Serum MYCN as a predictive biomarker of prognosis and therapeutic response in the prevention of hepatocellular carcinoma recurrence.

The proto-oncogene MYCN expression marked a cancer stem-like cell population in hepatocellular carcinoma (HCC) and served as a therapeutic target of acyclic retinoid (ACR), an orally administered vitamin A derivative that has demonstrated promising efficacy and safety in reducing HCC recurrence. This study investigated the role of MYCN as a predictive biomarker for therapeutic response to ACR and prognosis of HCC. MYCN gene expression in HCC was analyzed in the Cancer Genome Atlas and a Taiwanese cohort (N = 118). Serum MYCN protein levels were assessed in healthy controls (N = 15), patients with HCC (N = 116), pre- and post-surgical patients with HCC (N = 20), and a subset of patients from a phase 3 clinical trial of ACR (N = 68, NCT01640808). The results showed increased MYCN gene expression in HCC tumors, which positively correlated with HCC recurrence in non-cirrhotic or single-tumor patients. Serum MYCN protein levels were higher in patients with HCC, decreased after surgical resection of HCC, and were associated with liver functional reserve and fibrosis markers, as well as long-term HCC prognosis (>4 years). Subgroup analysis of a phase 3 clinical trial of ACR identified serum MYCN as the risk factor most strongly associated with HCC recurrence. Patients with HCC with higher serum MYCN levels after a 4-week treatment of ACR exhibited a significantly higher risk of recurrence (hazard ratio 3.27; p = .022). In conclusion, serum MYCN holds promise for biomarker-based precision medicine for the prevention of HCC, long-term prognosis of early-stage HCC, and identification of high-response subgroups for ACR-based treatment.

Color-coded Imaging of the Fate of Cancer-cell-derived Exosomes During Pancreatic Cancer Metastases in a Nude-mouse Model.

BACKGROUND/AIM: Tumor-derived exosomes play important roles in tumor metastases. In this report, we observed the fate of tumor-derived exosomes in pancreatic cancer metastatic nude-mouse models using color-coded imaging. MATERIALS AND METHODS: Mia-PaCa-2 human pancreatic cancer cells expressing red fluorescent protein (RFP) were transduced by exosome-specific pCT-CD63-green fluorescent protein (GFP) and injected in the spleen of nude mice. RESULTS: Four weeks after injection of these cells into the spleen, liver metastases developed and tumor-derived exosomes were observed within the metastatic cancer cells and in Kupffer cells. Furthermore, tumor-derived exosomes diffused to bone marrow and lung cells, especially macrophages, without any metastases present. CONCLUSION: In the present study, we visualized the distribution of cancer-derived exosomes for the first time at the cellular level, in a pancreatic-cancer metastatic model.

Reduced handgrip strength is predictive of poor survival among patients with liver cirrhosis: A sex-stratified analysis.

AIM: Handgrip strength (HGS) is a marker of sarcopenia and has been used to stratify an individual's risk of death. We aimed to assess the prognostic significance of HGS in patients with liver cirrhosis. METHODS: In this retrospective study, we collated data of 563 consecutive patients admitted to our hospital with cirrhosis (375 men). A dynamometer was used to measure HGS. Body composition (including skeletal muscle and adipose tissue volumes) was estimated using computed tomography. Predictors of mortality were identified using sex-stratified multivariate analyses. RESULTS: After adjustments for age, cirrhosis etiology, Child-Pugh score, and other confounding variables, HGS, but not body composition, was independently associated with mortality in male patients (hazard ratio [HR], 0.96; 95% confidence interval [CI], 0.94-0.99; P < 0.01) and female patients (HR, 0.91; 95% CI, 0.84-0.99; P = 0.02). Men with low HGS (<30 kg) had a higher risk of mortality (HR, 2.09; 95% CI, 1.39-3.17; P < 0.001), as did women with low (<15 kg) HGS (HR, 2.14; 95% CI, 1.16-4.01; P = 0.02). We could stratify the sex-specific risk of mortality in cirrhotic patients using HGS, regardless of coexistent hepatocellular carcinoma and the Child-Pugh class. CONCLUSIONS: Reduced HGS, rather than skeletal muscle and adipose tissue volumes, is associated with an increased risk of mortality in patients of both sexes with liver cirrhosis. Measurement of HGS is a simple, cost-effective, and appropriate bedside assessment for the prediction of survival in patients with cirrhosis.

