Essential dextrin structure as donor substrate for 4-α-glucanotransferase in glycogen debranching enzyme.

Glycogen debranching enzyme is a single polypeptide with distinct catalytic sites for 4-α-glucanotransferase and amylo-α-1,6-glucosidase. To allow phosphorylase to degrade the inner tiers of highly branched glycogen, 4-α-glucanotransferase converts the phosphorylase-limit biantennary branch G-G-G-G-(G-G-G-G↔)G-G- (G: D-glucose, hyphens: α-1,4-linkages; double-headed arrow: α-1,6-linkage) into the G-G-G-G-(G↔)G-G- residue, which is then subjected to amylo-α-1,6-glucosidase to release the remaining G↔ residue. However, while the essential side-chain structure of the 4-α-glucanotransferase donor substrate has been determined to be the G-G-G-G↔ residue (Watanabe, Y., et al. (2008) J. Biochem.  143, 435-440), its essential main-chain structure remains to be investigated. In this study, we probed the 4-α-glucanotransferase donor-binding region using novel fluorogenic dextrins Gm-(G4↔)G-Gn-F (F: 1-deoxy-1-[(2-pyridyl)amino]-D-glucitol) and maltohexaose (G6) as the donor and acceptor substrates, respectively. 4-α-Glucanotransferase exhibited maximum activity toward G4-(G4↔)G-F and G4-(G4↔)G-G-F, indicating that recognition of the G4-(G4↔)G- moiety was essential for full enzyme function. Notably, when the 4-α-glucanotransferase activity toward G4-(G4↔)G-G-F was taken as unity, those toward nonbranching dextrins were <0.001. This indicated that the disproportionation activities toward maltooligosaccharides (Gm) are abnormal behaviors of 4-α-glucanotransferase. Notably, however, these activities have been traditionally measured to identify the 4-α-glucanotransferase mutations causing glycogen storage disease type III. This study provides a basis for more accurate identification.

Usefulness of contrast-enhanced ultrasonography for biliary tract disease.

Conventional ultrasonography (US) for biliary tract disease shows high time and spatial resolution. In addition, it is simple and minimally invasive, and is selected as a first-choice examination procedure for biliary tract disease. Currently, contrast-enhanced US (CEUS), which facilitates the more accurate assessment of lesion blood flow in comparison with color and power Doppler US, is performed using a second-generation ultrasonic contrast agent. Such agents are stable and provide a timeline for CEUS diagnosis. Gallbladder lesions are classified into three types: gallbladder biliary lesion (GBL), gallbladder polypoid lesion (GPL), and gallbladder wall thickening (GWT). Bile duct lesions can also be classified into three types: bile duct biliary lesion (BBL), bile duct polypoid lesion (BDPL), and bile duct wall thickening (BDWT). CEUS facilitates the differentiation of GBL/BBL from tumorous lesions based on the presence or absence of blood vessels. In the case of GPL, it is important to identify a vascular stalk attached to the lesion. In the case of GWT, the presence or absence of a non-contrast-enhanced area, the Rokitansky-Aschoff sinus, and continuity of a contrast-enhanced gallbladder wall layer are important for differentiation from gallbladder cancer. In the case of BDWT, it is useful to evaluate the contour of the contrast-enhanced medial layer of the bile duct wall for differentiating IgG4-related sclerosing cholangitis from primary sclerosing cholangitis. CEUS for ampullary carcinoma accurately reflects histopathological findings of the lesion. Evaluating blood flow in the lesion, continuity of the gallbladder wall, and contour of the bile duct wall via CEUS provides useful information for the diagnosis of biliary tract disease.

Usefulness of endoscopic ultrasound-guided fine-needle biopsy for the diagnosis of autoimmune pancreatitis using a 22-gauge Franseen needle: a prospective multicenter study.

