Cancer-associated fibroblasts induce sorafenib resistance of hepatocellular carcinoma cells through CXCL12/FOLR1.

BACKGROUND: Due to the high drug resistance of hepatocellular carcinoma (HCC), sorafenib has limited efficacy in the treatment of advanced HCC. Cancer-associated fibroblasts (CAFs) play an important regulatory role in the induction of chemoresistance. This study aimed to clarify the mechanism underlying CAF-mediated resistance to sorafenib in HCC. METHODS: Immunohistochemistry and immunofluorescence showed that the activation of CAFs was enhanced in HCC tissues. CAFs and paracancerous normal fibroblasts (NFs) were isolated from the cancer and paracancerous tissues of HCC, respectively. Cell cloning assays, ELISAs, and flow cytometry were used to detect whether CAFs induced sorafenib resistance in HCC cells via CXCL12. Western blotting and qPCR showed that CXCL12 induces sorafenib resistance in HCC cells by upregulating FOLR1. We investigated whether FOLR1 was the target molecule of CAFs regulating sorafenib resistance in HCC cells by querying gene expression data for human HCC specimens from the GEO database. RESULTS: High levels of activated CAFs were present in HCC tissues but not in paracancerous tissues. CAFs decreased the sensitivity of HCC cells to sorafenib. We found that CAFs secrete CXCL12, which upregulates FOLR1 in HCC cells to induce sorafenib resistance. CONCLUSIONS: CAFs induce sorafenib resistance in HCC cells through CXCL12/FOLR1.

NIR-II fluorescence-guided liver cancer surgery by a small molecular HDAC6 targeting probe.

BACKGROUND: Hepatocellular carcinoma (HCC) is the sixth most common malignancy globally and ranks third in terms of both mortality and incidence rates. Surgical resection holds potential as a curative approach for HCC. However, the residual disease contributes to a high 5-year recurrence rate of 70%. Due to their excellent specificity and optical properties, fluorescence-targeted probes are deemed effective auxiliary tools for addressing residual lesions, enabling precise surgical diagnosis and treatment. Research indicates histone deacetylase 6 (HDAC6) overexpression in HCC cells, making it a potential imaging biomarker. This study designed a targeted small-molecule fluorescent probe, SeCF3-IRDye800cw (SeCF3-IRD800), operating within the Second near-infrared window (NIR-II, 1000-1700 nm). The study confirms the biocompatibility of SeCF3-IRD800 and proceeds to demonstrate its applications in imaging in vivo, fluorescence-guided surgery (FGS) for liver cancer, liver fibrosis imaging, and clinical samples incubation, thereby preliminarily validating its utility in liver cancer. METHODS: SeCF3-IRD800 was synthesized by combining the near-infrared fluorescent dye IRDye800cw-NHS with an improved HDAC6 inhibitor. Initially, a HepG2-Luc subcutaneous tumor model (n = 12) was constructed to investigate the metabolic differences between SeCF3-IRD800 and ICG in vivo. Subsequently, HepG2-Luc (n = 12) and HCCLM3-Luc (n = 6) subcutaneous xenograft mouse models were used to assess in vivo targeting by SeCF3-IRD800. The HepG2-Luc orthotopic liver cancer model (n = 6) was employed to showcase the application of SeCF3-IRD800 in FGS. Liver fibrosis (n = 6) and HepG2-Luc orthotopic (n = 6) model imaging results were used to evaluate the impact of different pathological backgrounds on SeCF3-IRD800 imaging. Three groups of fresh HCC and normal liver samples from patients with liver cancer were utilized for SeCF3-IRD800 incubation ex vivo, while preclinical experiments illustrated its potential for clinical application. FINDINGS: The HDAC6 inhibitor 6 (SeCF3) modified with trifluoromethyl was labeled with IRDy800CW-NHS to synthesize the small-molecule targeted probe SeCF3-IRD800, with NIR-II fluorescence signals. SeCF3-IRD800 was rapidly metabolized by the kidneys and exhibited excellent biocompatibility. In vivo validation demonstrated that SeCF3-IRD800 achieved optimal imaging within 8 h, displaying high tumor fluorescence intensity (7658.41 ± 933.34) and high tumor-to-background ratio (5.20 ± 1.04). Imaging experiments with various expression levels revealed its capacity for HDAC6-specific targeting across multiple HCC tumor models, suitable for NIR-II intraoperative imaging. Fluorescence-guided surgery experiments were found feasible and capable of detecting sub-visible 2 mm tumor lesions under white light, aiding surgical decision-making. Further imaging of liver fibrosis mice showed that SeCF3-IRD800's imaging efficacy remained unaffected by liver pathological conditions. Correlations were observed between HDAC6 expression levels and corresponding fluorescence intensity (R2 = 0.8124) among normal liver, liver fibrosis, and HCC tissues. SeCF3-IRD800 identified HDAC6-positive samples from patients with HCC, holding advantages for perspective intraoperative identification in liver cancer. Thus, the rapidly metabolized HDAC6-targeted small-molecule NIR-II fluorescence probe SeCF3-IRD800 holds significant clinical translational value. INTERPRETATION: The successful application of NIR-II fluorescence-guided surgery in liver cancer indicates that SeCF3-IRD800 has great potential to improve the clinical diagnosis and treatment of liver cancer, and could be used as an auxiliary tool for surgical treatment of liver cancer without being affected by liver pathology. FUNDING: This paper is supported by the National Natural Science Foundation of China (NSFC) (92,059,207, 62,027,901, 81,930,053, 81,227,901, 82,272,105, U21A20386 and 81,971,773), CAS Youth Interdisciplinary Team (JCTD-2021-08), the Zhuhai High-level Health Personnel Team Project (Zhuhai HLHPTP201703), and Guangdong Basic and Applied Basic Research Foundation under Grant No. 2022A1515011244.