Hypozincemia is associated with human hepatocarcinogenesis in hepatitis C virus-related liver cirrhosis.

AIM: Hypozincemia is associated with the progression of chronic liver diseases, but it is unknown whether hypozincemia promotes human hepatocarcinogenesis. Our aim is to evaluate the serum zinc levels in liver cirrhosis (LC) patients and clarify the relationship between the serum zinc levels and the development of hepatocellular carcinoma (HCC). METHODS: Cirrhotic patients without HCC (n = 299) were enrolled from 14 medical institutes in Japan as a multicenter prospective study (No. 2028). Of the 299 patients, 157 were included in the present study based on reliable and consistent serum zinc levels and no history of oral zinc supplementation. Clinical parameters associated with the development of HCC were determined. Furthermore, the cumulative incidence of HCC was analyzed using Kaplan-Meier methods and was calculated using the log-rank test. A Cox regression analysis was utilized for the multivariate analysis to evaluate the predictors of hepatocarcinogenesis. RESULTS: Thirty of 157 patients (19.1%) developed HCC during an observation period of 3 years. Serum zinc levels were significantly decreased in hepatitis C virus-related LC (C-LC) patients with HCC (0.0180). The risk factors for incidence of HCC were hypozincemia (0.0014), high α-fetoprotein (0.0080), low branched chain amino acids-to-tyrosine ratio (0.0128), or female sex (0.0228). Hypozincemia (hazard ratio 1.61, 0.0324) was the only significant predictor of hepatocarcinogenesis by multivariate Cox regression analysis. CONCLUSIONS: Hypozincemia is associated with hepatocarcinogenesis in C-LC patients.

Prognostic significance of minimal hepatic encephalopathy in patients with liver cirrhosis in Japan: A propensity score-matching analysis.

BACKGROUND AND AIM: Minimal hepatic encephalopathy (MHE) represents the mildest form of the hepatic encephalopathy spectrum. This study aimed to clarify the prognostic significance of MHE in cirrhotic patients. METHODS: This retrospective study evaluated 357 consecutive patients with liver cirrhosis. MHE was diagnosed using a neuropsychiatric test. A propensity score-matching analysis was employed to adjust significant differences in the baseline characteristics between patients with and without MHE. RESULTS: Of 269 eligible patients, 56 patients (21%) were diagnosed as having MHE. The Child-Pugh score, model for end-stage liver disease score, and serum ammonia levels were significantly increased, while serum albumin levels were reduced in patients with MHE. By contrast, no significant difference was found between the two groups in matched patients. During the median follow-up period of 13.4 months, 67 patients (24.9%) died. Overall survival rates were significantly lower in patients with MHE (median, 25.4 vs 48.8 months; P < 0.001). Multivariate analysis revealed that male sex (hazard ratio [HR], 1.78; 95% confidence interval [CI], 1.03-3.18; P = 0.038), stage III/IV hepatocellular carcinoma (HR, 6.32; 95% CI, 3.30-12.79; P < 0.001), the Child-Pugh score (HR, 1.35; 95% CI, 1.12-1.62; P = 0.002), and MHE (HR, 1.92; 95% CI, 1.09-3.29; P = 0.024) were independently associated with mortality in all patients as well as in matched patients. CONCLUSION: Minimal hepatic encephalopathy is associated with an increased risk of mortality in patients with liver cirrhosis, independent of hepatocellular carcinoma stage or Child-Pugh score.

Effect of loop diuretics on skeletal muscle depletion in patients with liver cirrhosis.

