Daisuke Ban

Identification of Pancreatic Cancer Stem Cells and Selective Toxicity of Chemotherapeutic Agents

BACKGROUND & AIMS Identification and purification of cancer stem cells (CSCs) could lead to new therapeutic targets, but their heterogeneous expansion is an obstacle to their study. We investigated whether it is possible to monitor pancreatic CSCs in real time, based on their intrinsic low level of proteasome activity. METHODS We engineered human pancreatic adenocarcinoma cells (PANC1, MIAPaCa2, BxPC3, and KLM1) to express a green fluorescent molecule fused to the degron of ornithine decarboxylase (Gdeg) from a retroviral vector; the fluorescent Gdeg accumulates in CSCs as a result of low activity of the 26S proteasome. Cells with high and low levels of fluorescence (Gdeg(high) and Gdeg(low)) were isolated by flow cytometry; tumor growth was analyzed in immunocompromised mice. We performed a screen for agents that were specifically toxic to pancreatic CSCs, in a synthetic lethal manner. RESULTS Gdeg(high) cells, but not Gdeg(low) cells, formed spheres and underwent asymmetric division-features of CSCs. Injection of as few as 10 Gdeg(high) cells led to tumor formation in mice. Gemcitabine was toxic to cultured Gdeg(low) cells, whereas Gdeg(high) cells were resistant. We observed that quercetin was toxic to Gdeg(high) cells in culture and in pre-established tumors grown from these cells in mice. Nuclear accumulation of β-catenin was detected in Gdeg(high), but not Gdeg(low), and lost after exposure to quercetin. CONCLUSIONS We used a fluorescence marker system for level of proteasome activity to identify pancreatic cancer cells with features of cancer stem cells. We identified quercetin as a compound that is specifically toxic to pancreatic CSCs.

Is celiac axis resection justified for T4 pancreatic body cancer

BACKGROUND The clinical impact of the distal pancreatectomy with en-bloc celiac axis resection for locally advanced pancreatic body cancer remains unclear. METHODS We reviewed the records of 13 patients who underwent distal pancreatectomy-celiac axis resection between 1991 and 2009, 58 patients who underwent distal pancreatectomy for pancreatic body cancer involving major vessels, the extrapancreatic neural plexus or other organs (T4 according to the Japanese stage classification) between 1991 and 2009, and 24 patients with unresectable locally advanced pancreatic cancer without distant metastases (unresectable group) between 2001 and 2009. The clinicopathologic factors and overall survival among the 3 groups were compared. RESULTS The distal pancreatectomy-celiac axis resection group was associated with a significantly higher incidence of morbidity (92% vs 60%, P = .03) and positive surgical margins (69% vs 26%, P = .003) than the distal pancreatectomy group; however, no survival difference was found between the 2 groups. No survivor has lived more than 3 years after operation in the distal pancreatectomy-celiac axis resection group. The distal pancreatectomy-celiac axis resection group had a significantly better prognosis than the unresectable group (median survival time, 20.8 vs 9.8 months; P = .01). CONCLUSION Aggressive resection for T4 pancreatic body cancer by distal pancreatectomy-celiac axis resection can be justified for otherwise unresectable tumors. The surgical indication should be evaluated carefully because of the higher incidence of morbidity and lower incidence of curability compared with distal pancreatectomy, as well as because there have been no long-term survivors so far.

