Atsushi Suetsugu

Imaging exosome transfer from breast cancer cells to stroma at metastatic sites in orthotopic nude-mouse models☆

Exosomes play an important role in cell-to-cell communication to promote tumor metastasis. In order to image the fate of cancer-cell-derived exosomes in orthotopic nude mouse models of breast cancer, we used green fluorescent protein (GFP)-tagged CD63, which is a general marker of exosomes. Breast cancer cells transferred their own exosomes to other cancer cells and normal lung tissue cells in culture. In orthotopic nude-mouse models, breast cancer cells secreted exosomes into the tumor microenvironment. Tumor-derived exosomes were incorporated into tumor-associated cells as well as circulating in the blood of mice with breast cancer metastases. These results suggest that tumor-derived exosomes may contribute to forming a niche to promote tumor growth and metastasis. Our results demonstrate the usefulness of GFP imaging to investigate the role of exosomes in cancer metastasis.

Sarcopenia impairs prognosis of patients with liver cirrhosis.

OBJECTIVE Sarcopenia is characterized by the loss of skeletal muscle mass, and is reported to appear in patients with liver cirrhosis (LC). The aim of this study was to investigate the prevalence of sarcopenia in patients with LC, and to test the association between sarcopenia and patient outcomes. We also analyzed the effect of branched-chain amino acid (BCAA) supplementation on sarcopenic LC. METHODS Clinical and blood biochemical data of 130 patients with LC who underwent abdominal computed tomography scan were analyzed in this retrospective study. The cross-sectional area of skeletal muscles was measured at the level of the third lumbar vertebra on the scan. The skeletal muscle index was calculated to identify sarcopenia. Cirrhotic patients who were treated with BCAA supplementation of 12 g/d for ≥ 1 y were defined as the BCAA group, and the effect of BCAA on sarcopenic LC was evaluated. RESULTS Sixty-eight percent of all patients (82% of men and 50% of women) were diagnosed with sarcopenia. Male sex (P = 0.01) and body mass index (P < 0.0001) were predictors of sarcopenia. The multivariate Cox proportional hazards model found BCAA supplementation (hazard ratio [HR], 0.38; P = 0.01), sarcopenia (HR, 3.03; P < 0.01), and Child-Pugh classes B (HR, 2.39; P = 0.03) and C (HR, 5.49; P < 0.001) to be independently associated with mortality. The mortality of sarcopenic LC was significantly higher than that of non-sarcopenic LC (P = 0.01). Moreover, BCAA supplementation improved the survival of sarcopenic patients in subgroup analysis (P < 0.01). CONCLUSIONS Sarcopenia is significantly associated with mortality in patients with LC. BCAA supplementation might be associated with improved survival of such patients.

Efficacy of Salmonella typhimurium A1-R versus chemotherapy on a pancreatic cancer patient-derived orthotopic xenograft (PDOX)

The aim of this study is to determine the efficacy of tumor‐targeting Salmonella typhimurium A1‐R (A1‐R) on pancreatic cancer patient‐derived orthotopic xenografts (PDOX). The PDOX model was originally established from a pancreatic cancer patient in SCID‐NOD mice. The pancreatic cancer PDOX was subsequently transplanted by surgical orthotopic implantation (SOI) in transgenic nude red fluorescent protein (RFP) mice in order that the PDOX stably acquired red fluorescent protein (RFP)‐expressing stroma for the purpose of imaging the tumor after passage to non‐transgenic nude mice in order to visualize tumor growth and drug efficacy. The nude mice with human pancreatic PDOX were treated with A1‐R or standard chemotherapy, including gemcitabine (GEM), which is first‐line therapy for pancreatic cancer, for comparison of efficacy. A1‐R treatment significantly reduced tumor weight, as well as tumor fluorescence area, compared to untreated control (P = 0.011), with comparable efficacy of GEM, CDDP, and 5‐FU. Histopathological response to treatment was defined according to Evanss criteria and A1‐R had increased efficacy compared to standard chemotherapy. The present report is the first to show that A1‐R is effective against a very low‐passage patient tumor, in this case, pancreatic cancer. The data of the present report suggest A1‐1 will have clinical activity in pancreatic cancer, a highly lethal and treatment‐resistant disease and may be most effectively used in combination with other agents. J. Cell. Biochem. 115: 1254–1261, 2014.

