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.

Platelet-mediated circulating tumor cell evasion from natural killer cell killing through immune checkpoint CD155-TIGIT.

BACKGROUND AND AIMS: Circulating tumor cells (CTCs) are precursors of cancer metastasis. However, how CTCs evade immunosurveillance during hematogenous dissemination remains unclear. APPROACH AND RESULTS: We identified CTC-platelet adhesions by single-cell RNA sequencing and multiplex immunofluorescence of blood samples from multiple cancer types. Clinically, CTC-platelet aggregates were associated with significantly shorter progression-free survival and overall survival in patients with HCC. In vitro, ex vivo, and in vivo assays demonstrated direct platelet adhesions gifted cancer cells with an evasive ability from NK cell killing by upregulating inhibitory checkpoint CD155 (PVR cell adhesion molecule), therefore facilitating distant metastasis. Mechanistically, CD155 was transcriptionally regulated by the FAK/JNK/c-Jun cascade in a platelet contact-dependent manner. Further competition assays and cytotoxicity experiments revealed that CD155 on CTCs inhibited NK-cell cytotoxicity only by engaging with immune receptor TIGIT, but not CD96 and DNAM1, another 2 receptors for CD155. Interrupting the CD155-TIGIT interactions with a TIGIT antibody restored NK-cell immunosurveillance on CTCs and markedly attenuated tumor metastasis. CONCLUSIONS: Our results demonstrated CTC evasion from NK-cell-mediated innate immunosurveillance mainly through immune checkpoint CD155-TIGIT, potentially offering an immunotherapeutic strategy for eradicating CTCs.

Proteomics identifies new therapeutic targets of early-stage hepatocellular carcinoma.

Hepatocellular carcinoma is the third leading cause of deaths from cancer worldwide. Infection with the hepatitis B virus is one of the leading risk factors for developing hepatocellular carcinoma, particularly in East Asia1. Although surgical treatment may be effective in the early stages, the five-year overall rate of survival after developing this cancer is only 50-70%2. Here, using proteomic and phospho-proteomic profiling, we characterize 110 paired tumour and non-tumour tissues of clinical early-stage hepatocellular carcinoma related to hepatitis B virus infection. Our quantitative proteomic data highlight heterogeneity in early-stage hepatocellular carcinoma: we used this to stratify the cohort into the subtypes S-I, S-II and S-III, each of which has a different clinical outcome. S-III, which is characterized by disrupted cholesterol homeostasis, is associated with the lowest overall rate of survival and the greatest risk of a poor prognosis after first-line surgery. The knockdown of sterol O-acyltransferase 1 (SOAT1)-high expression of which is a signature specific to the S-III subtype-alters the distribution of cellular cholesterol, and effectively suppresses the proliferation and migration of hepatocellular carcinoma. Finally, on the basis of a patient-derived tumour xenograft mouse model of hepatocellular carcinoma, we found that treatment with avasimibe, an inhibitor of SOAT1, markedly reduced the size of tumours that had high levels of SOAT1 expression. The proteomic stratification of early-stage hepatocellular carcinoma presented in this study provides insight into the tumour biology of this cancer, and suggests opportunities for personalized therapies that target it.

Identification of a DNA damage repair-related LncRNA signature for predicting the prognosis and immunotherapy response of hepatocellular carcinoma.

BACKGROUND: DNA damage repair (DDR) may affect tumorigenesis and therapeutic response in hepatocellular carcinoma (HCC). Long noncoding RNAs (LncRNAs) can regulate DDR and play a vital role in maintaining genomic stability in cancers. Here, we identified a DDR-related prognostic signature in HCC and explored its potential clinical value. METHODS: Data of HCC samples were obtained from the Cancer Genome Atlas (TCGA), and a list of DDR-related genes was extracted from the Molecular Signatures database (MSigDB). A DDR-related lncRNAs signature associated to overall survival (OS) was constructed using the least absolute shrinkage and selection operator-cox regression, and was further validated by the Kaplan-Meier curve and receiver operating characteristic curve. A nomogram integrating other clinical risk factors was established. Moreover, the relationships between the signature with somatic mutation, immune landscape and drug sensitivity were explored. RESULTS: The prognostic model of 5 DDR-related lncRNAs was constructed and classified patients into two risk groups at median cut-off. The low-risk group had a better OS, and the signature was an independent prognostic indicator in HCC. A nomogram of the signature combined with TNM stage was constructed. TP53 gene was more frequently mutated in the high-risk group. Marked differences in immune cells were observed, such as CD4 + T cells, NK cells and macrophages, between the two groups. Moreover, an increase in the expression of immune checkpoint molecules was found in the high-risk group. The low-risk group presented with a significantly higher response to sorafenib or cisplatin. Finally, potential value of this signature was validated in real-world HCC patients. CONCLUSION: Our findings provided a promising insight into DDR-related lncRNAs in HCC and a personalized prediction tool for prognosis and therapeutic response.