AIM: Sarcopenia, the loss of skeletal muscle mass, impairs prognosis of patients with liver cirrhosis. The aim of this study was to investigate the effect of loop diuretics, which are frequently used to treat hepatic edema/ascites, on skeletal muscle depletion and the prognosis in patients with liver cirrhosis. METHODS: This retrospective study evaluated 226 patients with liver cirrhosis. The skeletal muscle cross-sectional area at the level of the third lumbar vertebra was measured using computed tomography. The relative change in skeletal muscle area per year (ΔSMA) was calculated, and the association between ΔSMA and therapeutic dosage of loop diuretics was examined. RESULTS: The therapeutic dosage of loop diuretics was inversely correlated with ΔSMA by simple (r = -0.27, P < 0.0001) and multiple regression analyses (t = -3.07, P = 0.002). During a median follow-up period of 49 months, 82 patients died. Overall survival rates were lower in patients treated with loop diuretics at >20 mg than in those who received ≤20 mg (median, 66 vs. 97 months; P = 0.002). Multivariate analysis revealed that loop diuretics of >20 mg (hazard ratio [HR], 1.86; 95% confidence interval [CI], 1.03-3.24; P = 0.039) and ΔSMA of ≤-3.1% (HR, 3.87; 95% CI, 2.32-6.60; P < 0.0001) were independently associated with mortality. CONCLUSIONS: A higher dose of loop diuretic use was associated with more rapid skeletal muscle depletion and poor survival in patients with liver cirrhosis, independent of the severity of liver disease.

Color-coded Imaging of the Circulating Tumor Cell Microenvironment.

BACKGROUND/AIM: Circulating tumor cells (CTCs) may initiate metastasis. Some studies show that the number of CTCs and existence of CTC clusters can be prognostic. In the present study, our color-coded imaging nude mouse model of metastatic lymphoma was utilized to investigate the microenvironment of CTC clusters using fluorescent-protein imaging. MATERIALS AND METHODS: EL-4 mouse lymphoma cells expressing red fluorescent protein (RFP) were injected into the spleen of transgenic C57B/6-green fluorescent protein (GFP) mice. Three weeks later, the number of CTCs and CTC clusters both in heart blood and portal blood were quantified and characterized using confocal microscopy for color-coded imaging. RESULTS: There was no significant difference in the number of CTCs between heart and portal blood. CTC clusters comprised 8.8% of CTCs, determined by color-coded imaging. Heterotypic CTC clusters containing other types of cells were distinguishable from homotypic CTCs. Heterotypic CTC clusters comprising cancer cells and fibroblasts were more rare than homotypic ones. Heterotypic CTC clusters with fibroblasts were observed only in portal blood, not in heart blood. CONCLUSION: CTCs can have variable properties depending on the blood source. CTCs can form clusters, which may contain fibroblast that may play a role in promoting CTC metastasis. Our results demonstrate the concept of the CTC microenvironment (CME), which may play a critical role in CTC behavior, including of metastasis.

Color-coded Imaging Distinguishes Cancer Cells, Stromal Cells, and Recombinant Cancer-stromal Cells in the Tumor Microenvironment During Metastasis.

BACKGROUND/AIM: Our laboratory pioneered color-coded imaging of the tumor microenvironment (TME). We observed recruitment of cancer and stromal cells to the TME and recombination between cancer and stromal cells. The aim of the present study was to observe the dynamics of the TME by color-coded imaging during metastasis and in the formation of a pre-metastatic niche. MATERIALS AND METHODS: Red-fluorescent protein (RFP-expressing) mouse colon-cancer 26 cells were initially injected subcutaneously in green-fluorescent protein (GFP) nude mice. The resulting subcutaneous tumors were harvested and cultured. The cultured subcutaneous tumors contained RFP colon cancer cells, GFP stromal cells and recombinant cancer-stromal cells expressing yellow fluorescence. After 14 days culture, the cells were injected into the spleen. RESULTS: After splenic injection, colon-cancer 26 metastases were observed in the liver, ascites, and bone marrow. Using the Olympus FV1000 confocal microscope, the cells cultured from tumors and metastasis in each site were visualized. RFP colon-cancer cells, GFP stromal cells derived from host GFP nude mice, and recombinant yellow-fluorescent cells were observed in each organ. In addition, in the liver, areas with only GFP stromal cells were observed and assumed to be a pre-metastatic niche. CONCLUSION: Color-coded imaging demonstrated the dynamics of colon cancer and stromal cells at different metastatic sites including the formation of recombinant cancer-stromal cells.

Prevention of hepatocellular carcinoma by targeting MYCN-positive liver cancer stem cells with acyclic retinoid.

