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