Journal: Cancer Science

Article Title: Downregulation of 15‐hydroxyprostaglandin dehydrogenase by interleukin‐1β from activated macrophages leads to poor prognosis in pancreatic cancer

doi: 10.1111/cas.13467

Figure Lengend Snippet: Low 15‐hydroxyprostaglandin dehydrogenase (15‐ PGDH ) expression is implicated in poor pancreatic ductal adenocarcinoma ( PDAC ) prognosis. A, Workflow diagram of patients who underwent pancreatic resection and contributed samples for immunohistochemical ( IHC ) analysis. B, Representative IHC staining of 15‐ PGDH expression in 107 PDAC tissues. Scale bar = 100 μm. C,D, Relationship between 15‐ PGDH expression and relapse‐free survival (C) or overall survival (D) using the Kaplan‐Meier method

Article Snippet: The human PDAC cell lines PK‐8 and S2‐013 were obtained from the Japanese Collection of Research Bioresource Cell Bank (Ibaraki, Japan) and RIKEN Bioresource Center Cell Bank (Tsukuba, Japan).

Techniques: Expressing, Immunohistochemical staining, Immunohistochemistry

Journal: Cancer Science

Article Title: Downregulation of 15‐hydroxyprostaglandin dehydrogenase by interleukin‐1β from activated macrophages leads to poor prognosis in pancreatic cancer

doi: 10.1111/cas.13467

Figure Lengend Snippet: 15‐Hydroxyprostaglandin dehydrogenase (15‐ PGDH ) downregulation by interleukin‐1β ( IL ‐1β) enhances pancreatic ductal adenocarcinoma cell growth. A,B, Expression of HPGD (the gene coding 15‐ PGDH protein, upper panel) or 15‐ PGDH (lower panel) in PK ‐8 cells (A) or S2‐013 cells (B) after treatment with si RNA targeting 15‐ PGDH or with control si RNA , evaluated by quantitative RT ‐ PCR (upper panel) or Western blot analysis (lower panel). Data are presented as the treated/control cell ratio. C,D, PK ‐8 cells (C) or S2‐013 cells (D) transfected with si RNA s targeting 15‐ PGDH or with control si RNA were incubated for up to 96 hours and assayed for cell number; data are presented as the treated/control (time = 0) cell ratio. E,F, Expression of 15‐ PGDH in PK ‐8 cells or S2‐013 cells after IL ‐1β (E) or tumor necrosis factor‐α ( TNF ‐α) (F) treatment for 24 and 48 hours and distilled water treatment for 48 hours as a control was evaluated by Western blotting. G, Column graph showing relative 15‐ PGDH levels in PK ‐8 cells or S2‐013 cells after IL ‐1β and TNF ‐α treatment for 24 and 48 hours, and distilled water treatment for 48 hours as a control, were evaluated using ImageJ software. H, Expression of HPGD and IL 1B in six PDAC patients determined by quantitative RT ‐ PCR . Data were normalized to the ACTB mRNA level and are shown as the mean ± SD of three independent experiments. **P < .01

Article Snippet: The human PDAC cell lines PK‐8 and S2‐013 were obtained from the Japanese Collection of Research Bioresource Cell Bank (Ibaraki, Japan) and RIKEN Bioresource Center Cell Bank (Tsukuba, Japan).

Techniques: Expressing, Control, Quantitative RT-PCR, Western Blot, Transfection, Incubation, Software

Journal: Cancer Science

Article Title: Downregulation of 15‐hydroxyprostaglandin dehydrogenase by interleukin‐1β from activated macrophages leads to poor prognosis in pancreatic cancer

doi: 10.1111/cas.13467

Figure Lengend Snippet: Tumor‐associated macrophages are inversely correlated with pancreatic ductal adenocarcinoma ( PDAC ) cells harboring high 15‐hydroxyprostaglandin dehydrogenase (15‐ PGDH ) expression. A,B, Representative immunohistochemical ( IHC ) staining of 15‐ PGDH (upper panel) and CD 163 (lower panel) expression in high 15‐ PGDH (A) and low 15‐ PGDH (B) serial PDAC specimens. Scale bar = 200 μm. C, Graph showing Pearson's correlation between the expression of 15‐ PGDH and the number of CD 163‐positive cells in 107 PDAC patients. D, Schematic representation of the findings of this study. IL ‐1βR, interleukin‐1β receptor