Hepatocellular carcinoma (HCC) is a highly lethal cancer that has a high rate of recurrence, in part because of cancer stem cell (CSC)-dependent field cancerization. Acyclic retinoid (ACR) is a synthetic vitamin A-like compound capable of preventing the recurrence of HCC. Here, we performed a genome-wide transcriptome screen and showed that ACR selectively suppressed the expression of MYCN, a member of the MYC family of basic helix-loop-helix-zipper transcription factors, in HCC cell cultures, animal models, and liver biopsies obtained from HCC patients. MYCN expression in human HCC was correlated positively with both CSC and Wnt/ß-catenin signaling markers but negatively with mature hepatocyte markers. Functional analysis showed repressed cell-cycle progression, proliferation, and colony formation, activated caspase-8, and induced cell death in HCC cells following silencing of MYCN expression. High-content single-cell imaging analysis and flow cytometric analysis identified a MYCN+ CSC subpopulation in the heterogeneous HCC cell cultures and showed that these cells were selectively killed by ACR. Particularly, EpCAM+ cells isolated using a cell-sorting system showed increased MYCN expression and sensitivity to ACR compared with EpCAM- cells. In a long-term (>10 y) follow-up study of 102 patients with HCC, MYCN was expressed at higher levels in the HCC tumor region than in nontumor regions, and there was a positive correlation between MYCN expression and recurrence of de novo HCC but not metastatic HCC after curative treatment. In summary, these results suggest that MYCN serves as a prognostic biomarker and therapeutic target of ACR for liver CSCs in de novo HCC.

The Prevalence and Implication of Zinc Deficiency in Patients With Chronic Liver Disease.

BACKGROUND: Patients with liver cirrhosis often exhibit zinc deficiency. Although zinc is involved in many bioactivities, many aspects of clinical implications of zinc deficiency in liver cirrhosis remain unclear. We aimed to reveal the prevalence and implications of zinc deficiency in liver cirrhosis by assessing associations with parameters such as clinical symptoms and laboratory data. METHODS: In 235 cirrhosis patients enrolled at multiple medical institutions in 2009, we assessed how blood zinc levels were associated with their clinical symptoms, patients characteristics, and liver function test results. RESULTS: Blood zinc levels were most strongly correlated with blood albumin levels among the study parameters (r = 0.587, P < 0.0001). When blood albumin levels were ≤ 3.5 g/dL, blood zinc levels were < 70 µg/dL in 88% of patients. Additionally, significant correlations were observed with age (r = -0.253, P = 0.0014), aspartate aminotransferase levels (r = -0.254, P = 0.0020), total bilirubin levels (r = -0.222, P = 0.0053), prothrombin time (r = -0.255, P = 0.0029), branched-chain amino acid to tyrosine ratio (r = 0.357, P < 0.0001), Child-Pugh score (r = 0.469, P < 0.0001), ammonia levels (r = -0.246, P = 0.0028), and total cholesterol levels (r = 0.314, P < 0.0001). Blood zinc levels were significantly lower in patients with edema/ascites (P < 0.0001), those with hepatic encephalopathy (P = 0.0215), those receiving oral diuretics (P = 0.0045), and those receiving oral branched-chain amino acids (P < 0.0001) than in those without these conditions. CONCLUSIONS: Zinc deficiency is prevalent in cirrhosis patients, whereas nitrogen metabolic disorders, particularly hypoalbuminemia, can be an indicator of zinc deficiency. Thus, cirrhosis patients exhibiting a nitrogen metabolic disorder should be examined for the presence of zinc deficiency.

Visualizing the Tumor Microenvironment by Color-coded Imaging in Orthotopic Mouse Models of Cancer.

The tumor microenvironment (TME) contains stromal cells in a complex interaction with cancer cells. This relationship has become better understood with the use of fluorescent proteins for in vivo imaging, originally developed by our laboratories. Spectrally-distinct fluorescent proteins can used for color-coded imaging of the complex interaction of the tumor microenvironment in the living state using cancer cells expressing a fluorescent protein of one color and host mice expressing another-color fluorescent protein. Cancer cells engineered in vitro to express a fluorescent protein were orthotopically implanted into transgenic mice expressing a fluorescent protein of a different color. Confocal microscopy was then used for color-coded imaging of the TME. Color-coded imaging of the TME has enabled us to discover that stromal cells are necessary for metastasis. Patient-derived orthotopic xenograft (PDOX) tumors were labeled by first passaging them orthotopically through transgenic nude mice expressing either green, red, or cyan fluorescent protein in order to label the stromal cells of the tumor. The colored stromal cells become stably associated with the PDOX tumors through multiple passages in transgenic colored mice or non-colored mice. The fluorescent protein-expressing stromal cells included cancer-associated fibroblasts and tumor-associated macrophages. The cancer cells in PDOX models can also be labeled with a telomerase-dependent adenovirus containing the gene for green fluorescent protein. Using this model, specific cancer-cell or stromal-cell targeting by potential therapeutics can be visualized. Color-coded imaging enabled the visualization of apparent fusion of cancer and stromal cells. Color-coded imaging is a powerful tool visualizing the interaction of cancer and stromal cells during cancer progression and treatment.

