Hidenobu Kojima

SOX9 is a novel cancer stem cell marker surrogated by osteopontin in human hepatocellular carcinoma.

The current lack of cancer stem cell (CSC) markers that are easily evaluated by blood samples prevents the establishment of new therapeutic strategies in hepatocellular carcinoma (HCC). Herein, we examined whether sex determining region Y-box 9 (SOX9) represents a new CSC marker, and whether osteopontin (OPN) can be used as a surrogate marker of SOX9 in HCC. In HCC cell lines transfected with a SOX9 promoter-driven enhanced green fluorescence protein gene, FACS-isolated SOX9+ cells were capable of self-renewal and differentiation into SOX9− cells, and displayed high proliferation capacity in vitro. Xenotransplantation experiments revealed that SOX9+ cells reproduced, differentiated into SOX9− cells, and generated tumors at a high frequency in vivo. Moreover, SOX9+ cells were found to be involved in epithelial-mesenchymal transition (EMT) and activation of TGFb/Smad signaling. Gain/loss of function experiments showed that SOX9 regulates Wnt/beta-catenin signaling, including cyclin D1 and OPN. Immunohistochemistry of 166 HCC surgical specimens and serum OPN measurements showed that compared to SOX9− patients, SOX9+ patients had significantly poorer recurrence-free survival, stronger venous invasion, and higher serum OPN levels. In conclusion, SOX9 is a novel HCC-CSC marker regulating the Wnt/beta-catenin pathway and its downstream target, OPN. OPN is a useful surrogate marker of SOX9 in HCC.

Hepatic Resection for Hepatocellular Carcinoma with Tumor Thrombus in the Major Portal Vein.

Background: The prognosis of hepatocellular carcinoma (HCC) with tumor thrombus in the major portal vein has been extremely poor. We investigated the outcome of hepatic resection in HCC with major portal vein tumor thrombus (PVTT). Methods: We retrospectively evaluated 52 consecutive patients who underwent hepatic resection for HCC with tumor thrombi in the first branch or trunk of the portal vein. Factors related to disease-free survival (DFS) and overall survival (OS) were analyzed. Results: The median DFS and OS times were 8.9 and 27.6 months for the whole cohort, respectively. Multiple tumors (hazard ratio 2.12; 95% CI 1.11-4.33; p = 0.023), positive surgical margins (hazard ratio 2.45; 95% CI 1.19-4.81; p = 0.016), and non-adjuvant hepatic arterial infusion chemotherapy (HAIC; hazard ratio 2.07; 95% CI 1.11-3.90; p = 0.023) were independent risk factors for DFS. Non-adjuvant HAIC (hazard ratio 1.84; 95% CI 1.01-3.37; p = 0.047) was an independent risk factor for OS. Conclusions: Macroscopically curative resection seems to be of benefit to HCC patients with PVTT, even with tumor thrombi in the first branch or trunk of the portal vein. Adjuvant postoperative HAIC might improve DFS and OS in such patients.

Efficient recellularisation of decellularised whole-liver grafts using biliary tree and foetal hepatocytes

A whole-organ regeneration approach, using a decellularised xenogeneic liver as a scaffold for the construction of a transplantable liver was recently reported. Deriving suitable scaffolds was the first step towards clinical application; however, effective recellularisation remains to be achieved. This report presents a strategy for the improvement of the recellularisation process, using novel cell-seeding technique and cell source. We evaluated recellularised liver grafts repopulated through the portal vein or the biliary duct with mice adult hepatocytes or E14.5 foetal hepatocytes. More than 80% of the cells seeded through the biliary tree entered the parenchyma beyond the ductule-lining matrix barrier and distributed throughout the liver lobule. In contrast, about 20% of the cells seeded through the portal tree entered the parenchyma. The gene expression levels of foetal hepatocyte albumin, glucose 6-phosphatase, transferrin, cytokeratin 19, and gamma-glutamyl transpeptidase were increased in three-dimensional cultures in the native liver-derived scaffolds, and the activation of liver detoxification enzymes and formation of biliary duct-like structures were supported. The metabolic functions of liver grafts recellularised with different cell types were similar. These results suggest that biliary tree cell-seeding approach is promising, and that liver progenitor cells represent a good cell source candidate.