Visualization of stem cell features in human hepatocellular carcinoma reveals in vivo significance of tumor‐host interaction and clinical course

Hepatocellular carcinoma (HCC) is one of the most aggressive malignancies because of recurrence and/or metastasis even after curative resection. Emerging evidence suggests that tumor metastasis and recurrence might be driven by a small subpopulation of stemness cells, so‐called cancer stem cells (CSCs). Previous investigations have revealed that glioma and breast CSCs exhibit intrinsically low proteasome activity and that breast CSCs also reportedly contain a lower reactive oxygen species (ROS) level than corresponding nontumorigenic cells. Here we visualized two stem cell features, low proteasome activity and low intracellular ROS, in HCC cells using two‐color fluorescence activated cell sorting to isolate cells with stem cell features. These cells were then analyzed for their division behavior in normoxia and hypoxia, expression of stem cell markers, tumorigenicity, metastatic potential, specific gene expression signatures, and their clinical implications. A visualized small subpopulation of HCC cells demonstrated asymmetric divisions. Their remarkable tumorigenicity in nonobese diabetic/severe combined immunodeficient mice suggested the cancer initiation potential of these HCC CSCs. Comprehensive gene expression analysis revealed that chemokine‐related genes were up‐regulated in the CSCs subpopulation. Our identified HCC CSCs facilitated the migration of macrophages in vitro and demonstrated metastatic potential by way of recruitment of macrophages in vivo. In patients who undergo curative operation for HCC, the CSC‐specific gene signature in the liver microenvironment significantly correlates with recurrence. Conclusion: Based on these findings, the stem cell feature monitoring system proposed here is a promising tool to analyze the in vivo significance of CSC microenvironments in human HCCs. (HEPATOLOGY 2013;)

Efficacy of a Hepatectomy and a Tumor Thrombectomy for Hepatocellular Carcinoma with Tumor Thrombus Extending to the Main Portal Vein

IntroductionHepatocellular carcinoma (HCC) with major portal tumor thrombus has been considered to be a fatal disease. A thrombectomy remains the only therapeutic option that offer a chance of complete tumor removal avoiding acute portal vein obstruction. However, the efficacy of tumor thrombectomy in addition to hepatectomy has not been well evaluated.MethodsOf 979 patients who consecutively underwent initial HCC resection, 45 (4.6%) HCC patients with tumor invasion of the first branch of the portal vein (vp3) and tumor in the main portal trunk or the opposite-side portal branch (vp4) were retrospectively analyzed to evaluate the efficacy of hepatectomy and tumor thrombectomy.ResultsAlpha-fetoprotein, serosal invasion, and intrahepatic metastases were independently significant prognostic factors in all the 45 patients with vp3 or vp4 HCC. The 3- and 5-year survival rates in vp3 and vp4 group were 35.3% and 41.8%, and 21.2% and 20.9%, respectively. There were longer operative times and more intraoperative bleeding in patients with vp4, but no significant difference in mortality, morbidity, and survival between patients with vp3 and vp4.ConclusionHepatectomy and thrombectomy for vp4 could not only avoid acute portal occlusion due to tumor thrombus but provide a comparable survival benefit with hepatectomy for vp3.

Resection of a cancer developing in the remnant pancreas after a pancreaticoduodenectomy for pancreas head cancer

We report a rare case of a curative resection performed on a carcinoma developing in the remnant pancreas at 3 years 7 months after a pancreaticoduodenectomy for pancreatic cancer. A 63-year-old man underwent a pancreaticoduodenectomy for pancreatic cancer on November 1999. Because the celiac trunk was occluded by atherosclerosis, an aortohepatic bypass with a saphenous vein graft was performed simultaneously. In May 2003, tumor marker levels increased, and a tumor was detected in the remnant pancreas on computed tomography. There were no findings such as invasion into the surrounding tissue or distant metastasis, and therefore we removed the remnant pancreas in July 2003. Histopathologically, the tumor consisted of a well-differentiated tubular adenocarcinoma and was limited to the pancreas. Moreover, the anastomotic site of the pancreaticojejunostomy was negative for cancer, and some foci of papillary hyperplasia and goblet cell metaplasia of the pancreatic ductal epithelium, which was thought to be the precursor of the pancreatic cancer, were seen. These findings suggested that the tumor was a second primary cancer developing in the remnant pancreas. This case provided suggestive evidence for the development of pancreatic cancer, and the surgical procedure for a pancreaticoduodenectomy with occlusion of the celiac trunk is discussed.