Differential Organ-targeting and Cellular Characteristics of Metastatic Human Pancreatic Cancer Cell Lines in Mouse Models.

BACKGROUND/AIM: The lethal characteristic of pancreatic cancer is metastasis which is recalcitrant to currently-used chemotherapy. Our aim was to understand metastasis at the cellular level. We previously reported that multi-nucleate cells or spindle cells were more prominent in pancreatic cancer metastasis than in the primary tumor. In the present report, we investigated four representative human pancreatic cell lines for differences in cell morphology between the primary tumor and various metastatic organ targets for each cell line. MATERIALS AND METHODS: Human pancreatic cancer cell lines AsPC-1, Panc-1, KP2 and KP3 were used. Pancreatic cancer cells were injected into spleen of nude mice resulting in experimental metastasis to various organs which were observed at the cellular level when the organs were placed into culture. RESULTS: AsPC-1 and KP2 pancreatic cells formed many experimental liver metastases, in contrast to Panc-1 and KP3. Lung metastasis was only observed for AsPC-1. In the cultures established from the primary and metastatic tumors, multi-nucleate cells were found to be more prominent in the metastasis of the pancreatic cell lines with frequent metastasis, AsPC-1 and KP2. Spindle-like cells were observed prominently in AsPC-1 lung metastasis. CONCLUSION: Human pancreatic cancer cell lines have differential metastatic characteristics with regard to target organs and cell-morphology changes. Multi-nucleate and spindle cells may play an important role in pancreatic cancer metastasis to the liver and lung, respectively.

Prevalence of Sarcopenia and Its Relationship with Nutritional State and Quality of Life in Patients with Digestive Diseases.

This study aimed to clarify the prevalence of sarcopenia, which is characterized by loss of skeletal muscle mass and strength, and its relationship with nutritional state and quality of life (QOL) in patients with digestive diseases. This study enrolled 303 patients (187 men and 116 women with a median age of 70 y) having digestive diseases. Diseases were gastrointestinal in 99 patients, biliary/pancreatic in 93, and hepatic in 111. Skeletal muscle cross-sectional area was measured by abdominal computed tomography at the 3rd vertebra (L3) level. Sarcopenia was defined using the L3 skeletal muscle index and hand grip strength. Nutritional state was assessed by Subjective Global Assessment (SGA), Mini nutritional Assessment®-Short Form (MNA®-SF), anthropometry, and blood biochemistry. QOL was evaluated by Short Form-8TM. Prevalence of sarcopenia was 32.0% in overall cases. The value was 22.2% in gastrointestinal disease, 36.6% in biliary/pancreatic disease, and 36.9% in hepatic disease. Advanced age, male sex, and advanced cancer state were associated with development of sarcopenia. Patients with sarcopenia had a significantly worse nutritional state according to SGA and MNA®-SF than those without sarcopenia. In anthropometry, % arm muscle area, % calf circumference, and body mass index were significantly lower in the sarcopenic group. Serum albumin and hemoglobin were also significantly lower in the sarcopenia patients. QOL was more impaired in physical component subscales in sarcopenia patients than non-sarcopenia ones. Patients with digestive diseases frequently suffer from sarcopenia and this is associated with worsening of nutritional state and QOL in these patients.

Efficacy and safety of rifaximin in Japanese patients with hepatic encephalopathy: A phase II/III, multicenter, randomized, evaluator-blinded, active-controlled trial and a phase III, multicenter, open trial.