An SCD1-dependent mechanoresponsive pathway promotes HCC invasion and metastasis through lipid metabolic reprogramming.

Matrix stiffness promotes hepatocellular carcinoma (HCC) metastasis. This study examined the contribution of lipid metabolic reprogramming to matrix stiffness-induced HCC metastasis. HCC cells were cultured on mechanically tunable polyacrylamide gels and subjected to lipidomic analysis. The key enzyme that responded to matrix stiffness and regulated lipid metabolism was identified. The comparative lipidomic screening revealed that stearoyl-CoA desaturase 1 (SCD1) is a mechanoresponsive enzyme that reprogrammed HCC cell lipid metabolism. The genetic and pharmacological inhibition of SCD1 expression/activity altered the cellular lipid composition, which in turn impaired plasma membrane fluidity and inhibited in vitro invasive motility of HCC cells in response to high matrix stiffness. Knockdown of SCD1 suppressed HCC invasion and metastasis in vivo. Conversely, the overexpression of SCD1 or exogenous administration of its product oleic acid augmented plasma membrane fluidity and rescued in vitro invasive migration in HCC cells cultured on soft substrates, mimicking the effects imposed by high matrix stiffness. In human HCC tissues, collagen content, a marker of increasing matrix stiffness, and increased expression of SCD1 together predicted poor survival of HCC patients. An SCD1-dependent mechanoresponsive pathway that responds to increasing matrix stiffness in the tumor microenvironment promotes HCC invasion and metastasis through lipid metabolic reprogramming.

Pen Yan Jing Tablets Alleviates Pelvic Inflammatory Disease by Inhibiting Akt/NF-κB Pathway.

Purpose: Pen Yan Jing tablets (PYJ), a Chinese patent medicine, has being used for pelvic inflammatory disease (PID) effectively. This study was designed to explore the underlying mechanisms of PYJ for treating PID. Methods: A rat model of PID was established by mixed bacteria liquid plus mechanical damage. After PYJ treatment, the morphology of uteri and extent of pelvic adhesion were observed. The pathological changes were evaluated by hematoxylin-eosin (HE) staining. The protein expressions of CD68, intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), monocyte chemotactic protein-1 (MCP-1) and cyclooxygenase-2 (COX-2) were quantitated by immunohistochemistry. A cell model of lipopolysaccharide (LPS)-activated RAW 264.7 macrophages was performed. The cell proliferation and NO level were measured by CCK-8 and Griess method, respectively. The tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) levels were detected by ELISA. The protein kinase B (Akt)/nuclear factor kappa-B (NF-κB) pathway-related protein expressions were assayed by western blot or immunofluorescence. Results: PYJ alleviated pelvic adhesion and inflammatory lesions of uteri in PID rats. PYJ down-regulated protein expressions of ICAM-1, VCAM-1, MCP-1, COX-2, p-Akt, p-IκB kinaseα/ß (p-IKKα/ß), p-IκBα, p65, and p-p65 in uteri of PID rats. Moreover, PYJ medicated serum inhibited abnormal cell proliferation, NO release, levels of TNF-α and IL-6, nuclear translocation of p65, and protein expressions of p-Akt, p-p65 and p-IκBα in LPS-activated RAW 264.7 macrophages. Conclusions: Taken together, PYJ may alleviates PID through inhibiting Akt/NF-κB pathway.

Hsa_circ_0003945 promotes progression of hepatocellular carcinoma by mediating miR-34c-5p/LGR4/ß-catenin axis activity.