Modified Laparoscopic Anatomic Hepatectomy: Two-Surgeon Technique Combined with the Simple Extracorporeal Pringle Maneuver.

Laparoscopic anatomic hepatectomy (LAH) has become increasingly prevalent worldwide in recent years. However, LAH remains a challenging procedure due to the anatomical characteristics of the liver, with intraoperative hemorrhage being a primary concern. Intraoperative blood loss is the leading cause of conversion to open surgery; therefore, effective management of bleeding and hemostasis is crucial for a successful LAH. The two-surgeon technique is proposed as an alternative to the traditional single-surgeon approach, with potential benefits in reducing intraoperative bleeding during laparoscopic hepatectomy. However, there remains a lack of evidence to determine which mode of the two-surgeon technique yields superior patient outcomes. Besides, to our knowledge, the LAH technique, which involves the use of a cavitron ultrasonic surgical aspirator (CUSA) by the primary surgeon while an ultrasonic dissector by the second surgeon, has been rarely reported before. Herein, we present a modified, two-surgeon LAH technique, wherein one surgeon employs a CUSA while the other uses an ultrasonic dissector. This technique is combined with a simple extracorporeal Pringle maneuver and low central venous pressure (CVP) approach. In this modified technique, the primary and secondary surgeons utilize a laparoscopic CUSA and an ultrasonic dissectorconcurrently to achieve precise and expeditious hepatectomy. A simple extracorporeal Pringle maneuver, combined with the maintenance of low CVP, is employed to regulate the hepatic inflow and outflow in order to minimize intraoperative bleeding. This approach facilitates the attainment of a dry and clean operative field, which allows for the precise ligation and dissection of blood vessels and bile ducts. The modified LAH procedure is simpler and safer due to its effective control over bleeding as well as the seamless transition between the roles of primary and secondary surgeons. It holds great promise for future clinical applications.

Combined Treatment of Tanshinone I and Epirubicin Revealed Enhanced Inhibition of Hepatocellular Carcinoma by Targeting PI3K/AKT/HIF-1α.

Background: Epirubicin (EADM) is a common chemotherapeutic agent in hepatocellular carcinoma (HCC). The accumulation of hypoxia-inducible factor-1α (HIF-1α) is an important cause of drug resistance to EADM in HCC. Tanshinone I (Tan I) is an agent with promising anti-cancer effects alone or with other drugs. Some tanshinones mediate HIF-1α regulation via PI3K/AKT. However, the role of Tan I combined with EADM to reduce the resistance of HCC to EADM has not been investigated. Therefore, this study aimed to investigate the combined use of Tan I and EADM in HCC and the underlying mechanism of PI3K/AKT/HIF-1α. Methods: HCC cells were treated with Tan I, EADM, or the combined treatment for 48 hrs. Cell transfection was used to construct HIF-1α overexpression HCC stable cells. Cell viability, colony formation, and flow cytometric assays were used to detect the viability, proliferation, and apoptosis in HCC cells. Synergism between Tan I and EADM were tested by calculating the Bliss synergy score, positive excess over bliss additivism (EOBA), and the combination index (CI). Western blotting analyses were used to detect the levels of ß-actin, HIF-1α, PI3K p110α, p-Akt Thr308, Cleaved Caspase-3, and Cleaved Caspase-9. Toxicity parameters were used to evaluate the safety of the combination in mice. The xenograft model of mice was built by HCC stable cell lines, which was administrated with Tan I, EADM, or a combination of them for 8 weeks. Immunohistochemistry staining (IHC) was used to assess tumor apoptosis in mouse models. Results: Hypoxia could upregulate HIF-1α to induce drug resistance in HCC cancer cells. The combination of Tan I and EADM was synergistic. Although Tan I or EADM alone could inhibit HCC cancer cells, the combination of them could further enhance the cytotoxicity and growth inhibition by targeting the PI3K/AKT/HIF-1α signaling pathway. Furthermore, Tan I and EADM synergistically reversed HIF-1α-mediated drug resistance to inhibit HCC. The results of toxicity parameters showed that the combination was safe in mice. Meanwhile, animal models showed that Tan I not only improved the anti-tumor effect of EADM, but also reduced the drug reactions of EADM-induced weight loss. Conclusion: Our results suggested that Tan I could effectively improve the anti-tumor effect of EADM, and synergize EADM to reverse HIF-1α mediated resistance via targeting PI3K/AKT/HIF-1α signaling pathway.

