<i>In vivo</i> analysis of mice xenografted with MzChA-1 and MzChA-1_GR cells.

<p>NOD/SCID mice xenografted with MzChA-1 and MzChA-1_GR cells were employed as <i>in vivo</i> models. When the tumor volume was over 200 mm³, the mice were treated with the indicated drugs (GEM [125 mg/kg, once a week] and/or vorinostat [60 mg/kg, 5 consecutive days per week]). The prognostic value was evaluated by the Kaplan–Meier method and a log-rank test. (A) Survival curve for mice xenografted with MzChA-1 cells. (B) Survival curve for mice xenografted with MzChA-1_GR cells. (C) Immunohistochemistry for SMAD4 on the resected tumor specimens of the mice xenografted with MzChA-1_GR cells.</p

The mRNA expression of class I HDACs and HDAC activity in MzChA-1 and MzChA-1_GR cells.

<p>Values represent the mean ± S.D. *<i>P</i> < 0.05. All experiments were conducted at least three times. (A) Comparison of the expression of class I HDACs in MzChA-1 and MzChA-1_GR cells by qRT-PCR. (B) The effect of TGF-β1 on HDAC activity in MzChA-1 cells and the HDAC activity in MzChA-1_GR cells, activity was measured with the HDAC activity assay kit. (C) The changes in HDAC activity in MzChA-1 and MzChA-1_GR cells treated with TGF-β1 and vorinostat. In the experiments for panels (B) and (C), the cells were incubated with or without 5 ng/ml TGF-β1 or 100 nM vorinostat for 72 h.</p

Application of a laparoscopic device for cell-derived sheet transplantation on the liver in a porcine model

Cell-derived sheets are of global interest for regenerative therapy. Transplanting a sheet for abdominal organs requires a device for laparoscopic delivery to minimize invasiveness. Here, using a porcine model, we aimed to confirm the feasibility of a device developed to deliver sheets to the thoracic cavity in a laparoscopic transplantation procedure. We used the device to transplant human skeletal myoblast cell sheets onto the liver and measured extra-corporeal, intra-abdominal, and total procedure times for sheet transplantation. Tissues, including the liver and the sheet, were collected two days after transplantation and analyzed histologically. In all experiments (n = 27), all sheets were successfully placed at target locations. The mean (± standard deviation) extra-corporeal, intra-abdominal, and total procedure times were 44 ± 29, 33 ± 12, and 77 ± 36 s, respectively. We found no difference between the two surgeons in procedure times. Histological analyses showed no liver damage with the transplantation and that sheets were transplanted closely onto the liver tissue without gaps. We confirmed the feasibility of a simple universal device to transplant cell-derived sheets via laparoscopic surgery. This device could support a minimally invasive procedure for sheet transplantation.</p

A practical approach to pancreatic cancer immunotherapy using resected tumor lysate vaccines processed to express α-gal epitopes

<div><p>Objectives</p><p>Single-agent immunotherapy is ineffective against poorly immunogenic cancers, including pancreatic ductal adenocarcinoma (PDAC). The aims of this study were to demonstrate the feasibility of production of novel autologous tumor lysate vaccines from resected PDAC tumors, and verify vaccine safety and efficacy.</p><p>Methods</p><p>Fresh surgically resected tumors obtained from human patients were processed to enzymatically synthesize α-gal epitopes on the carbohydrate chains of membrane glycoproteins. Processed membranes were analyzed for the expression of α-gal epitopes and the binding of anti-Gal, and vaccine efficacy was assessed <i>in vitro</i> and <i>in vivo</i>.</p><p>Results</p><p>Effective synthesis of α-gal epitopes was demonstrated after processing of PDAC tumor lysates from 10 different patients, and tumor lysates readily bound an anti-Gal monoclonal antibody. α-gal(+) PDAC tumor lysate vaccines elicited strong antibody production against multiple tumor-associated antigens and activated multiple tumor-specific T cells. The lysate vaccines stimulated a robust immune response in animal models, resulting in tumor suppression and a significant improvement in survival without any adverse events.</p><p>Conclusions</p><p>Our data suggest that α-gal(+) PDAC tumor lysate vaccination may be a practical and effective new immunotherapeutic approach for treating pancreatic cancer.</p></div

The effect of TGF-β1 knockdown using TGF-β small interfering RNA (siRNA) on EMT and chemoresistance in MzChA-1_GR cells.

<p>MzChA-1_GR cells were transfected with scrambled oligonucleotide siRNA (negative control) or TGF-β1 siRNA. All experiments were conducted at least three times. Values represent the mean ± S.D. *<i>P</i> < 0.05. (A) Comparison of the expression of TGF-β1 in MzChA-1 and MzChA-1_GR cells. (B) The changes of EMT-related mRNA expression as a result of TGF-β siRNA transfection in MzChA-1_GR cells. (C) The effect of TGF-β siRNA transfection on chemoresistance in MzChA-1_GR cells. Growth inhibition assays were performed for transfected and non-transfected cells treated with GEM.</p

Inhibition of the nuclear translocation of SMAD4 by vorinostat in MzChA-1 cells.