Accumulating evidence suggests that circular RNAs (circRNAs) play essential roles in regulating cancer progression, but many circRNAs in hepatocellular carcinoma (HCC) remain unknown. Dysregulated circRNAs in HCC were identified through bioinformatics analysis of Gene Expression Omnibus data sets. Quantitative real-time PCR (qRT-PCR), Sanger sequencing, RNase R digestion and actinomycin D treatment were conducted to confirm the characterization of circRNAs. CCK-8, wound-healing and Transwell assays were performed to assess the functional roles of Hsa_circ_0003945 (Circ_0003945) in HCC cell lines. Subcellular fractionation and fluorescence in situ hybridization (FISH) were performed to locate Circ_0003945 in HCC cells. Dual-luciferase reporter assay was executed to verify the binding of Circ_0003945 to microRNAs (miRNAs) or the miRNAs to their target genes. In this study, we found that Circ_0003945 was upregulated in HCC tissue, and higher Circ_0003945 expression was positively correlated with tumour size and tumour stage. Furthermore, high plasma levels of circulating Circ_0003945 were confirmed in HCC patients compared with those in non-HCC groups. The functional experiments revealed that overexpression or knockdown of Circ_0003945 promoted or attenuated tumour growth and migration, respectively. Mechanistically, Circ_0003945 might exert as a miR-34c-5p sponge to upregulate the expression of leucine-rich repeat-containing G protein-coupled receptor 4 (LGR4), activating the ß-catenin pathway, and finally facilitating HCC progression. Additionally, a ß-catenin activator could reverse the effect of Circ_0003945 knockdown. In conclusion, Circ_0003945 exerts a tumour-promoting role in HCC cells by regulating the miR-34c-5p/LGR4/ß-catenin axis, which may be a potential target for HCC therapy.

Visualizing the Interplay of Lipid Droplets and Protein Aggregates During Aging via a Dual-Functional Fluorescent Probe.

Fluorescence imaging the interplay between lipid droplets (LDs) and protein aggregates (PAs) is extremely valuable for elucidating molecular mechanisms of aging. Here, we describe the first dual-functional fluorescent probe, LW-1, for simultaneously imaging LDs and PAs in distinct fluorescence channels to dissect interplaying roles between LDs and PAs during aging. Notably, based on an intriguing mechanism of hydrogen bonds regulating single bond rotation, LW-1 selectively detected LDs in a red channel. Meanwhile, based on another mechanism of the hydrogen bond regulating intramolecular charge transfer efficiency, probe LW-1 further detected PAs in an NIR channel. Practical applications showed that LW-1 was capable of concurrently detecting LDs and PAs in living cells. Moreover, simultaneously imaging LDs and PAs in intestine tissues of mice at different aging degrees was conducted. The results denoted that the PAs level in the intestine tissue increased dramatically with aging, accompanying the buildup of LDs. Significantly, the interplay between LDs and PAs during aging was observed. These evidences demonstrated that the PAs level was closely related with aging processes in intestine tissues, while LDs were formed correspondingly to interact with PAs, suggesting that excessive PAs can be loaded into LDs and then be removed by lipophagy.

Exosomal microRNAs in the DLK1-DIO3 imprinted region derived from cancer-associated fibroblasts promote progression of hepatocellular carcinoma by targeting hedgehog interacting protein.

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.

High serum soluble CD155 level predicts poor prognosis and correlates with an immunosuppressive tumor microenvironment in hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most prevalent malignancies with poor prognosis. There is no research about the clinical significance of serum soluble CD155 (sCD155) level for HCC. We aim to explore the prognostic and diagnostic value of sCD155 in HCC patients undergoing curative resection. METHODS: Serum sCD155 level in HCC patients was determined by enzyme-linked immunosorbent assay. The prognostic significance of sCD155 was evaluated by Cox regression and Kaplan-Meier analyses. CD155 expression and biomarkers of immune cells in HCC tissues were detected by immunohistochemistry staining. The diagnostic significance of sCD155 was evaluated using receiver operating characteristic curve. RESULTS: Serum sCD155 level was significantly increased in HCC patients and predicted poor prognosis. The prognostic value of sCD155 remained in low recurrent risk subgroups of HCC. Serum sCD155 level was positively related to CD155 expression in HCC tissues. High serum sCD155 level was associated with decreased numbers of CD8+ T cells and CD56+ NK cells and increased number of CD163+ M2 macrophages. Serum sCD155 level had better performance in distinguishing HCC patients from healthy donors and patients with chronic liver conditions than α-fetoprotein. Among patients with α-fetoprotein ≤ 20 ng/ml, serum sCD155 level could differentiate HCC patients from non-HCC patients. CONCLUSION: Serum sCD155 level represents a promising biomarker for diagnosis and prognosis of HCC. High serum sCD155 level may reflect an immunosuppressive tumor microenvironment in HCC.

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.

Determination of Triterpenoids and Phenolic Acids from Sanguisorba officinalis L. by HPLC-ELSD and Its Application.