Combined HIF-1α and SHH Up-Regulation Is a Potential Biomarker to Predict Poor Prognosis in Postoperative Hepatocellular Carcinoma.

BACKGROUND: Hypoxia-inducible factor-1α (HIF-1α) or sonic hedgehog (SHH) is associated with hepatocellular carcinoma (HCC) progression. Hypoxia inhibits ferroptosis, which induces cancer cell death. However, the correlation between the combined HIF-1α and SHH up-regulation with prognosis, and the association between SHH and ferroptosis remain unclear. This study aimed to investigate them. METHODS: We detected the expression of HIF-1α and SHH in HCC. Cox regression, clinical data, and Kaplan-Meier analyses were performed. In vitro cell experiments verified the relationship between HIF-1α and SHH, and observed the invasion of hypoxic HCC cells. The correlation between SHH and ferroptosis was also analyzed. RESULTS: HIF-1α and SHH expression levels were significantly correlated with HCC (p < 0.0001). HIF-1α and SHH expression levels were found to be associated with TNM stage (p = 0.0121, p = 0.0078, respectively), vascular invasion (p < 0.0001, p < 0.0001, respectively), and recurrence (p = 0.0212, p = 0.0392, respectively). The combined upregulation of HIF-1α and SHH was an independent factor for predicting the overall survival (OS) of patients with HCC (p = 0.003), who had the shortest OS (p = 0.0009). SHH paralleled the increase in HIF-1α expression, which promotes cancer cell invasion. The upregulation of SHH was related to the inhibition of the expression of ferroptosis-related factors (FANCD2, p < 0.0001 and FTH1, p = 0.0009) in HCC. CONCLUSION: Combined HIF-1α and SHH upregulation is a potentially poor prognosis indicator in patients with HCC because the upregulation of SHH inhibits ferroptosis in hypoxic cancer cells.

Efficacy of Near-Infrared Fluorescence-Guided Hepatectomy for the Detection of Colorectal Liver Metastases: A Randomized Controlled Trial.

BACKGROUND: The application of indocyanine green fluorescence-guided hepatectomy for liver metastases from colorectal cancer is in the preliminary stage of clinical practice; thus, its efficacy needs to be determined. This study compared the number of intrahepatic colorectal liver metastases detected intraoperatively and postoperative recovery data between patients who underwent traditional hepatectomy (nonindocyanine green group) and traditional hepatectomy plus intraoperative indocyanine green fluorescence imaging (indocyanine green group). STUDY DESIGN: Between January 2018 and March 2020, patients with potentially resectable colorectal liver metastases were randomly assigned to the nonindocyanine green or indocyanine green group. The number of intrahepatic colorectal liver metastases identified intraoperatively and based on postoperative recovery data were compared between both groups. RESULTS: Overall, we recruited 80 patients, among whom 72 eligible patients were randomly assigned. After allocation, 64 patients, comprising 32 in each group, underwent the allocated intervention and follow-up. Compared with the nonindocyanine green group, the mean number of intrahepatic colorectal liver metastases identified intraoperatively in the indocyanine green group was significantly greater (mean [standard deviation], 3.03 [1.58] vs 2.28 [1.35]; p = 0.045), the postoperative hospital stay was shorter (p = 0.012) and the 1-year recurrence rate was also lower (p = 0.017). Postoperative complications and 90-day mortality were comparable, with no statistical differences. CONCLUSIONS: Indocyanine green fluorescence imaging significantly increases the number of intrahepatic colorectal liver metastases identified and reduces postoperative hospital stay and 1-year recurrence rate without increasing hepatectomy-related complications and mortality rates.