BACKGROUND: Detailed histological evaluation is important in the diagnosis of autoimmune pancreatitis (AIP). However, it remains challenging to obtain adequate tissue from the pancreas. Recently, several reports have suggested the usefulness of endoscopic ultrasound-guided fine-needle biopsy (EUS-FNB) using the new "core" needles for acquiring pancreatic tissue. We aimed to investigate the usefulness of EUS-FNB for diagnosing AIP with one such needle, a 22-gauge Franseen needle. METHODS: Patients who met the imaging diagnostic criteria for AIP based on the International Consensus Diagnostic Criteria (ICDC) were enrolled in the study. All patients underwent EUS-FNB with a 22-gauge Franseen needle. Histological findings were evaluated based on the ICDC, and the detection rates of level 1 and level 1 or 2 histology were calculated. RESULTS: 56 patients from 11 different institutions were enrolled in the final analysis (55 suspected to have type 1 AIP and one with type 2 AIP). Lymphoplasmacytic infiltration, obliterative phlebitis, storiform fibrosis, and > 10 IgG4-positive cells per high-power field were detected in 55 (100 %), 24 (43.6 %), 40 (72.7 %), and 36 (65.5 %) of the 55 patients, respectively. The detection rates of level 1 and level 1 or 2 histology for AIP were 58.2 % (95 % confidence interval [CI] 44.1 % - 71.3 %) and 92.7 % (95 %CI 82.4 % - 98.0 %), respectively, which were apparently higher than our historical results (7.9 % [95 %CI 1.7 % - 21.4 %] and 62.2 % [95 %CI 46.5 % - 76.2 %], respectively) using a conventional needle. CONCLUSIONS: EUS-FNB with a 22-gauge Franseen needle demonstrated favorable detection rates which would be clinically beneficial for the histological diagnosis of AIP.

Comparison of 8- and 10-mm diameter fully covered self-expandable metal stents: A multicenter prospective study in patients with distal malignant biliary obstruction.

OBJECTIVES: The time to recurrent biliary obstruction (TRBO) of unresectable distal malignant biliary obstruction is generally thought to be longer when a self-expandable metal stent (SEMS) with a thicker inner diameter is used for drainage, but the dependence on the inner diameter using a fully covered SEMS (FCSEMS) is uncertain. The objective of this multicenter prospective study was to compare TRBO and adverse events, such as cholecystitis and pancreatitis, in treatment of patients with unresectable malignant biliary obstruction using 8- and 10-mm diameter FCSEMS. METHODS: Eighteen tertiary-care centers participated in the study. Patients were allocated to the 8- and 10-mm diameter groups. TRBO, non-inferiority of the 8-mm FCSEMS, overall survival time, frequency and type of adverse events, and non-recurrent biliary obstruction (RBO) rate at the time of death were compared between the two groups. RESULTS: Median TRBO did not differ significantly between the 8-mm (n = 102) and 10-mm (n = 100) groups (275 vs 293 days, P = 0.971). The hazard ratio of the 8- to 10-mm groups was 0.90 (80% confidence interval, 0.77-1.04; upper limit lower than the acceptable hazard ratio [1.33] of the null hypothesis). Based on these findings, the 8-mm diameter stent was determined to be non-inferior to the 10-mm diameter stent. Survival time, incidence of adverse events and non-RBO rate at the time of death did not differ significantly between the two groups. CONCLUSIONS: Time to RBO with an 8-mm diameter FCSEMS was non-inferior to that with a 10-mm diameter FCSEMS. This finding is important for development of future SEMS.

Randomized Phase II Study of Consecutive-Day versus Alternate-Day Treatment with S-1 as Second-Line Chemotherapy in Advanced Pancreatic Cancer.