A novel analytical method involving high-performance liquid chromatography with evaporative light scattering detection (HPLC-ELSD) was developed for simultaneous determination of 11 phenolic acids and 12 triterpenes in Sanguisorba officinalis L. Chromatographic separation was conducted with gradient elution mode by using a DiamonsilTM C18 column (250 mm × 4.6 mm, 5 µm) with the mobile phase of 0.1% acetic acid water (A) and methanol (B). The drift tube temperature of ELSD was set at 70 °C and the nitrogen cumulative flow rate was 1.6 L/min. The method was fully validated to be linear over a wide concentration range (R2 ≥ 0.9991). The precisions (RSD) were less than 3.0% and the recoveries were between 97.7% and 101.4% for all compounds. The results indicated that this method is accurate and effective for the determination of 23 functional components in Sanguisorba officinalis L. and could also be successfully applied to study the influence of processing method on those functional components in Sanguisorba officinalis L.

Establishment of a hepatocellular carcinoma patient-derived xenograft platform and its application in biomarker identification.

Using a method optimized in hepatocellular carcinoma (HCC), we established patient-derived xenograft (PDX) models with an increased take rate (42.2%) and demonstrated that FBS +10% dimethyl sulfoxide exhibited the highest tumor take rate efficacy. Among 254 HCC patients, 103 stably transplantable xenograft lines that could be serially passaged, cryopreserved and revived were established. These lines maintained the diversity of HCC and the essential features of the original specimens at the histological, transcriptome, proteomic and genomic levels. Tumor engraftment was associated with lack of encapsulation, poor tumor differentiation, large size and overexpression of cancer stem cell biomarkers, and was an independent predictor for overall survival and tumor recurrence after resection. To confirm the preclinical value of the PDX model in HCC treatment, several antitumor agents were tested in 16 selected PDX models. The results revealed a high degree of pharmacologic heterogeneity in the cohort, as well as heterogeneity to different agents in the same individual. The sorafenib responses observed between HCC patients and the corresponding PDXs were also consistent. After molecular characterization of the PDX models, we explored the predictive markers for sorafenib response and found that mitogen-activated protein kinase kinase kinase 1 (MAP3K1) might play an important role in sorafenib resistance and sorafenib response is impaired in patients with MAP3K1 downexpression. Our results indicated that PDX models could accurately reproduce patient tumors biology and could aid in the discovery of new treatments to advance in precision medicine.

Associating Liver Partition and Portal Vein Ligation for Staged Hepatectomy for Unresectable Hepatitis B Virus-related Hepatocellular Carcinoma: A Single Center Study of 45 Patients.

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.

FUN14 Domain-Containing 1-Mediated Mitophagy Suppresses Hepatocarcinogenesis by Inhibition of Inflammasome Activation in Mice.

Mitochondria lie at the heart of innate immunity, and aberrant mitochondrial activity contributes to immune activation and chronic inflammatory diseases, including liver cancers. Mitophagy is a selective process for removing dysfunctional mitochondria. The link between mitophagy and inflammation in tumorigenesis remains largely unexplored. We observed that FUN14 domain-containing 1 (FUNDC1), a previously characterized mitophagy receptor, accumulates in most human hepatocellular carcinomas (HCCs), and we thus explored the role of FUNDC1-mediated mitophagy in HCC initiation and progression in a mouse model in which HCC is induced by the chemical carcinogen, diethylnitrosamine (DEN). We showed that specific knockout of FUNDC1 in hepatocytes promotes the initiation and progression of DEN-induced HCC, whereas FUNDC1 transgenic hepatocytes protect against development of HCC. Hepatocyte-specific FUNDC1 ablation results in the accumulation of dysfunctional mitochondria and triggers a cascade of events involving inflammasome activation and hyperactivation of Janus kinase/signal transducer and activator of transcription signaling. Specifically, cytosolic mitochondrial DNA (mtDNA) release and caspase-1 activation are increased in FUNDC1-depleted hepatocytes. This subsequently results in the elevated release of proinflammatory cytokines, such as interleukin-1ß (IL1ß) and hyperproliferation of hepatocytes. Conclusion: Our results suggest that FUNDC1 suppresses HCC initiation by reducing inflammasome activation and inflammatory responses in hepatocytes, whereas up-regulation of FUNDC1 expression at the late stage of tumor development may benefit tumor growth. Our study thus describes a mechanistic link between mitophagic modulation of inflammatory response and tumorigenesis, and further implies that FUNDC1-mediated mitophagy and its related inflammatory response may represent a therapeutic target for liver cancer.

A Positive Feedback Loop Between Cancer Stem-Like Cells and Tumor-Associated Neutrophils Controls Hepatocellular Carcinoma Progression.