AIM: The efficacy and safety of rifaximin in the treatment of hepatic encephalopathy (HE) are widely known, but they have not been confirmed in Japanese patients with HE. Thus, two prospective, randomized studies (a phase II/III study and a phase III study) were carried out. METHODS: Subjects with grade I or II HE and hyperammonemia were enrolled. The phase II/III study, which was a randomized, evaluator-blinded, active-comparator, parallel-group study, was undertaken at 37 institutions in Japan. Treatment periods were 14 days. Eligible patients were randomized to the rifaximin group (1200 mg/day) or the lactitol group (18-36 g/day). The phase III study was carried out in the same patients previously enrolled in the phase II/III study, and they were all treated with rifaximin (1200 mg/day) for 10 weeks. RESULTS: In the phase II/III study, 172 patients were enrolled. Blood ammonia (B-NH3 ) concentration was significantly improved in the rifaximin group, but the difference between the two groups was not significant. The portal systemic encephalopathy index (PSE index), including HE grade, was significantly improved in both groups. In the phase III study, 87.3% of enrolled patients completed the treatment. The improved B-NH3 concentration and PSE index were well maintained from the phase II/III study during the treatment period of the phase III study. Adverse drug reactions (ADRs) were seen in 13.4% of patients who received rifaximin, but there were no severe ADRs leading to death. CONCLUSION: The efficacy of rifaximin is sufficient and treatment is well tolerated in Japanese patients with HE and hyperammonemia.

Alpha-glucosidase inhibitor use is associated with decreased colorectal neoplasia risk in patients with type 2 diabetes mellitus receiving colonoscopy: a retrospective study.

PURPOSE: The purpose of this study was to clarify the factors that influence the incidence of colorectal neoplasia in patients with type 2 diabetes mellitus (DM). STUDY DESIGN AND SETTING: Among a total of 1176 patients who underwent total colonoscopy at our hospital, we retrospectively analyzed 168 patients with type 2 DM. Univariate and multivariate logistic regression analyses were then performed to identify the risk factors associated with colorectal neoplasia. RESULTS: A multivariate analysis of these patients demonstrated that male gender (odds ratio [OR] = 4.04, 95% confidence interval [CI] = 1.67-10.37, p = 0.002), taking statins (OR = 4.59, 95% CI = 1.69-13.43, p = 0.003), taking alpha glucosidase inhibitor (α-GI) (OR = 0.35, 95% CI = 0.13-0.87, p = 0.023) and taking low-dose aspirin (LDA) (OR = 0.32, 95% CI = 0.10-0.95, p = 0.040) were independent factors associated with an increased (male gender and statins) or decreased (α-GI and LDA) risk of colorectal neoplasia. CONCLUSIONS: While male gender and taking statins are risk factors, taking α-GI as well as LDA may reduce the risk of colorectal neoplasia in patients with type2 DM.

Choline-Deficient-Diet-Induced Fatty Liver Is a Metastasis-Resistant Microenvironment.

BACKGROUND/AIM: Fatty liver disease is increasing in the developed and developing world. Liver metastasis from malignant lymphoma in the fatty liver is poorly understood. In a previous report, we developed color-coded imaging of the tumor microenvironment (TME) of the murine EL4-RFP malignant lymphoma during metastasis, including the lung. In the present report, we investigated the potential and microenvironment of the fatty liver induced by a choline-deficient diet as a metastatic site in this mouse lymphoma model. MATERIALS AND METHODS: C57BL/6-GFP transgenic mice were fed with a choline-deficient diet in order to establish a fatty liver model. EL4-RFP cells were injected in the spleen of normal mice and fatty-liver mice. Metastases in mice with fatty liver or normal liver were imaged with the Olympus SZX7 microscope and the Olympus FV1000 confocal microscope. RESULTS: Metastases of EL4-RFP were observed in the liver, ascites and bone marrow. Primary tumors were imaged in the spleen at the injection site. The fewest metastases were observed in the fatty liver. In addition, the fewest cancer-associated fibroblasts (CAFs) were observed in the fatty liver. CONCLUSION: The relative metastatic resistance of the fatty liver may be due to the reduced number of CAFs in the fatty livers. The mechanism of the effect of the choline-deficient diet is discussed.

Imaging the Role of Multinucleate Pancreatic Cancer Cells and Cancer-Associated Fibroblasts in Peritoneal Metastasis in Mouse Models.