Efficacy of Adjuvant Transarterial Chemoembolization after Radical Hepatectomy in Solitary Hepatocellular Carcinoma Patients: A Retrospective Study.

Background: More and more studies have suggested that hepatocellular carcinoma (HCC) patients with high-risk recurrence factors can benefit the most from postoperative adjuvant transarterial chemoembolization (PA-TACE) for its potential effect in delaying cancer recurrence. However, it remains unclear if solitary HCC (SHCC) patients particularly those without high-risk recurrence factors should also receive PA-TACE. This study aimed to analyze the efficacy of PA-TACE in them. Methods: Retrospectively, we enrolled 123 SHCC patients who either received radical hepatectomy alone (No TACE group, n = 39) or followed by PA-TACE (PA-TACE group, n = 84) in our institution. Prognostic risk factors, disease-free survival (DFS), and overall survival (OS) were analyzed using the Cox proportional hazard regression model, the Kaplan-Meier method, and the log-rank test. Results: Liver cirrhosis was the only independent risk factor for SHCC patients. Overall, the PA-TACE group had no improved OS (P = 0.977) but worse DFS compared with the No TACE group (P = 0.045). Consistently, in subgroup analysis, SHCC patients with negative microvascular invasion (MVI), tumor size ≤ 5 cm and preoperative alpha-fetoprotein (AFP) < 400 ng/ml had similar OS (P = 0.466, P = 0.864, P = 0.488, respectively) but even worse DFS (P = 0.035, P = 0.040, P = 0.019, respectively) than those in the No TACE group. Besides, there was no significant difference in DFS and OS between the two groups of SHCC patients with liver cirrhosis (P = 0.342, P = 0.941, respectively). Conclusions: PA-TACE may not improve the long-term survival of SHCC patients, but may even potentially promote their postoperative tumor recurrence, especially for those with MVI-negative, tumor size ≤ 5 cm, and preoperative AFP < 400 ng/ml.

Evidence of SARS-CoV-2 infection in gallbladder and aggravating cholecystitis to septic shock: a case report.

Coronavirus disease 2019 (COVID-19) has threatened human health worldwide and could lead to multiple organs injury. However, the impact on the virus infecting the biliary system, especially the gallbladder, has remained unclear and no pathological evidence has been reported yet. A case of SARS-CoV-2 infection in a gallbladder with cholecystitis, which progressed rapidly to sepsis and required an emergency operation was investigated and reported. Clinical specimens of the COVID-19 patient including serum, oropharyngeal swabs, sputum, bile, abdominal drainage fluid, urine, stool, and gallbladder tissue were collected and tested for SARS-CoV-2 RNA using a quantitative polymerase chain reaction (qPCR) assay. Fresh normal gallbladder tissue and gangrenous gallbladder tissue were also collected for further research including hematoxylin and eosin (HE), immunohistochemistry (IHC), and immunofluorescent (IF) staining, and compared with the gallbladder from the COVID-19 patient. The bile, as well as the serum, oropharyngeal swabs, sputum, abdominal drainage fluid, urine, and rectal swabs were consecutively negative for SARS-CoV-2 RNA. The viral host receptor angiotensin-converting enzyme 2 (ACE2) was highly expressed in gallbladder epithelial cells, and viral nucleocapsid protein (NP) was visualized in the cytoplasm of gallbladder epithelial cells. Immune cells including CD2, CD3, CD4, CD8, CD20, CD38, CD68, and MPO were positive in gangrenous gallbladder tissues without SARS-CoV-2 infection, and were relatively downregulated in SARS-CoV-2 infective gallbladder tissue. This study provided evidence of SARS-CoV-2 infection in the gallbladder and verified that the gallbladder was one of the target organs that SARS-CoV-2 could attack and damage using ACE2 as a cell receptor. Due to the immune dysregulation involved, more vigilant management and early assessment is needed for COVID-19 patients with the comorbidity of cholecystitis.

The impact of liver fibrosis on microvascular invasion and prognosis of hepatocellular carcinoma with a solitary nodule: a Surveillance, Epidemiology, and End Results (SEER) database analysis.