Oxidative stress pathways in noncancerous human liver tissue to predict hepatocellular carcinoma recurrence: A prospective, multicenter study†‡

The prediction of cancer recurrence holds the key to improvement of the postoperative prognosis of patients. In this study, the recurrence of early‐stage hepatocellular carcinoma (HCC) after curative hepatectomy was analyzed by the genome‐wide gene‐expression profiling on cancer tissue and the noncancerous liver tissue. Using the training set of 78 cases, the cytochrome P450 1A2 (CYP1A2) gene in noncancerous liver tissue was identified as the predictive candidate for postoperative recurrence (hazard ratio [HR], 0.447; 95% confidence interval [CI], 0.249‐0.808; P = 0.010). Multivariate analysis revealed the statistically significant advantage of CYP1A2 down‐regulation to predict recurrence (odds ratio, 0.534; 95% CI, 0.276‐0.916; P = 0.036), and the expression of CYP1A2 protein was confirmed immunohistochemically. An independently multi‐institutional cohort of 211 patients, using tissue microarrays, validated that loss of expression of CYP1A2 in noncancerous liver tissue as the only predictive factor of recurrence after curative hepatectomy for early‐stage HCC (HR, 0.480; 95% CI, 0.256‐0.902; P = 0.038). Gene set‐enrichment analysis revealed close association of CYP1A2 down‐regulation with oxidative stress pathways in liver tissue (P < 0.001, false discovery rate [FDR] = 0.042; P = 0.006, FDR = 0.035). Our results indicate these pathways as the molecular targets to prevent recurrence, as well as the potential prediction of the super high‐risk population of HCC using liver tissue. (HEPATOLOGY 2011;54:1273–1281)

Dominant Expression of DCLK1 in Human Pancreatic Cancer Stem Cells Accelerates Tumor Invasion and Metastasis

Patients with pancreatic cancer typically develop tumor invasion and metastasis in the early stage. These malignant behaviors might be originated from cancer stem cells (CSCs), but the responsible target is less known about invisible CSCs especially for invasion and metastasis. We previously examined the proteasome activity of CSCs and constructed a real-time visualization system for human pancreatic CSCs. In the present study, we found that CSCs were highly metastatic and dominantly localized at the invading tumor margins in a liver metastasis model. Microarray and siRNA screening assays showed that doublecortin-like kinase 1 (DCLK1) was predominantly expressed with histone modification in pancreatic CSCs with invasive and metastatic potential. Overexpression of DCLK1 led to amoeboid morphology, which promotes the migration of pancreatic cancer cells. Knockdown of DCLK1 profoundly suppressed in vivo liver metastasis of pancreatic CSCs. Clinically, DCLK1 was overexpressed in the metastatic tumors in patients with pancreatic cancer. Our studies revealed that DCLK1 is essential for the invasive and metastatic properties of CSCs and may be a promising epigenetic and therapeutic target in human pancreatic cancer.

Pancreatic ducts as an important route of tumor extension for acinar cell carcinoma of the pancreas.

Acinar cell carcinoma (ACC) of the pancreas is very rare, which usually grows expansively. Recently, a variant of ACC with predominant growth in the pancreatic ducts has been proposed, and is speculated to have potentially less aggressive behavior. The aim of this study was to investigate how the pancreatic duct system is related to the growth and extension of ACC. We reviewed the detailed gross and histologic features of 13 cases of ACC, of which 7 (54%) showed intraductal polypoid growth (IPG) of the tumor in the large pancreatic ducts with a mean IPG length of 24.8 mm. Tumors with IPG were found to spread characteristically along the pancreatic ducts as extending polypoid projections, filling the ducts and destroying the duct walls, although tumors did not tend to extend beyond the pancreatic parenchyma. Comparison of the clinicopathologic characteristics showed that ACC with IPG had less infiltrative features including lymphatic, venous, and neural invasion, formation of tumor thrombus in the portal vein, nodal metastasis, and invasion beyond the pancreas to the surrounding organs; death in only 1 case (14%) of ACC with IPG was the result of ACC itself. In contrast, ACC without IPG frequently showed more infiltrative growth, and was the cause of death in 50% of patients with this type of tumor. Intraductal dissemination of ACC in pancreatic ducts was proven in 1 case of ACC with IPG. These findings suggest that a significant proportion of ACC shows IPG, which is potentially linked to less aggressive clinicopathologic characteristics.

