were enrolled into training (n=117) or validation (n=192) cohorts, then classified as CTC-positive (CTC≥1) or CTC-negative (CTC=0). A standardized pathologic sampling method was used in the training cohort to quantify microvascular invasion (mVI) and the farthest mVI from the tumor (FMT). FINDINGS: CTC number positively correlated with mVI counts (r=0.655, P<0.001) and FMT (r=0.495, P<0.001). The CTC-positive group had higher mVI counts (P=0.032) and greater FMT P=0.008) than the CTC-negative group. In the CTC-positive group, surgical margins of >1 cm independently protected against early recurrence (training cohort, P=0.004; validation cohort, P=0.001) with lower early recurrence rates (training cohort, 20.0% vs. 65.1%, P=0.005; validation cohort, 36.4% vs. 65.1%, P=0.003) compared to surgical margins of ≤1 cm. No differences in postoperative liver function were observed between patients with margins >1 cm vs. ≤1 cm. Surgical margin size minimally impacted early postoperative HCC recurrence in CTC-negative patients when using 0.5 cm or 1 cm as the threshold. INTERPRETATIONS: Preoperative CTC status predicts mVI severity in HCC patients and is a potential factor for determining optimal surgical margin size to ensure disease eradication and conserve liver function. A surgical margin of >1 cm should be achieved for patients with positive CTC. FUNDING: A full list of funding bodies that contributed to this study can be found in the Acknowledgement section.