Takanori Ochiai

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.

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;)

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.

Liver metastasis from rectal cancer with prominent intrabile duct growth.

Intrabiliary growth of liver metastases from colorectal cancer has rarely been studied. A surgically resected case of a metastatic liver tumor with prominent intrabiliary growth derived  from  rectal  cancer  is  reported.  The  patient  was  a 62‐year‐old man who had received a low anterior resection for rectal cancer in March 2000. He was re‐admitted due to obstructive jaundice in January 2003, and was diagnosed with hepatic malignancy in segment II of the liver with an intrabiliary tumor extending from the intrahepatic bile duct of segment II to the common hepatic duct. He underwent a left hepatectomy, a partial resection of segment VI, and an extrahepatic bile duct resection with reconstruction of the biliary tract. In the resected specimen, there were whitish tumors of 3 cm and 1.5 cm in diameter in segments II and VI, respectively, and an intrabiliary tumor originating from the main tumor in segment II extended to the common hepatic duct. Both the liver tumors and the intrabiliary tumor consisted of a well‐ to moderately differentiated adenocarcinoma, which showed the same histological features as the rectal cancer. The immunohistochemical findings strongly supported that these tumors, including the intrabiliary growth, were liver metastasis from the rectal cancer. The intrabiliary invasion and growth of metastatic liver tumors has generally been overlooked, notwithstanding their frequently observed biological behavior. The present case is informative, and further investigation into this type of metastatic liver tumor may be warranted.

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)

ENDOCYTOSCOPIC OBSERVATION OF ESOPHAGEAL SQUAMOUS CELL CARCINOMA

The endocytoscopy system (ECS), adapted for clinical use in 2003, is an ultra‐high‐power magnifying endoscope that allows observations at the cell level. ECS is based on the technology of light‐contact microscopy. The most evident use of ECS is for real‐time, high‐resolution diagnosis of nuclear abnormalities, mainly in patients with esophageal cancer. Up to now, three different types of ECS have been available. This diagnostic tool makes it possible to omit histological examination of biopsy samples in approximately 84% of esophageal squamous cell carcinoma, as evidence for both an increase of cell density and nuclear abnormalities is considered to be convincing proof that a lesion is malignant. Here we describe the features of ECS and the background that led to its development, and review the published literature pertaining to the observation of esophageal neoplasms using ECS.

Fournier's gangrene: report of six cases.

Abstract Fourniers gangrene (FG) is a fatal infectious disease with necrotic fasciitis of the external genitalia. This disease persists to this day in spite of recent advances in antibiotics. Although fewer than 100 cases have been reported in Japan, we have treated six cases in the last 4 years. The patients consisted of five men and one woman, with an average age of 47.5 years. All patients received surgical treatment including incisions, aggressive debridement, drainage, irrigation, and antibiotic therapy. Two patients, who suffered from underlying diseases of diabetic nephropathy and inclusion body myositis, died. These findings confirm the fact that FG requires a prompt diagnosis and immediate surgical treatment.

Treatment strategy for blunt hepatic trauma: analysis of 183 consecutive cases.

BACKGROUND/AIMS Non-operative management of hemodynamically stable trauma has proven successful; however laparotomy for hemodynamically unstable patients is still insufficient. We evaluated the results of treating blunt hepatic injury and appraised the appropriate surgical procedures. METHODOLOGY We analyzed the demographics, vital status, and severity of hepatic and concomitant organ injuries of 183 consecutive patients with blunt hepatic injuries between January 2001 and December 2008, retrospectively. RESULTS Twenty five of 183 patients died before the treatment was selected. The initial management was operative for 24 and non-operative for 134, 15 of whom later required laparotomy. Of the 134 treated non-operatively, 2 died after arterial embolization for pelvic fractures. Twelve patients died postoperatively: 6 of the hepatic injury and 6 of concomitant organ injuries. Considering Liver Injury Scale of operated patients, there was no liver-related death with grades I-III; however, liver-related mortality of grades IV and V was 37.5%. The incidence of liver-related deaths after anatomical resection was 0% of patients with grade IV, but 50% of patients with grade V, despite anatomical resection being the only effective procedure for grade V. CONCLUSIONS The results of anatomical resection for grade IV is satisfactory, but additional strategies are still required for grade V.

The difficulty of laparoscopic liver resection

Grading of difficulty is needed for laparoscopic liver resection (LLR). Indications for LLR are expanding worldwide from minor to major resections, particularly in institutions having surgeons with advanced skills. If the degrees of surgical difficulty were defined, it would serve as a useful guide when introducing LLR and stepping up to the more advanced LLR. As no previous study has addressed the degrees of difficulty of various LLR procedures, we devised a practical scoring system for this purpose. We extracted the following five factors from preoperative information to score difficulty levels: (1) tumor location, (2) extent of liver resection, (3) tumor size, (4) proximity to major vessels, and (5) liver function. This difficulty index is comprised of the cumulative score for the five individual factors. There has not yet been a standard definition of difficulty. Our proposed scoring system might be a practical means of assessing the difficulty of LLR procedures. However, this system must be prospectively validated.

Current status and limitations of the newly developed endocytoscope GIF‐Y0002 with reference to its diagnostic performance for common esophageal lesions

Objectives:  To investigate both neoplastic and non‐neoplastic lesions of the esophagus and to clarify the features of the surface cell morphology using a newly developed endocytoscope, the GIF‐Y0002.