Journal: bioRxiv

Article Title: TMEM Doorway Mediated Metastasis in Pancreatic Ductal Adenocarcinoma by Tie2 Signaling

doi: 10.64898/2025.12.05.692608

Figure Lengend Snippet: (A) Immunofluorescence imaging of Tie2Hi/VEGFAHi macrophages in TMEM doorways in sequential sections. Endothelial cells (EC), macrophage (M) and tumor tissue (TC). In the merge panel, a mask for IBA1 macrophage was created in ImageJ ® for a better visualization of Tie2. Scale bar,10μm. (B and C) Quantification of Tie2+ macrophages in tumor area and withim TMEM doorway area of KPCY T-low (B) and KPC (C) tumors. -total macrophages. -Tie2 positive macrophages. (D) Western blot analysis of phospho-Tie2 immunoprecipitated from extracts of KPC, KPCY T-low, RAW 264.7 or 3B-11 cells treated with Rebastinib +/- Angiopoietin 2. (E) Experimental design of the subluminal to luminal trans-endothelial migration (iTEM) assay. Macrophages = red. Tumor cells = green. The tumor cells (green) are considered transendothelial migrating cells when they are crossing the endothelial layer (pink). (F) Representative image of iTEM assay from confocal microscope. Macrophages = red. Tumor cells = green. Endothelial layer = pink. Quantitation of trans-endothelial migration of KPCY T-low (G) and KPC (H). In KPCY T-low and KPC, we observed an increase of transendothelial migration from 3 cells/field to 12.65 cells/field and 6 cells/field to 16.65 cells/field respectively, in the presence of macrophages (p<0.05), following by a decrease to 8.46 cells/field and 8.46 cells/field after rebastinib treatment (p<0.05). Co-culture with macrophages significantly increased migration, which was inhibited by the Tie2 inhibitor rebastinib. Data are presented as mean ± SEM; (n ≥ 10 per group). *P < 0.05, by one-way ANOVA with Tukey’s post hoc test.

Article Snippet: KPC ( Cat.#: 153600: Cancer tools.org ) , KPCY T-low (6419c5) , or KPCY C-EMT 3077 (provided by Ben Z. Stanger) , murine macrophage cell line RAW264.7 (ATCC-provided by Dianne Cox), and murine endothelial cells 3B11 (ATCC - provided by Camille Duran) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (cat# SH30243, Hyclone, GE Healthcare Life Sciences, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS) (cat# S11550, Atlanta Biologicals, Flowery Branch, GA, USA).

Techniques: Immunofluorescence, Imaging, Western Blot, Immunoprecipitation, Migration, Microscopy, Quantitation Assay, Co-Culture Assay

Journal: bioRxiv

Article Title: TMEM Doorway Mediated Metastasis in Pancreatic Ductal Adenocarcinoma by Tie2 Signaling

doi: 10.64898/2025.12.05.692608

Figure Lengend Snippet: (A) panel 1 shows TMEM doorways, visualized by immunohistochemistry (IHC) staining for Mena, Iba-1, and endomucin. The three cells of the TMEM doorway (contained in black circle, with the three cells forming the TMEM doorway indicated with the yellow triangle) are the TMEM doorway endothelial cell (TEC, endomucin stained in blue), TMEM doorway macrophage (TM, Iba1 stained in brown), and TMEM doorway tumor cell (TTC, Mena stained in pink). Panels 2-4 show different staining channels of the sequential tissue section 2 aligned with the IHC stained section (panel 1) with immunofluorescence (IF) with antibodies against endomucin (panel 3 yellow), dextran (panel 4 pink) and nuclear stain DAPI (blue). Active versus inactive TMEM doorways were distinguished by the presence of extravascular dextran staining (panel), which indicates that the vessel had a TMEM doorway-associated vascular opening (TAVO). Scale bars=20μm. (B-H) Mice were treated with control chow or ∼0.44 mg/day of rebastinib for 3 weeks. (B and D) Quantification of TMEM doorway density in 10 high-power fields (HPFs; no significant difference) in KPCY T-low (B) and KPC tumors (D). (C and E) Immunofluorescence measurement of extravascular dextran levels in Tie2 + TMEM doorways indicate significant inhibition of TMEM doorways by rebastinib inKPCY T – low tumors (C) and KPC tumors (E). TMEM doorways were identified in the IF-stained sections as described above. Active TMEM doorways were identified by the presence of extravascular dextran which is not present in inactive TMEM doorways. *p<0.05. (F) Disseminated tumor cells in the liver stained with p53 (red). Increased magnification of field of the p53 positive cells is shown in the yellow box. The left bottom is a representative image of control and right bottom of rebastinib treated group in magnified fields. (G-H) Quantification of DTCs in the liver of animals as compared to the control with rebastinib treated groups showing significant inhibition of DTC levels. (G) KPCY T-low (H) KPC where each dot is a separate mouse. *p<0.05, analyzed by Student’s t-test. N=8.