OBJECTIVE: To evaluate the efficacy and safety of alternate-day administration of S-1 as second-line chemotherapy for unresectable pancreatic cancer in a multicenter, randomized, phase II study. METHODS: Patients with histologically proven, unresectable pancreatic cancer treated with chemotherapy not including S-1 as first-line therapy were randomly assigned to receive either daily or alternate-day treatment with S-1. The primary end point was overall survival (OS), and the secondary end points were progression-free survival (PFS), time to treatment failure (TTF), response rate, and adverse events. RESULTS: A total of 77 patients were enrolled, of which 75 were included in the final analysis. The median OS was 4.5 months in the daily group and 4.4 months in the alternate-day group (HR 1.178; 95% CI 0.741-1.875), with no significance in PFS and TTF. The response rate was 2.8% in the daily group and 0% in the alternate-day group. Grade 3 or higher adverse events occurred with significantly higher incidence in the daily group (47.2 vs. 25.6%, p = 0.044). CONCLUSION: As a second-line chemotherapy for unresectable pancreatic cancer, although the efficacy in both groups was comparable and we can expect fewer toxicities with alternate-day administration of S-1, the noninferiority of alternate-day treatment to daily treatment with S-1 was not verified.

A Rare Case of Cryopyrin-associated Periodic Syndrome in an Elderly Woman with NLRP3 and MEFV Mutations.

We herein report a case of a 75-year-old woman who presented with a low-grade fever, repeated cold-induced urticaria, and painful leg edemas with neutrocytosis. Because her mother also had cold-induced urticaria and her skin lesions histologically showed neutrophilic dermatitis, we suspected that she had familial cold autoinflammatory syndrome, a subtype of cryopyrin-associated periodic syndromes. Sequencing of the NLRP3 and MEFV genes revealed that she carried both the p.A439V missense mutation and p.E148Q homozygous mutation, which is commonly detected in familial Mediterranean fever patients. The administration of colchicine reduced the frequency and severity of her skin rash and leg edema.

[Gastric tuberculosis in an elderly patient:a case report].

A woman in her 70s presented to our hospital with epigastric pain, back pain, and weight loss. Esophagogastroduodenoscopy was performed, and numerous protuberances, which were suspected to be submucosal tumors, were found at the gastric corpus. The patient was diagnosed with gastric tuberculosis based on the biopsy results of these protuberances. Histopathological analysis demonstrated non-caseating epithelioid granuloma. A positive culture for Mycobacterium tuberculosis was also obtained on gastric juice analysis and confirmed using polymerase chain reaction assay. In the rapidly aging population in Japan, our findings emphasize on the importance of differentiating gastrointestinal tuberculosis, including gastric tuberculosis, from other diseases. This case may provide information about the development of gastric tuberculosis.

Natural history of pancreatic cystic lesions: A multicenter prospective observational study for evaluating the risk of pancreatic cancer.

BACKGROUND AND AIM: The aim of this study is to elucidate the natural history of pancreatic cystic lesions (PCLs), including branch duct-type intraductal papillary mucinous neoplasm (BD-IPMN), via midterm follow-up analysis of a multicenter prospective observational study (NSPINAL study). METHODS: From July 2011 to October 2016, 881 patients with PCLs were enrolled in NSPINAL study, and 664 patients with > 12 months of follow up were analyzed. Every patient was asymptomatic, and endoscopic ultrasound was performed at the initial diagnosis to exclude high-risk individuals. Follow up included endoscopic ultrasound, computed tomography, or magnetic resonance imaging at least once a year. Serial morphological changes and the pancreatic cancer (PC) incidence, including malignant progression of PCLs, were evaluated. RESULTS: The 664 patients (358 men) were followed for a median of 33.5 months (interquartile range 29). The cyst and main pancreatic duct sizes were 16.6 ± 9.3 and 2.3 ± 1.0 mm, respectively. Morphologically, 518 cases were multilocular, 137 were unilocular, and 9 had a honeycomb pattern; 269 cases involved multifocal lesions. Ninety-six patients (14.5%) showed worsening progression on imaging. There were two resectable and four unresectable cases of pancreatic ductal adenocarcinoma and three cases of malignant BD-IPMN. The 3-year risk of developing PC was 1.2%. The standardized incidence ratio for PC among PCLs was 10.0 (95% confidence interval 3.5-16.5), and the standardized incidence ratio among BD-IPMN was 16.6 (95% confidence interval 5.1-28.1). Multivariate analysis showed that development of symptoms and worsening progression were significant predictors of PC. CONCLUSIONS: Malignant progression of PCLs, including PC development, is not uncommon. Patients with PCLs should be carefully monitored to detect pancreatic ductal adenocarcinoma at early stages.