BACKGROUND/AIM: The interaction between pancreatic-cancer cells and stromal cells in the tumor microenvironment (TME) is of particular importance in cancer progression and metastasis. The present report demonstrates the role of cancer-associated fibroblasts (CAFs) and multinucleate pancreatic-cancer cells in peritoneal metastasis. MATERIALS AND METHODS: An orthotopic mouse model of pancreatic cancer was established with the human pancreatic cancer cell line BxPC3, which stably expresses green fluorescent protein (GFP). RESULTS: BxPC3-GFP cells formed peritoneal metastases by week 18 after orthotopic implantation. Using an Olympus FV1000 confocal microscope, multi-nucleated cancer cells were frequently observed in the peritoneal metastases. The primary pancreatic tumor and peritoneal-metastases were harvested, cultured and then transplanted subcutaneously. Subcutaneous tumors established from peritoneal-metastatic cells were larger than subcutaneous tumors established from primary-tumor cells. Subcutaneous tumors of each type were subsequently cultured in vitro. CAFs were observed growing out from the tumors established from peritoneal-metastatic cells, but not the tumors established from the primary cancer. CONCLUSION: The results of the present study suggest that multi-nucleated cancer cells and CAFs were related to peritoneal metastasis of pancreatic cancer.

Erratum: Tanaka, T. et al. Cimetidine and Clobenpropit Attenuate Inflammation-Associated Colorectal Carcinogenesis in Male ICR Mice. Cancers, 2016, 8, 25.

n/a.

The Inhibition of Indoleamine 2,3-Dioxygenase Accelerates Early Liver Regeneration in Mice After Partial Hepatectomy.

BACKGROUND AND AIM: The inflammatory response accelerates early liver regeneration after liver injury and resection. Recent studies have demonstrated that indoleamine 2,3-dioxygenase-1 (IDO1) suppresses the activation of inflammatory cells and induces immune tolerance. In this study, we examined the role of IDO1 in liver regeneration after partial hepatectomy (PHx). METHODS: WT or IDO1-knockout (IDO1-KO) mice received 70% PHx. The liver-body weight ratio after PHx was measured and hepatocyte growth was assessed by immunostaining. The expression of cell cycle genes and pro-inflammatory cytokines in the liver was analyzed by quantitative RT-PCR. In addition, 1-methyl-DL-tryptophan (1-MT), which is an IDO1 inhibitory agent, was given to WT mice and the liver-body weight ratio was measured after PHx. RESULTS: The liver-body weight ratio was significantly increased in IDO1-KO mice compared with that in WT mice after PHx. More Ki-67-positive cells were present in IDO1-KO mice than in WT mice after PHx. The expression of cell cycle genes (cyclin D1, cyclin E) and pro-inflammatory cytokines (IL-1ß, TNF-α and IL-6) was up-regulated in the remnant liver of IDO1-KO mice compared with WT mice. Moreover, treatment with 1-MT promoted liver regeneration. CONCLUSION: IDO1 deficiency promoted early liver regeneration after PHx, indicating that IDO1 suppresses the production of inflammatory cytokines and subsequently inhibits hepatocyte proliferation during liver regeneration.

Genetic Recombination Between Stromal and Cancer Cells Results in Highly Malignant Cells Identified by Color-Coded Imaging in a Mouse Lymphoma Model.

The tumor microenvironment (TME) promotes tumor growth and metastasis. We previously established the color-coded EL4 lymphoma TME model with red fluorescent protein (RFP) expressing EL4 implanted in transgenic C57BL/6 green fluorescent protein (GFP) mice. Color-coded imaging of the lymphoma TME suggested an important role of stromal cells in lymphoma progression and metastasis. In the present study, we used color-coded imaging of RFP-lymphoma cells and GFP stromal cells to identify yellow-fluorescent genetically recombinant cells appearing only during metastasis. The EL4-RFP lymphoma cells were injected subcutaneously in C57BL/6-GFP transgenic mice and formed subcutaneous tumors 14 days after cell transplantation. The subcutaneous tumors were harvested and transplanted to the abdominal cavity of nude mice. Metastases to the liver, perigastric lymph node, ascites, bone marrow, and primary tumor were imaged. In addition to EL4-RFP cells and GFP-host cells, genetically recombinant yellow-fluorescent cells, were observed only in the ascites and bone marrow. These results indicate genetic exchange between the stromal and cancer cells. Possible mechanisms of genetic exchange are discussed as well as its ramifications for metastasis. J. Cell. Biochem. 118: 4216-4221, 2017. © 2017 Wiley Periodicals, Inc.