<p>In each experiment, cells were incubated with or without 5 ng/ml of TGF-β1 and 100 nM of vorinostat for 72 h. Scale bars: 100 μm. All experiments were conducted at least three times. (A) The change in SMAD4 expression in whole cell lysates (left panel) and in the nucleus (right panel) caused by TGF-β and vorinostat. (B) ChIP assay showing expression of SNAI1, SNAI2, ZEB1, ZEB2, and TWIST, which are regulatory elements of CDH1. (C) Immunofluorescence of SMAD4 (green) was performed in MzChA-1 cells. The effect of TGF-β1 and vorinostat on SMAD4 nuclear translocation were investigated. Nuclear staining (blue) was performed with Hoechst.</p

Influence of vorinostat on chemoresistant BTC cells.

<p>In each experiment, MzChA-1_GR cells were incubated with or without 100 nM vorinostat and TFK-1_GR cells with or without 300nM vorinostat for 72 h. Values represent the mean ± S.D. *<i>P</i> < 0.05. Scale bars: 100 μm. All experiments were conducted at least three times. (A) Representative morphological change induced by vorinostat in MzChA-1_GR cells. Immunofluorescence for CDH1 (red) was performed in MzChA-1 cells. Nuclear staining (blue) was performed with Hoechst. (B) The effect of vorinostat on the EMT-related mRNAs expression in MzChA-1_GR cells. (C) The effect of vorinostat on chemoresistance in MzChA-1_GRcells. Growth inhibition assays were performed for MzChA-1_GR cells treated with GEM. (D) Representative morphological changes induced by vorinostat in TFK-1_GR cells. Immunofluorescence for CDH1 (red) was performed in TFK-1_GR cells. Nuclear staining (blue) was performed with Hoechst. (E) The effect of vorinostat on the EMT-related mRNAs expression in TFK-1_GR cells. (F) The effect of vorinostat on chemoresistance in TFK-1_GR cells. Growth inhibition assays were performed for TFK-1_GR cells treated with GEM.</p

The effect of TGF-β1 and vorinostat on SMAD2, SMAD3, and JNK in MzChA-1.

<p>In each experiment, cells were incubated with or without 5 ng/ml of TGF-β1 and 100 nM of vorinostat for 72 h. All experiments were conducted at least three times. The changes in SMAD2, p-SMAD2, SMAD3, and p-SMAD3 expression in whole cell lysates (left panel) and in the nucleus (right panel) are shown. The expression of JNK and p-JNK in whole cell lysates are also shown (left panel).</p

Immunohistological findings of original PDAC tumors obtained from patients treated with or without neoadjuvant chemoradiotherapy.

<p>H&E stained sections of PDAC tumors clearly demonstrate viable PDAC cells in the tumor treated without NACRT. However, grade IIa destruction of PDAC cells (Evans classification) was detected in the tumor treated with NACRT. Expression of MUC1 and mesothelin was observed in PDAC tumors treated with or without NACRT. The expression levels of these TAAs were similar between PDAC tumors treated with or without NACRT. Representative images of four individual patients are shown. Scale bars = 100 μm.</p

Influence of vorinostat on TGF-β1-induced EMT and chemoresistance in BTC cell lines.

<p>In each experiment, the cells were incubated with or without 5 ng/ml of TGF-β1 and 100 nM of vorinostat for MzChA-1 cells and 300 nM of vorinostat for TFK-1 cells for 72 h. Values represent the mean ± S.D. *<i>P</i> < 0.05. Scale bars: 100 μm. All experiments were conducted at least three times. (A) Representative cell morphological changes induced by TGF-β and vorinostat in MzChA-1 cells. Immunofluorescence for CDH1 (red) was performed in MzChA-1 cells. Nuclear staining (blue) was performed with Hoechst. (B) The effect of vorinostat on the EMT-related mRNAs expression in MzChA-1 cells. (C) The effect of vorinostat on chemoresistance induced by TGF-β1 exposure in MzChA-1 cells. The growth inhibition assays were performed for MzChA-1 cells treated with GEM. (D) Representative cell morphological changes induced by TGF-β and vorinostat in TFK-1 cells. Immunofluorescence for CDH1 (red) was performed in TFK-1 cells. Nuclear staining (blue) was performed with Hoechst. (E) The effect of vorinostat on the EMT-related mRNAs expression in TFK-1 cells. (F) The effect of vorinostat on chemoresistance induced by TGF-β1 exposure in TFK-1 cells. Growth inhibition assays were performed for TFK-1 cells treated with GEM.</p