Article Snippet: The human PDAC cell lines PK‐8 and S2‐013 were obtained from the Japanese Collection of Research Bioresource Cell Bank (Ibaraki, Japan) and RIKEN Bioresource Center Cell Bank (Tsukuba, Japan).

Techniques: Expressing, Immunohistochemical staining, Immunohistochemistry

Journal: International journal of molecular sciences

Article Title: Complexing the Oncolytic Adenoviruses Ad∆∆ and Ad-3∆-A20T with Cationic Nanoparticles Enhances Viral Infection and Spread in Prostate and Pancreatic Cancer Models.

doi: 10.3390/ijms23168884

Figure Lengend Snippet: Figure 1. Cationic AuNP combined with Ad5wtGFP enhanced viral uptake in PCa and PDAC cell lines. (A) PCa cells were infected with Ad5wtGFP at 10, 100 and 500 ppc (PC3) and 10 and 50 ppc (22Rv) in the presence or absence of 0.5 pmol AuNPs. (B) PDAC cells were infected with Ad5wtGFP at 100 ppc and the stellate PS1 cells at 500 ppc in the presence or absence of 0.2 pmol AuNPs; samples were analysed 24 and 48 h after infection. (A,B) The EGFP signal was measured as an indication of infection levels by flow cytometry, averages ±SD, n = 3, ** p < 0.01, *** p < 0.001. (C) Fluorescent images of Panc04.03, PT45 and PS1 cells 24 and 48 h after infection with Ad5wtGFP at 50 ppc with and without AuNP at 0.2 pmol, representative of 3 studies. Images were taken at 10× magnification (Olympus IX70 microscope, Evident Europe GmbH, Stansted, UK).

Article Snippet: Human prostate cancer PC3 and 22Rv1 (ATCC, LGC Standards, UK), and human pancreatic ductal adenocarcinoma (PDAC) Panc04.03 (ATCC) and PT45 (Prof H. Kalthoff, Comprehensive Cancer Centre, Campus Kiel, Kiel, Germany) cell lines were used in the study.

Techniques: Infection, Cytometry, Microscopy

Journal: International journal of molecular sciences

Article Title: Complexing the Oncolytic Adenoviruses Ad∆∆ and Ad-3∆-A20T with Cationic Nanoparticles Enhances Viral Infection and Spread in Prostate and Pancreatic Cancer Models.

doi: 10.3390/ijms23168884

Figure Lengend Snippet: Figure 2. Ad∆∆-induced cell killing is enhanced by AuNPs in PCa and PDAC cells. (A) Dose–response to Ad∆∆in PC3 and 22Rv cells with and without 0.1 pmol AuNPs. Cell viability determined by MTS assay. (B) EC50-values (left panel) and the relative decreases in EC50-values (right panel) in each cell line compared to Ad∆∆alone. (C) PC3 cells treated with fixed doses of Ad∆∆at 500 ppc or 750 ppc and/or mitoxantrone (450 nM) with and without AuNPs at 0.1 pmol. Cell viability was measured by MTS assay 4d after infection, ** p < 0.01 (Ad/AuNP vs. Ad) and $$ p < 0.01 (Ad/M vs. Ad). (D) EC50-values generated from Ad∆∆dose–response curves ± AuNPs at 0.2 pmol (left panel) and the corresponding relative decreases in Panc04.03, PT45 and PS1 cells compared to Ad∆∆alone. (A–D) Averages ± SD, n = 3, * p < 0.05, ** p < 0.01.