Article Snippet: KPC ( Cat.#: 153600: Cancer tools.org ) , KPCY T-low (6419c5) , or KPCY C-EMT 3077 (provided by Ben Z. Stanger) , murine macrophage cell line RAW264.7 (ATCC-provided by Dianne Cox), and murine endothelial cells 3B11 (ATCC - provided by Camille Duran) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (cat# SH30243, Hyclone, GE Healthcare Life Sciences, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS) (cat# S11550, Atlanta Biologicals, Flowery Branch, GA, USA).

Techniques: Immunohistochemistry, Staining, Immunofluorescence, Control, Inhibition

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: Oncotarget

Article Title: Perspectives of TGF-β inhibition in pancreatic and hepatocellular carcinomas

doi:

Figure Lengend Snippet: At the cellular level, TGF-β induces proliferation and survival of PDAC cells in the late phase of PDAC carcinogenesis (after SMAD4 inactivation), and promotes epithelial-to-mesenchymal transition (EMT), invasion, and metastasis. At the microenvironment level, TGF-β is a key mediator of the dialogue between cancer and stellate cells (fibrotic cells), involved in the production of a dense fibrotic stroma and the resulting low vascularization of PDAC. TGF-β also deregulates the immune microenvironment toward immunosuppression and inappropriate inflammation.

Article Snippet: Consistent with this, Bachem et al.[ ] showed that PDAC cell lines stimulated PSC proliferation.

Techniques:

Journal: Journal of Gastroenterology

Article Title: Increased expression of PHD3 represses the HIF-1 signaling pathway and contributes to poor neovascularization in pancreatic ductal adenocarcinoma

doi: 10.1007/s00535-014-1030-3

Figure Lengend Snippet: Immunohistological staining for the expressions of CD105 and CD44 in surgical specimens from PDAC and HCC patients. a The expression of CD105, a marker for neovascularization, was extremely low in the PDAC specimens compared to the HCC specimens. The quantitative data and results of the statistical analysis between the groups are shown in the lower bar graph. There was a significant difference with the vascularity between PDAC and HCC. b A higher expression of CD44, a marker for cancer stem cells was observed in the PDAC specimens compared to the HCC specimens. The quantitative data and results of the statistical analysis between the groups are shown in the lower bar graph. c There was a significant negative correlation between the expressions of CD105 and CD44 in each specimens, for both PDAC ( p < 0.01, left ) and HCC ( p < 0.001, right )

Article Snippet: Two PDAC lines (HS766T and Panc-1) and two HCC cell lines (HepG2 and HuH7) purchased from the Japanese Collection of Research Bioresources (Osaka, Japan) were used for the experiments.

Techniques: Staining, Expressing, Marker

Journal: Journal of Gastroenterology

Article Title: Increased expression of PHD3 represses the HIF-1 signaling pathway and contributes to poor neovascularization in pancreatic ductal adenocarcinoma

doi: 10.1007/s00535-014-1030-3

Figure Lengend Snippet: Immunohistological staining for the expressions of HIF-1α, PHD3, and Siah2 in surgical specimens from PDAC and HCC patients. a HIF-1α was positively stained in the nuclei of cancer cells in HCC specimens, but was negatively or extremely rarely stained in the PDAC specimens, and a semiquantitative analysis showed a significant difference between the groups ( p < 0.05). b PHD3 was positively stained in many cancer cells in the PDAC specimens, but was negatively or weakly stained in the HCC specimens ( p < 0.001). c The expression of Siah2 was negative or weak in both the PDAC and HCC specimens, but the semiquantitative analysis showed a significantly higher expression in the HCC specimens ( p < 0.05)

Article Snippet: Two PDAC lines (HS766T and Panc-1) and two HCC cell lines (HepG2 and HuH7) purchased from the Japanese Collection of Research Bioresources (Osaka, Japan) were used for the experiments.