BACKGROUND: The pathogenesis of non-cirrhotic hepatocellular carcinoma (HCC) with a high recurrence remains controversial, while microvascular invasion (MVI) is highly suggestive of tumor recurrence. This study aimed to investigate the effects of liver fibrosis on MVI and prognosis in HCC. METHODS: Based on the data of HCC in the Surveillance, Epidemiology, and End Results (SEER) database [2004-2015], multivariate logistic regression was used for correlation analysis. Survival was analyzed by Log-Rank test and Cox regression, and decision curve analysis and receiver operating characteristic curves were established to evaluate alternative diagnostic and prognostic strategies. RESULTS: The study included 1,492 patients with MVI (17.8%) or without MVI (82.2%) for HCC with a solitary nodule. Liver fibrosis was significantly correlated with the occurrence of MVI, and the risk of MVI in patients with a fibrosis score F5-6 was lower than in those with a score of F0-4 (OR =0.651, 95% CI: 0.492-0.860). Combining liver fibrosis could improve the prediction performance of MVI risk models, but liver fibrosis was less associated with survival outcomes in comparison with other tumor characteristics. CONCLUSIONS: Lower liver fibrosis correlated with a higher risk of MVI in HCC with a solitary nodule and was a good indicator for improving the performance of MVI risk models. However, it was not a prognostic sensitive indicator.

A new method of near-infrared fluorescence image-guided hepatectomy for patients with hepatolithiasis: a randomized controlled trial.

BACKGROUND: Hepatectomy is a definitive treatment for hepatolithiasis because it simultaneously removes intrahepatic duct (IHD) stones and biliary tract strictures together with the involved liver region en bloc. Unlike cystic or solid liver tumors, hepatolithiasis is usually associated with alterations of anatomical structures and perihepatic adhesions because of chronic recurrent inflammation. This complicates identification of the target hepatic region and location of biliary strictures. METHODS: To determine the efficacy of near-infrared fluorescence (NIRF) imaging using indocyanine green (ICG), we performed a comparative trial and developed a white-light and near-infrared dual-channel image-guided device (DPM-I) for both open and endoscopic surgery. Forty-four eligible patients were randomly assigned to Group A (NIRF imaging) or Group B (traditional hepatectomy). We injected ICG via peripheral veins for patients in Group A. RESULTS: The NIRF imaging method was associated with less blood loss (OR 1.004, 95% CI 0.999-1.010; P = 0.016), briefer hospitalization (OR 1.336, 95% CI 1.016-1.756; P = 0.001), lower rates of margins with dilated bile ducts (OR 1.278, 95% CI 1.030-1.585; P = 0.023), lower postoperative white blood cell counts (OR 1.262, 95% CI 0.931-1.712; P = 0.038), lower procalcitonin levels (OR 1.316, 95% CI 1.020-1.513; P = 0.002), and lower alanine aminotransferase levels (OR 1.013, 95% CI 1.003-1.023; P = 0.002) compared with traditional hepatectomy. CONCLUSIONS: These data demonstrate the efficacy of NIRF imaging with ICG using DPM-I for treating hepatolithiasis.

HIF-2α upregulation mediated by hypoxia promotes NAFLD-HCC progression by activating lipid synthesis via the PI3K-AKT-mTOR pathway.

Non-alcoholic fatty liver disease (NAFLD) is a relevant risk factor for developing hepatocellular carcinoma (HCC). Steatohepatitic HCC (SH-HCC), characterized by HCC with steatosis, is influenced by lipid metabolism disorders. A hypoxic microenvironment is common in HCC and affects lipid metabolism. However, whether hypoxia-induced HIF-2α upregulation exacerbates lipid accumulation to contribute to SH-HCC progression remains unclear. In this study, we demonstrated that HIF-2α was elevated in tissues from NAFLD-HCC patients and was associated with survival. Under hypoxic conditions, upregulated HIF-2α was accompanied by lipid accumulation and PI3K-AKT-mTOR pathway activation. HIF-2α knockdown (KD) in steatotic HCC ameliorated triglyceride accumulation and steatosis. HIF-2α-KD steatotic HCC showed minimal lipid synthesis in a hypoxic environment, which contributes to a reduction in malignant behaviours. However, treatment with MHY1485 restored these behaviours. STAM mice, a mouse model that develops NAFLD-HCC, exhibit more rapid tumour progression upon exposure to hypoxia. STAM mice treated with INK-128 presented abrogated mTOR expression and tumour progression under hypoxic conditions with lower triglycerides and steatosis. In conclusion, in a hypoxic microenvironment, HIF-2α upregulation promotes steatotic HCC progression by activating lipid synthesis via the PI3K-AKT-mTOR pathway. Therefore, HIF-2α can be a biomarker and target in developing specific therapeutic measures for NAFLD-HCC patients.