Comparison of efficacy of Salmonella typhimurium A1-R and chemotherapy on stem-like and non-stem human pancreatic cancer cells

The XPA1 human pancreatic cancer cell line is dimorphic, with spindle stem-like cells and round non-stem cells. We report here the in vitro IC50 values of stem-like and non-stem XPA1 human pancreatic cells cells for: (1) 5-fluorouracil (5-FU), (2) cisplatinum (CDDP), (3) gemcitabine (GEM), and (4) tumor-targeting Salmonella typhimurium A1-R (A1-R). IC50 values of stem-like XPA1 cells were significantly higher than those of non-stem XPA1 cells for 5-FU (P = 0.007) and CDDP (P = 0.012). In contrast, there was no difference between the efficacy of A1-R on stem-like and non-stem XPA1 cells. In vivo, 5-FU and A1-R significantly reduced the tumor weight of non-stem XPA1 cells (5-FU; P = 0.028; A1-R; P = 0.011). In contrast, only A1-R significantly reduced tumor weight of stem-like XPA1 cells (P = 0.012). The combination A1-R with 5-FU improved the antitumor efficacy compared with 5-FU monotherapy on the stem-like cells (P = 0.004). The results of the present report indicate A1-R is a promising therapy for chemo-resistant pancreatic cancer stem-like cells.

Multi-color palette of fluorescent proteins for imaging the tumor microenvironment of orthotopic tumorgraft mouse models of clinical pancreatic cancer specimens

Pancreatic‐cancer‐patient tumor specimens were initially established subcutaneously in NOD/SCID mice immediately after surgery. The patient tumors were then harvested from NOD/SCID mice and passaged orthotopically in transgenic nude mice ubiquitously expressing red fluorescent protein (RFP). The primary patient tumors acquired RFP‐expressing stroma. The RFP‐expressing stroma included cancer‐associated fibroblasts (CAFs) and tumor‐associated macrophages (TAMs). Further passage to transgenic nude mice ubiquitously expressing green fluorescent protein (GFP) resulted in tumors that acquired GFP stroma in addition to their RFP stroma, including CAFs and TAMs as well as blood vessels. The RFP stroma persisted in the tumors growing in the GFP mice. Further passage to transgenic nude mice ubiquitously expressing cyan fluorescent protein (CFP) resulted in tumors acquiring CFP stroma in addition to persisting RFP and GFP stroma, including RFP‐ and GFP‐expressing CAFs, TAMs and blood vessels. This model can be used to image progression of patient pancreatic tumors and to visually target stroma as well as cancer cells and to individualize patient therapy. J. Cell. Biochem. 113: 2290–2295, 2012.

Spatial–temporal FUCCI imaging of each cell in a tumor demonstrates locational dependence of cell cycle dynamics and chemoresponsiveness

The phase of the cell cycle can determine whether a cancer cell can respond to a given drug. We report here on the results of monitoring of real-time cell cycle dynamics of cancer cells throughout a live tumor intravitally using a fluorescence ubiquitination cell cycle indicator (FUCCI) before, during, and after chemotherapy. In nascent tumors in nude mice, approximately 30% of the cells in the center of the tumor are in G0/G1 and 70% in S/G2/M. In contrast, approximately 90% of cancer cells in the center and 80% of total cells of an established tumor are in G0/G1 phase. Similarly, approximately 75% of cancer cells far from (>100 µm) tumor blood vessels of an established tumor are in G0/G1. Longitudinal real-time imaging demonstrated that cytotoxic agents killed only proliferating cancer cells at the surface and, in contrast, had little effect on quiescent cancer cells, which are the vast majority of an established tumor. Moreover, resistant quiescent cancer cells restarted cycling after the cessation of chemotherapy. Our results suggest why most drugs currently in clinical use, which target cancer cells in S/G2/M, are mostly ineffective on solid tumors. The results also suggest that drugs that target quiescent cancer cells are urgently needed.

Color-coding cancer and stromal cells with genetic reporters in a patient-derived orthotopic xenograft (PDOX) model of pancreatic cancer enhances fluorescence-guided surgery

Precise fluorescence-guided surgery (FGS) for pancreatic cancer has the potential to greatly improve the outcome in this recalcitrant disease. To achieve this goal, we have used genetic reporters to color code cancer and stroma cells in a patient-derived orthotopic xenograft (PDOX) model. The telomerase-dependent green fluorescent protein (GFP)-containing adenovirus OBP-401 was used to label the cancer cells of a pancreatic cancer PDOX. The PDOX was previously grown in a red fluorescent protein (RFP) transgenic mouse that stably labeled the PDOX stroma cells bright red. The color-coded PDOX model enabled FGS to completely resect the pancreatic tumors including stroma. Dual-colored FGS significantly prevented local recurrence, which bright-light surgery or single-color FGS could not. FGS, with color-coded cancer and stroma cells has important potential for improving the outcome of recalcitrant-cancer surgery.