Identification of keratin19-positive cancer stem cells associating human hepatocellular carcinoma using 18F-fluorodeoxyglucose positron emission tomography.

Purpose: The current lack of tools for easy assessment of cancer stem cells (CSC) prevents the development of therapeutic strategies for hepatocellular carcinoma (HCC). We previously reported that keratin 19 (K19) is a novel HCC-CSC marker and that PET with 18F-fluorodeoxyglucose (18F-FDG) is an effective method for predicting postoperative outcome in hepatocellular carcinoma. Herein, we examined whether K19+ HCC-CSCs can be tracked using 18F-FDG-PET. Experimental Design: K19 and glucose transporter-1 (GLUT1) expression was evaluated by IHC in 98 hepatocellular carcinoma patients who underwent 18F-FDG-PET scans before primary tumor resection. Standardized uptake values (SUV) for primary tumors and tumor-to-nontumor SUV ratios (TNR) were calculated using FDG accumulation levels, and values were compared among K19+/K19− patients. Using hepatocellular carcinoma cell lines encoding with a K19 promoter–driven enhanced GFP, 18F-FDG uptake and GLUT1 expression were examined in FACS-isolated K19+/K19− cells. Results: In hepatocellular carcinoma patients, K19 expression was significantly correlated with GLUT1 expression and FDG accumulation. ROC analyses revealed that among preoperative clinical factors, TNR was the most sensitive indicator of K19 expression in hepatocellular carcinoma tumors. In hepatocellular carcinoma cells, FACS-isolated K19+ cells displayed significantly higher 18F-FDG uptake than K19− cells. Moreover, gain/loss-of-function experiments confirmed that K19 regulates 18F-FDG uptake through TGFβ/Smad signaling, including Sp1 and its downstream target GLUT1. Conclusions:18F-FDG-PET can be used to predict K19 expression in hepatocellular carcinoma and should thereby aid in the development of novel therapeutic strategies targeting K19+ HCC-CSCs. Clin Cancer Res; 23(6); 1450–60. ©2016 AACR.

Establishment of practical recellularized liver graft for blood perfusion using primary rat hepatocytes and liver sinusoidal endothelial cells

Tissue decellularization produces a three‐dimensional scaffold that can be used to fabricate functional liver grafts following recellularization. Inappropriate cell distribution and clotting during blood perfusion hinder the practical use of recellularized livers. Here we aimed to establish a seeding method for the optimal distribution of parenchymal and endothelial cells, and to evaluate the effect of liver sinusoidal endothelial cells (LSECs) in the decellularized liver. Primary rat hepatocytes and LSECs were seeded into decellularized whole‐liver scaffolds via the biliary duct and portal vein, respectively. Biliary duct seeding provided appropriate hepatocyte distribution into the parenchymal space, and portal vein–seeded LSECs simultaneously lined the portal lumen, thereby maintaining function and morphology. Hepatocytes co‐seeded with LSECs retained their function compared with those seeded alone. Platelet deposition was significantly decreased and hepatocyte viability was maintained in the co‐seeded group after extracorporeal blood perfusion. In conclusion, our seeding method provided optimal cell distribution into the parenchyma and vasculature according to the three‐dimensional structure of the decellularized liver. LSECs maintained hepatic function, and supported hepatocyte viability under blood perfusion in the engineered liver graft owing to their antithrombogenicity. This recellularization procedure could help produce practical liver grafts with blood perfusion.