Potential Five-MicroRNA Signature Model for the Prediction of Prognosis in Patients with Wilms Tumor.

BACKGROUND Wilms tumor (WT) is the most common type of pediatric renal malignancy, and is associated with poor prognosis. The aim of the present study was to identify microRNA (miRNA) signatures which might predict prognosis and categorize WTs into high- and low-risk subgroups. MATERIAL AND METHODS The miRNA expression profiles of WT patients and normal samples were obtained from the Therapeutically Applicable Research to Generate Effective Treatment database. Differentially expressed miRNAs between WT patients and normal samples were identified using the EdgeR package. Subsequently, correlations between differentially expressed miRNAs and the prognosis of overall survival were analyzed. Enrichment analyses for the targeted mRNAs were conducted via the Database for Annotation, Visualization, and Integration Discovery. RESULTS A total of 154 miRNAs were identified as differentially expressed in WT. Of those, 18 miRNAs were associated with overall survival (P<0.05). A prognostic signature of 5 differentially expressed miRNAs (i.e., has-mir-149, has-mir-7112, has-mir-940, has-mir-1248, and has-mir-490) was constructed to classify the patients into high- and low-risk subgroups. The targeted mRNAs of these prognostic miRNAs were primarily enriched in Gene Ontology terms (i.e., protein autophosphorylation, protein dephosphorylation, and stress-activated MAPK cascade) and the Kyoto Encyclopedia of Genes and Genomes signaling pathways (i.e., MAPK, AMPK, and PI3K-Akt). CONCLUSIONS The 5-miRNA signature model might be useful in determining the prognosis of WT patients. As a promising prediction tool, this prognosis signature might serve as a potential biomarker for WT patients.

Nuclear GAPDH is vital for hypoxia-induced hepatic stellate cell apoptosis and is indicative of aggressive hepatocellular carcinoma behavior.

Background/aim: Hepatic stellate cells (HSCs) are critical determinants of liver tumor behavior such as vascular invasion, cell proliferation and migration. The apoptosis of HSCs can inhibit tumor growth and contribute to repressing hepatocellular carcinoma (HCC) progression. Our study aims to investigate the impact of nuclear glyceraldehyde-3-phosphate dehydrogenase (GAPDH) on HSCs under hypoxic conditions and the association of nuclear GAPDH with HCC patient outcomes and tumor progression. Patients and methods: Following stable cell passage, 0.3% O2 was used to induce hypoxia. Cell proliferation and apoptosis were analyzed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assays and flow cytometry, respectively. Proteins expression were detected by extracting nuclear and cytoplasmic proteins and performing Western blots. GAPDH nuclear translocation was blocked by the agent deprenyl. Immunohistochemical staining for GAPDH was investigated in 137 HCC tissue samples from our center. An analysis of the clinicopathological features, Kaplan-Meier analysis and Cox proportional hazards regression analysis were applied. Results: MTT assays and flow cytometry analyses showed that the nuclear accumulation of GAPDH led to the apoptotic death of HSCs, while blockade of this process with deprenyl significantly decreased apoptosis. Western blots revealed that deprenyl inhibited the nuclear translocation of GAPDH. An analysis of the immunohistochemical staining of HSCs in HCC tissue samples (137) revealed that nuclear GAPDH expression was significantly positively correlated with HIF-1α expression. Overall survival (OS) and time-to-recurrence (TTR) estimated by Kaplan-Meier analyses showed that patients with high HIF-1α or low nuclear GAPDH levels in HSCs had significantly poorer prognosis compared with patients with low HIF-1α or high nuclear GAPDH expression in HSCs. Moreover, patients with combined high HIF-1α/low nuclear GAPDH expression in HSCs had the worst prognosis. The Cox regression analysis revealed that the combination of nuclear GAPDH/HIF-1α expression in HSCs was an independent prognostic factor for OS and TTR in HCC patients. Conclusions: These findings provide a novel mechanism underlying the involvement of intranuclear GAPDH in hypoxia-induced HSCs apoptosis and a correlation between nuclear GAPDH levels and the clinical prognosis, which may prompt the development of a novel therapeutic strategy for HCC.

A novel 12-marker panel of cancer-associated fibroblasts involved in progression of hepatocellular carcinoma.