Article Snippet: Human prostate cancer PC3 and 22Rv1 (ATCC, LGC Standards, UK), and human pancreatic ductal adenocarcinoma (PDAC) Panc04.03 (ATCC) and PT45 (Prof H. Kalthoff, Comprehensive Cancer Centre, Campus Kiel, Kiel, Germany) cell lines were used in the study.

Techniques: MTS Assay, Infection, Generated

Journal: International journal of molecular sciences

Article Title: Complexing the Oncolytic Adenoviruses Ad∆∆ and Ad-3∆-A20T with Cationic Nanoparticles Enhances Viral Infection and Spread in Prostate and Pancreatic Cancer Models.

doi: 10.3390/ijms23168884

Figure Lengend Snippet: Figure 3. Ad∆∆replication is enhanced in the presence of AuNP and efficiently eliminates PDAC cells in three-dimensional co-cultures with PS1 stellate cells. (A) PC3 and 22 Rv cells were infected with Ad∆∆at 100 ppc in the presence or absence of 0.1 pmol AuNPs. PC3 cells were also treated with mitoxantrone (450 nM) and infected with Ad∆∆with and without preincubation with 0.5 pmol AuNP. (B) Panc04.03 and PT45 cells were infected at 100 pc and PS1 cells at 500 ppc with and without AuNPs at 0.2 pmol and analysed 48 h after infection. (A,B) Viral replication was determined by TCID50 assays 48 and 72 h (Pca cells) and 48 h (PDAC cells) after infection, averages ± SD, n = 3, * p < 0.05, ** p < 0.01. (C) Co-cultures of Panc04.03:PS1 (1:2) cells infected with Ad5wt (1000 or 2000 ppc) in the presence or absence of AuNP (0.5 pmol), H/E staining. Cells were cultured for 3d prior to infection with virus ± AuNPs, fixed and processed for IHC 5d post-infection, 8d-old cultures, 10× magnification, representative of three biological repeats. (D) Confocal images of co- cultures detailed in (C). Localisation of virus detected by GFP-labelled secondary antibody to the E1A-antibody (green) and nuclear DAPI stain (blue) (10× magnification).

Article Snippet: Human prostate cancer PC3 and 22Rv1 (ATCC, LGC Standards, UK), and human pancreatic ductal adenocarcinoma (PDAC) Panc04.03 (ATCC) and PT45 (Prof H. Kalthoff, Comprehensive Cancer Centre, Campus Kiel, Kiel, Germany) cell lines were used in the study.

Techniques: Infection, Staining, Cell Culture, Virus

Journal: International journal of molecular sciences

Article Title: Complexing the Oncolytic Adenoviruses Ad∆∆ and Ad-3∆-A20T with Cationic Nanoparticles Enhances Viral Infection and Spread in Prostate and Pancreatic Cancer Models.

doi: 10.3390/ijms23168884

Figure Lengend Snippet: Figure 4. Ad-3∆-A20T infection and replication is enhanced in the presence of AuNP in PDAC cells. (A) Panc04.03 and PT45 were infected with Ad-3∆-A20T-GFP (100 ppc) and PS1 (500 ppc) in the presence or absence of 0.2 pmol AuNPs; samples were analysed 24 and 48 h after infection by flow cytometry for GFP expression (left panel), averages ± SD, n = 3, ** p < 0.01, *** p < 0.001. Lower panel: representative fluorescent images of PS1 cells 24 h and 48 h after infection as in (A), images were taken at 10× magnification (Olympus IX70 microscope). (B) Replication rate in Panc04.03, PT45 and PS1 cells, determined by qPCR for viral genome copies. Cells were infected as above with Ad∆∆and Ad-3∆-A20T in the presence or absence of AuNPs at 0.2 pmol and viral DNA quantified after 24, 48 and 72 h. Data presented from one experiment in triplicates relative to the respective virus alone at 24 h, averages ± SEM.

Article Snippet: Human prostate cancer PC3 and 22Rv1 (ATCC, LGC Standards, UK), and human pancreatic ductal adenocarcinoma (PDAC) Panc04.03 (ATCC) and PT45 (Prof H. Kalthoff, Comprehensive Cancer Centre, Campus Kiel, Kiel, Germany) cell lines were used in the study.