Tumor-associated neutrophils (TANs) play a crucial role in tumor development and progression in the cancer microenvironment. Despite increased understanding of TAN contributions to hepatocellular carcinoma (HCC) progression and prognosis, the direct interaction between TANs and HCC cells is not fully understood. In this study, we tested the effect of TANs on HCC cells in vitro and in vivo and investigated the mechanism of interaction between them. Our results showed that TANs secreted bone morphogenetic protein 2 and transforming growth factor beta 2 and triggered microRNA 301b-3p (miR-301-3p) expression in HCC cells, subsequently suppressed gene expression of limbic system-associated membrane protein (LSAMP) and CYLD lysine 63 deubiquitinase (CYLD), and increased stem cell characteristics in HCC cells. These TAN-induced HCC stem-like cells were hyperactive in nuclear factor kappa B signaling, secreted higher levels of chemokine (C-X-C motif) ligand 5 (CXCL5), and recruited more TAN infiltration, suggesting a positive feedback loop. In clinical HCC samples, increased TANs correlated with elevated miR-301b-3p, decreased LSAMP and CYLD expression, and increased nuclear p65 accumulation and CXCL5 expression, all of which predicted patient outcome. Conclusion: Our work identified a positive feedback loop governing cancer stem-like cells and TANs in HCC that controls tumor progression and patient outcome.

Comparison of the pharmacokinetic profiles of 13 phenolic acids and 6 triterpenes in normal and leukopenia rats after oral administration of Sanguisorba officinalis L. extract by LC-MS/MS.

A selective, accurate, and efficient liquid chromatography-tandem mass spectrometry method was developed for the simultaneous determination of 13 phenolic acids. Additionally, for more comprehensively determining the chemical constituents in Sanguisorba officinalis L. extract, a previously developed method was employed for the simultaneous determination of six triterpenes. Thus, two methods were used to ensure the comprehensiveness and reliability of this study. Based on these methods, the pharmacokinetic profiles of the 13 phenolic acids and 6 triterpenes in normal and leukopenia rats after oral administration of S. officinalis L. extract were compared for the first time in the present study. Quantitative detection of the 13 phenolic acids and 6 triterpenes was performed using the multiple reaction monitoring mode with the electrospray ion source in negative and positive electrospray ionization, respectively. Chromatographic separation was performed on an Agilent Eclipse Plus C18 RRHD column (50 × 2.1 mm, 1.8 µm) using gradient elution with a mobile phase composed of methanol-0.1% aqueous formic acid. The pharmacokinetic results demonstrated that the pharmacokinetic characteristics of the 19 analytes in leukopenia rats differed significantly from those determined in normal rats, which could provide a helpful reference for the clinical application of S. officinalis L. in the prevention and treatment of leucopenia.

Genomic sequencing identifies WNK2 as a driver in hepatocellular carcinoma and a risk factor for early recurrence.

BACKGROUND & AIMS: Early recurrence of hepatocellular carcinoma (HCC) after curative resection is common. However, the association between genetic mechanisms and early HCC recurrence, especially in Chinese patients, remains largely unknown. METHODS: We performed whole-genome sequencing (49 cases), whole-exome sequencing (18 cases), and deep targeted sequencing (115 cases) on 182 primary HCC samples. Focusing on WNK2, we used Sanger sequencing and qPCR to evaluate all the coding exons and copy numbers of that gene in an additional 554 HCC samples. We also explored the functional effect and mechanism of WNK2 on tumor growth and metastasis. RESULTS: We identified 5 genes (WNK2, RUNX1T1, CTNNB1, TSC1, and TP53) harboring somatic mutations that correlated with early tumor recurrence after curative resection in 182 primary HCC samples. Focusing on WNK2, the overall somatic mutation and copy number loss occurred in 5.3% (39/736) and 27.2% (200/736), respectively, of the total 736 HCC samples. Both types of variation were associated with lower WNK2 protein levels, higher rates of early tumor recurrence, and shorter overall survival. Biofunctional investigations revealed a tumor-suppressor role of WNK2: its inactivation led to ERK1/2 signaling activation in HCC cells, tumor-associated macrophage infiltration, and tumor growth and metastasis. CONCLUSIONS: Our results delineate genomic events that characterize Chinese HCCs and identify WNK2 as a driver of early HCC recurrence after curative resection. LAY SUMMARY: We applied next-generation sequencing and conducted an in-depth genomic analysis of hepatocellular carcinomas from a Chinese patient cohort. The results delineate the genomic events that characterize hepatocellular carcinomas in Chinese patients and identify WNK2 as a driver associated with early tumor recurrence after curative resection.

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.

Sphere-forming culture enriches liver cancer stem cells and reveals Stearoyl-CoA desaturase 1 as a potential therapeutic target.

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