Identification of keratin 19-positive cancer stem cells associating human hepatocellular carcinoma using CYFRA 21-1

The current lack of an easily measurable surrogate marker of cancer stem cells (CSCs) prevents the clinical application of CSCs for hepatocellular carcinoma (HCC). We previously reported that keratin 19 (K19) is a novel HCC‐CSC marker associated with transforming growth factor beta (TGFβ)/Smad signaling, and that K19+ HCC‐CSCs could be a new therapeutic target of TGFβ receptor 1 inhibitor LY2157299. In this study, we examined whether K19+ HCC‐CSCs can be tracked using cytokeratin 19 fragment CYFRA 21‐1. In 147 HCC patients who underwent curative resection and evaluated K19 expression by immunohistochemistry, preoperative serum CYFRA 21‐1 levels were significantly higher in K19+ patients than in K19− patients (P < 0.01). Receiver operating characteristic analyses revealed that serum CYFRA 21‐1 was the statistically significant and the most sensitive predictor of tumor K19 expression among preoperative laboratory test values (P < 0.001). In HCC cells encoding with a K19 promoter‐driven enhanced green fluorescent protein, fluorescence‐activated cell sorting (FACS)‐isolated K19+ cells displayed significantly higher levels of supernatant CYFRA 21‐1 than K19− cells (P < 0.01). Gain/loss of K19 function experiments confirmed that CYFRA 21‐1 levels were regulated by K19 function in HCC cells. Furthermore, CYFRA 21‐1 levels reflected the treatment efficacy of LY2157299 in K19+ cells. In conclusion, CYFRA 21‐1 can be used to predict K19 expression in HCC, and should thereby aid in the development of novel therapeutic strategies targeting K19+ HCC‐CSCs.

CD90 expression in human intrahepatic cholangiocarcinoma is associated with lymph node metastasis and poor prognosis: YAMAOKA et al.

Intrahepatic cholangiocarcinoma (ICC) is the second most common primary liver cancer. However, its prognosis remains poor. Expression of cluster of differentiation 90 (CD90) has been identified as an indicator of poor prognosis in many cancers. Here, we examined the importance of CD90 expression in ICC.

Generation of non-viral, transgene-free hepatocyte like cells with piggyBac transposon

Somatic cells can be reprogrammed to induced hepatocyte-like cells (iHeps) by overexpressing certain defined factors in direct reprogramming techniques. Of the various methods to deliver genes into cells, typically used genome-integrating viral vectors are associated with integration-related adverse events such as mutagenesis, whereas non-integrating viral vectors have low efficiency, making viral vectors unsuitable for clinical application. Therefore, we focused on developing a transposon system to establish a non-viral reprogramming method. Transposons are unique DNA elements that can be integrated into and removed from chromosomes. PiggyBac, a type of transposon, has high transduction efficiency and cargo capacity, and the integrated transgene can be precisely excised in the presence of transposase. This feature enables the piggyBac vector to achieve efficient transgene expression and a transgene-free state, thus making it a promising method for cell reprogramming. Here, we attempted to utilize the piggyBac transposon system to generate iHeps by integrating a transgene consisting of Hnf4a and Foxa3, and successfully obtained functional iHeps. We then demonstrated removal of the transgene to obtain transgene-free iHeps, which still maintained hepatocyte functions. This non-viral, transgene-free reprogramming method using the piggyBac vector may facilitate clinical applications of iHeps in upcoming cell therapy.

A novel three-dimensional culture system maintaining the physiological extracellular matrix of fibrotic model livers accelerates progression of hepatocellular carcinoma cells

Liver fibrosis is characterized by the progressive accumulation of extracellular matrix (ECM) and is a strong predictor of hepatocellular carcinoma (HCC) development and progression. However, the effect of ECM in fibrotic livers on HCC cells is poorly understood. The aims of this study were to create a new culture system that retained the natural ECM of fibrotic model livers and to establish whether natural ECM regulated the characteristics of HCC cells. Using an organ decellularization technique, we created a new culture system that preserved the tissue-specific ECM of fibrotic model livers from CCl4-treated rats. The content of ECM in fibrotic model liver scaffolds was increased and the ECM microstructure was distorted. Quantitative polymerase chain reaction and immunofluorescence assays of HCC cells cultured in fibrotic model liver scaffolds for 7 days showed an epithelial-mesenchymal transition phenotype. Moreover, the ECM of fibrotic model livers promoted proliferation and chemoresistance of HCC cells. These results showed a novel effect of natural ECM in fibrotic model livers on the malignant behaviour of HCC cells. This new culture system will be useful for both understanding the cell biology of fibrotic livers and developing novel anti-cancer drugs.