Techniques: Infection, Cytometry, Expressing, Microscopy, Virus

Journal: BMC Medicine

Article Title: Effect of chimeric antigen receptor T cells against protease-activated receptor 1 for treating pancreatic cancer

doi: 10.1186/s12916-023-03053-9

Figure Lengend Snippet: Endogenous PAR1 expression in different human pancreatic ductal adenocarcinoma (PDAC) cells. Six human PDAC cell lines, namely MIA PaCa-2, HPAF-II, SU.8686, Capan-1, ASPC-1, and CFPAC-1, were cultured for PAR1 level screening. A RT-PCR analysis of endogenous PAR1 mRNA levels (*** p < 0.001). B Western blot analysis of PAR1 expression (~ 66 kDa) in whole-cell lysates among the six cell lines (*** p < 0.001). C The mean fluorescence intensity (MFI) of PAR1 surface expression by tumor cells was determined by a flow cytometric analysis using a phycoerythrin (PE)-anti-PAR1 antibody (Ab) versus an isotype control. Propidium iodide (PI) levels were used to examine apoptotic cells. D Immunocytofluorescence (IF) analysis of PAR1 expression patterns in PDAC cells using antihuman PAR1 with signal enhancement through m-IgGκ BP-FITC labeling (left panel). Quantified statistics of green fluorescent protein-positive (GFP + ) to DAPI. + cell ratio of IF results are also shown (right panel). Individual scale bars are shown. All data are presented as the mean ± SD. of three experiments. *** p < 0.001

Article Snippet: Human PDAC cell lines (MIA PaCa-2, SU.8686, HPAF-II, Capan-1, ASPC-1, and CFPAC-1), normal human cell lines (WS1, Hs181.Tes, MRC-5, and Hs67), and 293 T cells were purchased from American Type Culture Collection (ATCC; Manassas, VA, USA) and grown in Dulbecco’s modified Eagle’s medium (DMEM; Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) at 37 °C with 5% CO 2 incubation.

Techniques: Expressing, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Western Blot, Fluorescence, Control, Labeling

Journal: BMC Medicine

Article Title: Effect of chimeric antigen receptor T cells against protease-activated receptor 1 for treating pancreatic cancer

doi: 10.1186/s12916-023-03053-9

Figure Lengend Snippet: Suppression of PAR1-expressing MIA PaCa-2 and CFPAC-1 cells by PAR1CAR-T cells in vitro. A A standard 24-h MTT cytotoxicity assay using three replicates ( n > 3) with increasing effector/tumor (E/T; effector: PAR1CAR-T cells) ratios of 0, 0.1, 1, 5, 10, and 20 against pancreatic ductal adenocarcinoma (PDAC) cell lines of MIA PaCa-2, CFPAC-1, and HPAF-II. Cytotoxic activities were compared to those of non-transduced CD3 + T-cell-treated cells, and mock-transduced T-cell-treated cells served as the control PAR1CAR-T cells ( n > 3; * p < 0.05 and *** p < 0.001). B Real-time monitoring of cytotoxic activities used for comparison between non-transduced CD3 + T cell-treated and mock-transduced T cell-treated cells, and 1% Triton-X-100-treated cells served as a positive control. Real-time monitoring of PAR1CAR-T-cell-treated cells revealed specific growth inhibition of PAR1-expressing CFPAC-1 (low levels; n > 3; * p < 0.05 and ** p < 0.01) and MIA PaCa-2 cells (high levels; n > 3; * p < 0.05, ** p < 0.01, and *** p < 0.001) compared to PAR1 non-expressing HPAF-II cells, as observed using the x-CELLigence System. Data are presented as the mean ± SD of three independent experiments

Article Snippet: Human PDAC cell lines (MIA PaCa-2, SU.8686, HPAF-II, Capan-1, ASPC-1, and CFPAC-1), normal human cell lines (WS1, Hs181.Tes, MRC-5, and Hs67), and 293 T cells were purchased from American Type Culture Collection (ATCC; Manassas, VA, USA) and grown in Dulbecco’s modified Eagle’s medium (DMEM; Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) at 37 °C with 5% CO 2 incubation.