A case of a patient with granulocyte-colony stimulating factor-producing pancreatic cancer who responded to nab-paclitaxel plus gemcitabine.

A 67-year-old male patient presented with an irregular mass involving the pancreatic body and tail with multiple liver/lymph node metastases. A biopsy indicated the presence of a poorly differentiated adenocarcinoma. Fever and increased white blood cell count, C-reactive protein levels, and granulocyte-colony stimulating factor (G-CSF) levels led to the diagnose of G-CSF-producing pancreatic cancer. The patient did not respond to FOLFIRINOX therapy (leucovorin, fluorouracil, irinotecan, and oxaliplatin), but nab-paclitaxel plus gemcitabine treatment was effective, resulting in tumor shrinkage and reduced G-CSF levels. After the fifth course of this therapy, exacerbation was noted, and the patient died of primary cancer 6 months after initiating the therapy. Here we report the case of this patient with G-CSF-producing pancreatic cancer who responded to chemotherapy.

Analysis of the risk factors for severity in post endoscopic retrograde cholangiopancreatography pancreatitis: The indication of prophylactic treatments.

AIM: To determine the risk factors of severe post endoscopic retrograde cholangiopancreatography pancreatitis (sPEP) and clarify the indication of prophylactic treatments. METHODS: At our hospital, endoscopic retrograde cholangiopancreatography (ERCP) was performed on 1507 patients from May 2012 to December 2015. Of these patients, we enrolled all 121 patients that were diagnosed with post endoscopic retrograde PEP. Fourteen of 121 patients diagnosed as sPEP were analyzed. RESULTS: Forty-one patients had contrast media remaining in the pancreatic duct after completion of ERCP. Seventy-one patients had abdominal pain within three hours after ERCP. These were significant differences for sPEP (P < 0.05). The median of Body mass index, the median time for ERCP, the median serum amylase level of the next day, past histories including drinking and smoking, past history of pancreatitis, sphincter of Oddi dysfunction, whether emergency or not, expertise of ERCP procedure, diverticulum nearby Vater papilla, whether there was sphincterotomy or papillary balloon dilation, pancreatic duct cannulation, use of intra-ductal ultrasonography enforcement, and transpapillary biopsies had no significant differences with sPEP. CONCLUSION: Contrast media remaining in the pancreatic duct and the appearance of abdominal pain within three hours after ERCP were risk factors of sPEP.

Nab-paclitaxel+gemcitabine therapy for adenosquamous carcinoma of the pancreas: an autopsy case.

A male patient aged over 60 years presented with abdominal pain. A solid lesion measuring 7cm was detected in the pancreatic body and tail, along with periaortic lymphadenopathy. Endoscopic ultrasound-guided fine-needle aspiration suggested squamous cell carcinoma. Nab-paclitaxel+gemcitabine therapy was effective;however, tumor progression was noted after the completion of the fourth course, and the patient died from the primary cancer 7 months after the initial consultation. Autopsy led to a definitive diagnosis of adenosquamous carcinoma of the pancreas. Non-resected adenosquamous carcinoma of the pancreas treated by chemotherapy is rare. Here, we report such an example in the present case study.

Acute lymphoblastic leukemia in a girl with Wilson's disease.