BACKGROUND/AIM: Cancer-associated fibroblasts (CAFs) are important factors in the progression of hepatocellular carcinoma (HCC). But the characterization of these cells remains incomplete. This study aims to identify a panel of markers for CAFs that are associated with HCC progression. MATERIALS AND METHODS: The sequencing data and clinicopathological characteristics of 366 patients were obtained from the Cancer Genome Atlas (TCGA) database (366 HCC tissues and there were 50/366 cases with corresponding normal liver tissues). In vitro validation of the markers was performed by quantitative real-time PCR using the hepatic stellate cell line LX2 induced by the HCC cell line Huh7. The activation of LX2 was confirmed by α-smooth muscle actin and fibroblast activation protein, using quantitative real-time PCR and immunofluorescence staining. In vivo detections of the 12 markers were done in 40 tissue samples (30 HCC and 10 normal). RESULTS: We successfully identified 12 CAF markers from TCGA data: FGF5, CXCL5, IGFL2, MMP1, ADAM32, ADAM18, IGFL1, FGF8, FGF17, FGF19, FGF4, and FGF23. The 12-marker panel was associated with the pathological and clinical progressions of HCC. All 12 markers were upregulated in vitro. In vivo expressions of these markers were paralleled with those in TCGA data. CONCLUSION: A 12-marker panel of CAFs in HCC is identified, which is associated with both pathological and clinical progressions of cancer.

The Expression of FAP in Hepatocellular Carcinoma Cells is Induced by Hypoxia and Correlates with Poor Clinical Outcomes.

Fibroblast activation protein (FAP) is a serine protease that has been reported in fibroblasts and some carcinoma cells, which correlates with poor patient outcomes. FAP can be induced under hypoxia which is also vital in the malignant behaviors of cancer cells. However, the role of FAP and its correlation with hypoxia has not been investigated in HCC cancer cells. In tissues from post-surgical HCC patients in our center, we adopted immunohistochemistry staining (IHC), western blot and quantitative RT-PCR to detect the expression levels of FAP and the hypoxia related marker, hypoxia inducible factor 1α (HIF-1α). X-tile software was used for the determination of high and low expression of FAP and HIF-1α after the IHC analysis. Clinicopathological analysis, Kaplan-Meier analysis and Cox regression model were performed. In-vitro experiments were performed to confirm the relationship between FAP and hypoxia in HCC cancer cell lines (HepG2, Huh7 and MHCC97H). Results revealed that expression levels of FAP and HIF-1α were significantly correlated (Pearson r2 = 0.2753, p < 0.0001) in IHC analysis of the 138-patient cohort. Western blot and quantity RT-PCR indicated parallel changes in 11 post-surgical fresh frozen tissues. The HIF-1α and FAP expression were associated with serum AFP, TNM, tumor size and vascular invasion. Cox regression analysis showed that HIF-1α/ FAP combination were the independent predictor for overall survival (OS) and time-to-recurrence (TTR) in post-surgical HCC patients. Kaplan-Meier analyses revealed that the patient with high levels of HIF-1α, FAP and combined HIF-1α/FAP had the shortest OS and TTR. In-vitro experiments showed that FAP was increased in hypoxic HCC cancer cell lines in parallel with that of HIF-1α and three EMT markers (E-cadherin, Snail and TWIST). In conclusion, the up-regulation of FAP in HCC cancer cells under hypoxia can be indicative of poor prognosis in patients.

Portal vein arterialization promotes liver regeneration after extended partial hepatectomy in a rat model.

In the current study, we sought to establish a novel rat model of portal vein arterialization (PVA) and evaluate its impact on liver regeneration after extended partial hepatectomy (PH). A total of 105 Sprague-Dawley rats were randomly assigned to three groups: 68% hepatectomy (the PH group), portal arterialization after 68% hepatectomy (the PVA group), and right nephrectomy only (the control group). Liver regeneration rate (LRR), 5-bromo-2-deoxyuridine (BrdU) labeling index, and liver functions were assessed on postoperative day 2, 7, 14 and 28. The 28-day survival rates were compared among the three groups. The 28-day survival rates were similar in all groups (P  =  0.331), and the anastomotic patency was 100%. The LRR in the PVA group was significantly higher than that of the PH group within postoperative 14 days (P < 0.05). The PVA and PH group had increased serum alanine aminotransferase levels (232 ± 61 U/L and 212 ± 53 U/L, respectively) compared with the control group (101 ± 13 U/L) on postoperative day 2, whereas from postoperative day 7 to day 28 there were no differences among the three groups. Serum albumin values were higher after the PVA procedure within postoperative day 14, which gradually became comparable on postoperative day 28 among the three groups. The peaks of BrdU labeling index appeared on postoperative day 2 in all rats, and the PVA procedure was associated with increased BrdU labeling index from postoperative day 7 to 28. The 28-day survival of the PVA rats was comparable. Our findings demonstrate that the PVA procedure utilizing portal vein trunk-renal artery microvascular reconstruction promotes remnant liver regeneration and confers beneficial effects on maintaining and even optimizing liver function after extended partial hepatectomy in rats.