Contrast‐enhanced intraoperative ultrasonography for vascular imaging of hepatocellular carcinoma: Clinical and biological significance

Abnormal tumor vascularity is one of the typical features of hepatocellular carcinoma (HCC). In this study, the significance of contrast‐enhanced intraoperative ultrasonography (CEIOUS) images of HCC vasculature was evaluated by clinicopathological and gene expression analyses. We enrolled 82 patients who underwent curative hepatic resection for HCC with CEIOUS. Clinicopathological and gene expression analyses were performed according to CEIOUS vasculature patterns. CEIOUS images of HCC vasculatures were classified as reticular HCC or thunderbolt HCC. Thunderbolt HCC was significantly correlated with higher alpha‐fetoprotein levels, tumor size, histological differentiation, portal vein invasion, and tumor‐node‐metastasis stage, and these patients demonstrated a significantly poorer prognosis for both recurrence‐free survival (P = 0.0193) and overall survival (P = 0.0362) compared with patients who had reticular HCC. Gene expression analysis revealed that a rereplication inhibitor geminin was significantly overexpressed in thunderbolt HCCs (P = 0.00326). In vitro knockdown of geminin gene reduced significantly the proliferation of human HCC cells. Immunohistochemical analysis confirmed overexpression of geminin protein in thunderbolt HCC (P < 0.0001). Multivariate analysis revealed geminin expression to be an independent factor in predicting poor survival in HCC patients (P = 0.0170). Conclusion: CEIOUS vascular patterns were distinctly identifiable by gene expression profiling associated with cellular proliferation of HCC and were significantly related to HCC progression and poor prognosis. These findings might be clinically useful as a determinant factor in the postoperative treatment of HCC. (HEPATOLOGY 2013)

Decreased Mrp2-Dependent Bile Flow in the Post-Warm Ischemic Rat Liver

BACKGROUND The link between microcirculatory disturbance and hepatocellular dysfunction remains unknown. The present study was designed to examine the key event of warm ischemia reperfusion (WIR) injury with subsequent cholestasis. METHODS A left lobar 70% ischemia and reperfusion rat model was used in this study. The portal vein and hepatic artery to the left lateral lobe of the liver were subjected to 20 min of warm ischemia followed by 60 min of reperfusion to collect bile and to measure its constituents. RESULTS The hepatocellular injury was increased significantly in livers exposed to WIR, as judged by serum alanine aminotransferase. This event coincided with decreased bile production and biliary concentration of glutathione (GSH), suggesting impaired bile salts-independent bile flow, while biliary phospholipids and bile salts were not decreased. Additionally, hepatic adenosine triphosphate and GSH were not decreased after WIR. Since the biliary GSH, which is a major driving force for bile salts-independent bile flow, is excreted from hepatocytes into the bile via multidrug resistance protein 2 (Mrp2), we examined whether intracellular localization of Mrp2 occurred. Immunohistochemical analyses revealed hepatocellular Mrp2 was retrieved from bile canalicular membrane into the pericanalicular cytoplasm in the post-warm ischemic livers. Microcirculatory disturbance in livers exposed to 20 min of warm ischemia improved to levels comparable to controls. CONCLUSION Mrp2 internalization, observed in this study, may play an important determinant of cholestasis in the post-warm ischemic livers.