Wilson's disease (WD) is an autosomal recessive defect in cellular copper transportation. Although acute lymphoblastic leukemia (ALL) is the most common form of childhood malignancy, only two cases of ALL associated with WD have been reported to date. One patient died of relapse and infection, and the other died of neutropenic sepsis during the treatment. We here describe the case of a 10-year-old girl with WD and ALL. Adverse events of chemotherapy, including liver toxicity and severe myelosuppression, necessitated adjustments in the chemotherapy doses. After completion of the treatment, the patient has remained in remission from ALL without progression of liver damage for 2 years. Severe treatment-related toxicity should be considered in chemotherapy for patients with WD.

[Comparative study on the efficacy and safety of continuous versus twice-daily 3-hour infusion of cyclosporine A in pediatric hematopoietic stem cell transplantation].

Cyclosporine (CsA) is widely used for graft-versus-host disease (GVHD) in pediatric hematopoietic stem cell transplantation (HSCT); however, the optimal schedule of its administration has not been established. We retrospectively compared the time-course of blood CsA levels and the incidence of CsA-associated adverse effects, grade II-IV acute GVHD, and chronic GVHD among 26 pediatric HSCT recipients who were receiving CsA by continuous infusion (CIF) (n=8) or by 3h infusion twice-daily (3TD) (n=18). In the 3TD group, the target level of the C3 value, which is strongly co-related with the area under the concentration-time curve, was maintained without any serious adverse effects in most patients. No significant differences were observed in the incidence of grade II-IV acute GVHD and chronic GVHD between CIF and 3TD groups. 3TD in combination with C3 monitoring enables safe and easy control of CsA blood levels and is thought to be useful for GVHD prophylaxis in pediatric HSCT.

Endoscopic ultrasound-guided fine needle aspiration in the differentiation of type 1 and type 2 autoimmune pancreatitis.

AIM: To investigate the usefulness of endoscopic ultra-sound-guided fine needle aspiration (EUS-FNA) in the differentiation of autoimmune pancreatitis (AIP). METHODS: We retrospectively reviewed 47 of 56 AIP patients who underwent EUS-FNA and met the Asian diagnostic criteria. On 47 EUS-FNA specimens, we evaluated the presence of adequate material and characteristic features of lymphoplasmacytic sclerosing pancreatitis (LPSP) and idiopathic duct-centric pancreatitis (IDCP) mentioned in the International Consensus Diagnostic Criteria and examined if these findings make a contribution to the differential diagnosis of type 1 and type 2 AIP. A disposable 22-gauge needle was used for EUS-FNA. RESULTS: Adequate specimens including pancreatic tissue for differentiating AIP from cancer were obtained from 43 of 47 patients who underwent EUS-FNA. EUS-FNA was performed from the pancreatic head in 21 cases, which is known to be technically difficult when performed by core biopsy; there was no significant difference in the results compared with pancreatic body-tail. Nine of 47 patients met level 1 findings of LPSP and 5 patients met level 2 findings of LPSP. No one met level 1 findings of IDCP, but 3 patients met level 2 findings of IDCP. Of 10 seronegative cases, 2 cases were diagnosed with "definitive type 1 AIP", and 3 cases were diagnosed with "probable type 2 AIP" when considering both the level 2 histological findings and response to steroids. CONCLUSION: EUS-FNA is useful in the differentiation of type 1 and type 2 AIP, particularly in seronegative cases.

Malignant transformation of branch duct-type intraductal papillary mucinous neoplasms of the pancreas based on contrast-enhanced endoscopic ultrasonography morphological changes: focus on malignant transformation of intraductal papillary mucinous neoplasm itself.