Techniques: Staining, Expressing

Journal: Journal of Gastroenterology

Article Title: Increased expression of PHD3 represses the HIF-1 signaling pathway and contributes to poor neovascularization in pancreatic ductal adenocarcinoma

doi: 10.1007/s00535-014-1030-3

Figure Lengend Snippet: The expression of CD44 in PDAC (HS766T and Panc-1) and HCC (HepG2 and HuH7) cell lines. Cells were cultured in 20 % O 2 or 1 % O 2 for 24 h, and the expression of the CSC marker CD44 was then estimated by flow cytometry and compared to cancer stem cell markers by hypoxic stimulation. The expression of CD44 was much higher in the PDAC cell lines compared to the HCC cell lines, but the 24-h incubation in 1 % O 2 did not change the expression of CD44 in any of the cell lines. The green line shows the expression of CD44 in hypoxia (1 % O 2 for 24 h) and the purple area shows it in normoxia (20 % O 2 for 24 h)

Article Snippet: Two PDAC lines (HS766T and Panc-1) and two HCC cell lines (HepG2 and HuH7) purchased from the Japanese Collection of Research Bioresources (Osaka, Japan) were used for the experiments.

Techniques: Expressing, Cell Culture, Marker, Flow Cytometry, Incubation

Journal: Journal of Gastroenterology

Article Title: Increased expression of PHD3 represses the HIF-1 signaling pathway and contributes to poor neovascularization in pancreatic ductal adenocarcinoma

doi: 10.1007/s00535-014-1030-3

Figure Lengend Snippet: Western-blot analysis of the expressions of PHD3 and HIF-1α in the PDAC (HS766T and Panc-1) and HCC (HepG2 and HuH7) cell lines. a The expression of PHD3 was clearly detected in both of the PDAC cell lines, but negatively or very dimly in the two HCC cell lines cultured in the 20 or 1 % O 2 conditions. Although comparable expression levels of HIF-1α were detected between the PDAC and HCC cell lines under 20 % O 2 , the hypoxic stimulation by 1 % O 2 significantly enhanced the expression of HIF-1α only in the two HCC cell lines, but only slightly upregulated the expression in the PDAC cell lines. b The semiquantitative data are shown

Article Snippet: Two PDAC lines (HS766T and Panc-1) and two HCC cell lines (HepG2 and HuH7) purchased from the Japanese Collection of Research Bioresources (Osaka, Japan) were used for the experiments.

Techniques: Western Blot, Expressing, Cell Culture

Journal: Journal of Gastroenterology

Article Title: Increased expression of PHD3 represses the HIF-1 signaling pathway and contributes to poor neovascularization in pancreatic ductal adenocarcinoma

doi: 10.1007/s00535-014-1030-3

Figure Lengend Snippet: VEGF secretion from the PDAC (HS766T and Panc-1) and HCC (HepG2 and HuH7) cell lines. The level of VEGF in conditioned medium was measured by ELISA. The secretion of VEGF was comparable between the PDAC and HCC cell lines under 20 % O 2 . Hypoxic stimulation by 1 % O 2 significantly enhanced the VEGF secretion from both of the HCC cell lines ( p < 0.05), but no significant enhancement was observed in the PDAC cell lines

Article Snippet: Two PDAC lines (HS766T and Panc-1) and two HCC cell lines (HepG2 and HuH7) purchased from the Japanese Collection of Research Bioresources (Osaka, Japan) were used for the experiments.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Journal of Gastroenterology

Article Title: Increased expression of PHD3 represses the HIF-1 signaling pathway and contributes to poor neovascularization in pancreatic ductal adenocarcinoma

doi: 10.1007/s00535-014-1030-3

Figure Lengend Snippet: Knockdown of PHD3 in the PDAC cell line HS766T. a Knockdown of PHD3 was done by siRNA, and the expression levels of PHD3 and HIF-1α were estimated by Western blot. PHD3 knockdown did not change the expression of HIF-1α when the cells were cultured in 20 % O 2 , but the expression of HIF-1α was clearly enhanced when the cells were cultured in 1 % O 2 . b The semiquantitative data in HIF-1α are shown. c Hypoxic stimulation by 1 % O 2 significantly enhanced the VEGF secretion from HS766T cells treated by siPHD3 ( p < 0.05)

Article Snippet: Two PDAC lines (HS766T and Panc-1) and two HCC cell lines (HepG2 and HuH7) purchased from the Japanese Collection of Research Bioresources (Osaka, Japan) were used for the experiments.

Techniques: Knockdown, Expressing, Western Blot, Cell Culture