Techniques: Expressing, In Vitro, Cytotoxicity Assay, Control, Comparison, Positive Control, Inhibition

Journal: BMC Medicine

Article Title: Effect of chimeric antigen receptor T cells against protease-activated receptor 1 for treating pancreatic cancer

doi: 10.1186/s12916-023-03053-9

Figure Lengend Snippet: Transforming growth factor (TGF)-β-mediated PAR1 upregulation enhances pancreatic ductal adenocarcinoma (PDAC) cell responsiveness to PAR1CAR-T-cell-specific suppression. A Human PDAC cell lines (HPAF-II, CFPAC-1, and MIA PaCa-2) were exposed to TGF-β (18 ng/mL), and cells were collected at indicated times over 48 h. PAR1 expression was measured by flow cytometry. Results revealed original and enhanced levels of PAR1 by quantifying the mean fluorescent intensity (MFI) (left panel), expression fold-changes (right panel), and cell fold-changes (middle panel) over incubation times. B Standard 24-h cytotoxic activities of PAR1CAR-T cells toward tumor cells were measured using MTT assays with increasing effector/tumor (E/T) ratios of 0, 0.1, 1, 5, 10, and 20 against HPAF-II, CFPAC-1, and MIA PaCa-2 cells following 18 ng/mL TGF-β stimulation (18 ng/mL) for 48 h. Cytotoxic activities were compared to those of non-transduced CD3 + T-cell-treated cells, and mock-transduced T-cell-treated cells served as control PAR1CAR-T cells ( n > 3; * p < 0.05 and *** p < 0.001, respectively). Results are the mean ± SD of three independent experiments

Article Snippet: Human PDAC cell lines (MIA PaCa-2, SU.8686, HPAF-II, Capan-1, ASPC-1, and CFPAC-1), normal human cell lines (WS1, Hs181.Tes, MRC-5, and Hs67), and 293 T cells were purchased from American Type Culture Collection (ATCC; Manassas, VA, USA) and grown in Dulbecco’s modified Eagle’s medium (DMEM; Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) at 37 °C with 5% CO 2 incubation.

Techniques: Expressing, Flow Cytometry, Incubation, Control

Journal: BMC Medicine

Article Title: Effect of chimeric antigen receptor T cells against protease-activated receptor 1 for treating pancreatic cancer

doi: 10.1186/s12916-023-03053-9

Figure Lengend Snippet: Relationship between transforming growth factor (TGF)-β-modulated PAR1 and regulatory T cell (Treg) function and pancreatic ductal adenocarcinoma (PDAC) cell response to PAR1CAR-T cell targeting. A Western blotting results of 24-h stimulation with TGF-β on the MIAPaCa-2 and HPAF-II PDAC cell lines. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as an internal control. B Analysis of tissue factor (TF) and thrombin expressions in individual cell lines treated for 24 h with the TGF-β growth factor. C Effect of adding cancer-associated fibroblasts (CAFs) and different phenotypes of cytokine-independent T cells on cell viability according to different treatments. Different co-culture combinations also resulted in various tumor-derived TGF-β levels. D A schematic diagram shows the role of immuno-mediated TGF-β affecting Treg function and transformation in PDAC treated with PAR1CART cells

Article Snippet: Human PDAC cell lines (MIA PaCa-2, SU.8686, HPAF-II, Capan-1, ASPC-1, and CFPAC-1), normal human cell lines (WS1, Hs181.Tes, MRC-5, and Hs67), and 293 T cells were purchased from American Type Culture Collection (ATCC; Manassas, VA, USA) and grown in Dulbecco’s modified Eagle’s medium (DMEM; Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) at 37 °C with 5% CO 2 incubation.

Techniques: Western Blot, Control, Co-Culture Assay, Derivative Assay, Transformation Assay