The impact of laparoscopic converted to open colectomy on short-term and oncologic outcomes for colon cancer.

PURPOSE: This study was designed to evaluate the impact of laparoscopic converted to open colectomy on short-term and oncologic outcomes and to identify risk factors for long-term survival in patients undergoing colectomy for non-metastatic colon cancer. METHODS: A prospective database of consecutive operations for non-metastatic colon cancer was reviewed. Patients were grouped as conversion (CONV) group, completed laparoscopic resection (LAP) group, or open resection (OPEN) group. The clinical and perioperative parameters, pathologic features, and oncologic outcomes were collected. Univariate analysis was performed for comparing these data. Patients without evidence of recurrence at last follow-up or still alive at the end of study period were censored. Kaplan-Meier curves were utilized to analyze survival. A multivariate analysis was performed to identify predictors of poor disease-free survival (DFS) and overall survival (OS). RESULTS: The conversion rate was 15.2 %. The most common reason for conversion was locally advanced cancer (45.5 %). Converted patients were associated with a longer operative time (188 ± 29.1 min, P < 0.001), greater blood loss (147 ± 14 mL, P < 0.001), and a higher rate of intra-operative complications (15.2 %, P = 0.042) compared to the completely laparoscopic or open patients. Days to flatus, early ambulation, and length of hospitalization were significantly shorter in completed laparoscopic resection (LAP) group (P < 0.001); however, the outcomes were comparable between conversion (CONV) and open resection (OPEN) groups. The incidence of wound infection was significantly higher in the OPEN group than in the LAP group (P = 0.005), whereas there were no significant differences observed between the CONV group and the OPEN group (P = 1.000) or between the LAP group and the CONV group (P = 0.073). The 5-year DFS in CONV patients (46.5 %) was comparable to LAP patients (55.5 %, P = 0.138) and OPEN patients (59.1 %, P = 0.113). Moreover, there were no significant differences noted in terms of the 5-year OS in the CONV group (56.7 %) compared to the LAP group (67.3 %, P = 0.317) or the OPEN group (66.3 %, P = 0.420). The multivariate analysis showed that pT3-4 cancer (P < 0.001) and poor differentiation (P < 0.001) were independent predictors of both lower OS and lower DFS, whereas leakage (P = 0.008) and lack of adjuvant chemotherapy (P = 0.023) were independent risk factors only of lower DFS. CONCLUSION: Conversion to open colectomy from an initial laparoscopic approach does not worsen the long-term survival in patients with non-metastatic colon cancer.

[Predicting value of dynamic alteration of blood neutrophil/lymphocyte ratio on recurrence-free survival in patients with advanced colon cancer after operation and chemotherapy].

OBJECTIVE: To evaluate whether neutrophil-lymphocyte ratio(NLR) predicts risk of recurrence in patients with advanced colon cancer undergoing curative resection followed by adjuvant chemotherapy. METHODS: A total of 149 patients with advanced colon cancer undergoing curative resection followed by adjuvant chemotherapy(FOLFOX6 protocol) were included. NLR was calculated preoperatively and before chemotherapy. The changes in NLR and the predictive value of NLR for prognosis were analyzed. RESULTS: The NLR of 149 patients was 2.8±1.5. NLR of 3.5 was identified according to the ROC curve. NLR<3.5 and NLR≥3.5 were classified as low and high NLR group, respectively. The 5-year recurrence-free survival(RFS) of patients with high preoperative NLR(n=22) was significantly worse than that of those with low preoperative NLR(n=127)(50.9% vs. 76.4%, P=0.025). The difference of 5-year RFS between high pre-chemotherapy NLR group(n=34) and low pre-chemotherapy NLR group(n=115) was statistically significant(50.1% vs. 71.4%, P=0.032). The 5-year RFS was 79.5% in patients with low preoperative NLR converting to high pre-chemotherapy NLR(n=16), similar to the group with high pre-chemotherapy group(P=0.077). The 5-year RFS was 17.7% in patients with high preoperative NLR reverting to low pre-chemotherapy NLR(n=12), similar to the group with low pre-chemotherapy group(P=0.978). There was significant difference in 5-year RFS between the postoperatively elevated group and postoperatively decreased group(P=0.036). CONCLUSION: An elevated blood NLR may be a biomarker of poor RFS in patients with advanced colon cancer after curative resection and chemotherapy.