Acid sphingomyelinase regulates glucose and lipid metabolism in hepatocytes through AKT activation and AMP-activated protein kinase suppression

Acid sphingomyelinase (ASM) regulates the homeostasis of sphingolipids, including ceramides and sphingosine‐1‐phosphate (S1P). Because sphingolipids regulate AKT activation, we investigated the role of ASM in hepatic glucose and lipid metabolism. Initially, we overexpressed ASM in the livers of wild‐type and diabetic db/db mice by adenovirus vector (Ad5ASM). In these mice, glucose tolerance was improved, and glycogen and lipid accumulation in the liver were increased. Using primary cultured hepatocytes, we confirmed that ASM increased glucose uptake, glycogen deposition, and lipid accumulation through activation of AKT and glycogen synthase kinase‐3β. In addition, ASM induced up‐regulation of glucose transporter 2 accompanied by suppression of AMP‐activated protein kinase (AMPK) phosphorylation. Loss of sphingosine kinase‐1 (SphK1) diminished ASM‐mediated AKT phosphorylation, but exogenous S1P induced AKT activation in hepatocytes. In contrast, SphK1 deficiency did not affect AMPK activation. These results suggest that the SphK/S1P pathway is required for ASM‐mediated AKT activation but not for AMPK inactivation. Finally, we found that treatment with high‐dose glucose increased glycogen deposition and lipid accumulation in wild‐type hepatocytes but not in ASM_/_ cells. This result is consistent with glucose intolerance in ASM_/_ mice. In conclusion, ASM modulates AKT activation and AMPK inactivation, thus regulating glucose and lipid metabolism in the liver.—Osawa, Y., Seki, E., Kodama, Y., Suetsugu, A., Miura, K., Adachi, M., Ito, H., Shiratori, Y., Banno, Y., Olefsky, J. M., Nagaki, M., Moriwaki, H., Brenner, D. A., Seishima, M. Acid sphingomyelinase regulates glucose and lipid metabolism in hepatocytes through AKT activation and AMP‐activated protein kinase suppression. FASEBJ. 25, 1133‐1144 (2011). www.fasebj.org

l-Tryptophan-mediated Enhancement of Susceptibility to Nonalcoholic Fatty Liver Disease Is Dependent on the Mammalian Target of Rapamycin

Nonalcoholic fatty liver disease is one of the most common liver diseases. l-Tryptophan and its metabolite serotonin are involved in hepatic lipid metabolism and inflammation. However, it is unclear whether l-tryptophan promotes hepatic steatosis. To explore this issue, we examined the role of l-tryptophan in mouse hepatic steatosis by using a high fat and high fructose diet (HFHFD) model. l-Tryptophan treatment in combination with an HFHFD exacerbated hepatic steatosis, expression of HNE-modified proteins, hydroxyproline content, and serum alanine aminotransaminase levels, whereas l-tryptophan alone did not result in these effects. We also found that l-tryptophan treatment increases serum serotonin levels. The introduction of adenoviral aromatic amino acid decarboxylase, which stimulates the serotonin synthesis from l-tryptophan, aggravated hepatic steatosis induced by the HFHFD. The fatty acid-induced accumulation of lipid was further increased by serotonin treatment in cultured hepatocytes. These results suggest that l-tryptophan increases the sensitivity to hepatic steatosis through serotonin production. Furthermore, l-tryptophan treatment, adenoviral AADC introduction, and serotonin treatment induced phosphorylation of the mammalian target of rapamycin (mTOR), and a potent mTOR inhibitor rapamycin attenuated hepatocyte lipid accumulation induced by fatty acid with serotonin. These results suggest the importance of mTOR activation for the exacerbation of hepatic steatosis. In conclusion, l-tryptophan exacerbates hepatic steatosis induced by HFHFD through serotonin-mediated activation of mTOR.

Rapid skeletal muscle wasting predicts worse survival in patients with liver cirrhosis

Sarcopenia impairs the outcome of patients with liver cirrhosis independently of liver function reserves. The aim of this study was to investigate whether the rate of skeletal muscle wasting predicts mortality in cirrhotic patients.