OBJECTIVES: The natural history of branch duct-type intraductal papillary mucinous neoplasms (BD-IPMNs) of the pancreas remains unclear. We conducted a retrospective long-term follow-up study for malignant transformation (MT) of BD-IPMNs focusing on morphological changes. METHODS: The subjects consisted of 142 patients who underwent contrast-enhanced endoscopic ultrasonography for initial diagnosis from January 2001 with more than 12 months of follow-up. The MT rate, including the co-occurrence of invasive ductal cancer, was evaluated by univariate and multivariate analysis. In addition, on the basis of morphological changes in patients who underwent surgery, the predictive factors for malignant IPMNs were evaluated. RESULTS: Median follow-up term was 42.5 months (range, 12-105 months). Thirty patients who exhibited morphological changes underwent surgery. Malignant transformation occurred in 9 patients (6.3%), and 5-year MT rate was 10.7%. The co-occurrence of invasive ductal cancer was seen in 5 patients. Multivariate analysis showed that the existence of mural nodules at initial diagnosis and involvement of main pancreatic duct were significant predictors of MT of BD-IPMN. CONCLUSIONS: Malignant transformation of BD-IPMN is not rare. The observation of morphological changes of main pancreatic duct and nodules, mainly on contrast-enhanced endoscopic ultrasonography, is practical and useful for predicting MT of BD-IPMN itself.

Dynamic quantitative evaluation of contrast-enhanced endoscopic ultrasonography in the diagnosis of pancreatic diseases.

OBJECTIVES: This study aimed to investigate the usefulness of contrast-enhanced endoscopic ultrasonography (EUS) with time-intensity curve (TIC) in differentiating pancreatic diseases. METHODS: Patients who underwent contrast-enhanced EUS between January 2007 and June 2009 were analyzed retrospectively, including 48 with pancreatic ductal cancer (PC), 14 with autoimmune pancreatitis (AIP), 13 with mass-forming pancreatitis (MFP), and 16 with pancreatic endocrine tumor (PET). After intravenous injection of contrast agent, contrast imaging pattern, TIC-based quantitative evaluation, and diagnostic ability of EUS in combination with TIC to diagnose benignancy or malignancy were assessed. RESULTS: Hypovascular and heterogeneous pattern (42/48) in PC, isovascular and homogenous (21/27) in AIP and MFP, and hypervascular and rapid stained (16/16) in PET were observed. The echo intensity reduction rate from the peak at 1 minute was the greatest in PC followed by MFP, AIP, and PET (P < 0.05). The diagnostic accuracies based on contrast imaging pattern (84.0%) and TIC (88.0%) were higher than those based on B-mode imaging (82.6%) and dynamic computed tomography (81.3%). In EUS in combination with TIC, sensitivity, specificity, and accuracy rose up to 95.8%, 92.6%, and 94.7%, respectively. CONCLUSIONS: Contrast-enhanced EUS with the dynamic quantitative analysis preparing TIC increased the diagnostic accuracy for pancreatic diseases.

Neutrophil differentiation from human-induced pluripotent stem cells.

Induced pluripotent stem (iPS) cells are of potential value not only for regenerative medicine, but also for disease investigation. The present study describes the development of a neutrophil differentiation system from human iPS cells (hiPSCs) and the analysis of neutrophil function and differentiation. The culture system used consisted of the transfer of hiPSCs onto OP9 cells and their culture with vascular endothelial growth factor (VEGF). After 10 days, TRA 1-85(+) CD34(+) VEGF receptor-2 (VEGFR-2)(high) cells were sorted and co-cultured with OP9 cells in the presence of hematopoietic cytokines for 30 days. Floating cells were collected and subjected to morphological and functional analysis. These hiPSC-derived neutrophils were similar to peripheral blood mature neutrophils in morphology, contained functional neutrophil specific granules, and were equipped with the basic functions such as phagocytosis, superoxide production, and chemotaxis. In the process of differentiation, myeloid cells appeared sequentially from immature myeloblasts to mature segmented neutrophils. Expression patterns of surface antigen, transcription factors, and granule proteins during differentiation were also similar to those of granulopoiesis in normal bone marrow. In conclusion, differentiation of mature neutrophils from hiPSCs was successfully induced in a similar process to normal granulopoiesis using an OP9 co-culture system. This system may be applied to elucidate the pathogenesis of various hematological diseases that affect neutrophils.

A case of solid-pseudopapillary neoplasm, focusing on contrast-enhanced endoscopic ultrasonography.

We used contrast-enhanced endoscopic ultrasonography (CE-EUS) to diagnose a case of solid-pseudopapillary neoplasm (SPN) in an adult man. A 58-year-old man was referred to our hospital because of a pancreatic mass found by positron emission tomography (PET) using (18)F-fluorodeoxyglucose (FDG) during a medical checkup in 2009. Trans-abdominal ultrasonography revealed a 24-mm hypoechoic mass at the pancreatic tail with calcification inside. Multiphasic computed tomography (CT) showed a solid mass with delayed enhancement. EUS revealed a hypoechoic mass without lateral shadowing, and neither a septum nor cystic component was detected. CE-EUS using Sonazoid(®) showed a hypovascular mass compared with the surrounding pancreatic parenchyma, and the inside of the mass was enhanced like an alveolus nest, suggesting pseudopapillary change. Diagnosis of a solid-pseudopapillary neoplasm and a concomitant small neuroendocrine tumor was made by distal pancreatectomy.

Ring/marker chromosome derived from chromosome 7 in childhood acute megakaryoblastic leukemia with monosomy 7.

In some cases of childhood acute megakaryoblastic leukemia (AMKL), G-band analysis reveals supernumerary ring/marker chromosomes along with monosomy 7. However, their origin and relevance are poorly understood. We experienced three patients with AMKL, one of whom had Down's syndrome, whose blasts at the first visit exhibited both monosomy 7 and a ring/marker chromosome. For one case, precise molecular-cytogenetic techniques revealed that the ring chromosome was derived from a chromosome 7. It was strongly suggested that the ring chromosome was derived from a chromosome 7 in another case. The ring or one of the 2 marker chromosomes was derived from a chromosome 7 in the other case. All patients responded well to initial induction therapy. While it is not clear whether the ring/marker chromosome 7 affects the long-term prognosis of acute myeloid leukemia with monosomy 7, it may be of prognostic relevance to distinguish pure monosomy 7 from monosomy 7 with a ring/marker chromosome 7. For this purpose, conventional G-banding could be complemented with additional techniques such as spectral karyotyping or fluorescence in situ hybridization, which characterize the aberration in more detail. These methods may be useful for determining the optimal treatment and for elucidating the etiology of AMKL itself.

Mesenchymal mode of migration participates in pulmonary metastasis of mouse osteosarcoma LM8.

The outcomes of osteosarcoma patients still remain poor because of intractable pulmonary metastasis. We previously established a highly metastatic osteosarcoma cell line, LM8 from Dunn mouse osteosarcoma by in vivo selection. We herein aimed to clarify the characteristic biological features related with high metastatic potential and new target molecules to suppress pulmonary metastasis of osteosarcoma, using this syngeneic spontaneous metastatic model. LM8 cells acquired fibroblastic morphology with striking filopodia on the cell surface. Immunostaining showed faint stress fiber formation and peripherally localized integrin ß1, and biochemical analyses showed the activated Cdc42 and autophosphorylation of focal adhesion kinase (FAK) in LM8 cells when compared to Dunn cells. LM8 cells had activated motility in single cell migration mode. LM8 migration was increased by a Rho-associated kinase (ROCK) inhibitor, Y-27632, while decreased by Cdc42 silencing using RNA interference system. We found that a clinically approved camptothecin analog, irinotecan suppressed the migration, Cdc42 activity, and autophosphorylation of FAK, and attenuated integrin ß1 distribution selectively in LM8 cells. Daily oral administration of irinotecan significantly reduced the rate and size of pulmonary metastasis in syngeneic C3H mice. The fibroblastic morphology and activated cell migration with the dependency on Cdc42 but not Rho-ROCK signaling pathway argued that LM8 moved in mesenchymal mode of cell migration. This activated mesenchymal migration was a key component of the pulmonary metastasis of LM8 cells. The inhibition of mesenchymal migration by irinotecan, in addition to its cytotoxic effects, might be effective in preventing pulmonary metastasis of osteosarcoma.