Journal: Oncotarget

Article Title: TCGA data and patient-derived orthotopic xenografts highlight pancreatic cancer-associated angiogenesis.

doi: 10.18632/oncotarget.3233

Figure Lengend Snippet: Figure 4: STAT3 is active in PDAC tumor endothelia and enhances HDAC9 expression to promote endothelial proliferation. (A) p-STAT3 (red) is abundant in the nuclei of VE-Cadherin-positive vessels (green, outlined) and surrounding stromal cells (arrowheads) in KRC (top), EUS-PDOX tumors (middle), and human PDACs (bottom). (B) KRC CM markedly increases p-STAT3 levels in ECs, which is blocked by ruxolitinib [100 nM]. (C) KRC CM significantly enhances STAT3 luciferase reporter activity in ECs (top), which is blocked by ruxolitinib [100 nM] or a STAT3-targeting shRNA (shRNA#2). Immunoblotting (lower panel) shows the knockdown efficiency of STAT3-targeting shRNAs. ERK2 confirms equivalent lane loading. Shown in (B–C) are representative immunoblots from three independent experiments. (D) CM from KRC cells significantly enhances EC proliferation, but in the presence of ruxolitinib ([100 nM], left) or in ECs transduced with a STAT3-targeting shRNA#2 (right) CM fails to enhance EC proliferation. (E) Hdac9 mRNA levels are significantly decreased in ECs transduced with STAT3-targeting shRNA#2 (open bar). (F) CM from KRC cells significantly increases Hdac9 mRNA levels in ECs, but in the presence of shRNA#2 or ruxolitinib [100 nM] CM fails to up-regulate Hdac9. (G) CM fails to stimulate the proliferation of ECs transduced with shRNA#2, but when these ECs are transfected with an Hdac9 cDNA construct, CM significantly enhances EC proliferation. (H) HDAC9 (red) is abundant in the nuclei of CD31-positive vessels (green, outlined) and in surrounding stromal (arrowheads) and cancer cells (arrows) in EUS-PDOX (middle) and KRC PDACs (bottom) as evidenced by co- localization with DAPI (blue) in CD31-cadherin-positive vessels (outlined). (I) Compared with SVEC4–10 ECs, Hdac9 and Cd34 are significantly increase in KRC tumor-derived ECs, whereas Ck19 is absent in both. Shown in (A) and (H) are representative images from three KRC or EUS-PDOX tumors, or the TMA. Scale bars, 50 μm. Data in (C–G, I) are mean ± SEM. *P < 0.05, and **P < 0.01.

Article Snippet: An HDAC9 cDNA construct (Origene) was transfected into ECs using Lipofectamine 2000 (Life Technologies) per manufacturer’s recommendations.

Techniques: Expressing, Luciferase, Activity Assay, shRNA, Western Blot, Knockdown, Transduction, Transfection, Construct, Derivative Assay

Journal: Oncotarget

Article Title: TCGA data and patient-derived orthotopic xenografts highlight pancreatic cancer-associated angiogenesis.

doi: 10.18632/oncotarget.3233

Figure Lengend Snippet: Figure 5: Ruxolitinib suppresses mPDAC progression and prolongs survival of KRC mice. (A–B) H&Es show that KRC pancreata often exhibit ADM, PanIN and mPDAC at postnatal month 1 (A), and that vehicle-treated mice display abundant lesions and mPDAC, whereas ruxolitinib-treated pancreata are mostly normal and only display small foci of ADM (B) Shown are representative images from two mice per group. Right panels are high magnification images of boxed areas. (C) Nuclear p-STAT3 (left) is abundant in KRC mPDACs (top) and ADM (middle) in vehicle-treated mice, whereas ADM in ruxolitinib-treated mice (bottom) have weak p-STAT3 immunoreactivity. mPDACs and ADM in vehicle-treated mice also have abundant ECs and are highly proliferative as evidenced by the presence CD31 and p-Histone H3 immunoreactivity, respectively. ADM in ruxolitinib-treated mice have few CD31-positive ECs, and p-Histone H3 is mostly absent. (D) VE-cadherin-positive ECs (green) in vehicle-treated KRC mice harbor nuclear p-STAT3 (top panels, red, arrows), whereas ECs in ruxolitinib-treated mice lack nuclear p-STAT3 (arrowheads). CD31-positive ECs vehicle-treated KRC mice also exhibit strong, nuclear HDAC9 immunoreactivity (bottom panel, red, arrows) that is markedly attenuated in ECs in ruxolitinib-treated mice (arrowheads). All images were acquired using the same exposure time. Scale bars in (A–D), 50 μm. (E) Kaplan-Meier analysis shows that compared to vehicle (red line), ruxolitinib (blue line) significantly (P = 0.018) prolongs survival of KRC mice. Dashed line indicates that 2 ruxolitinib-treated mice were alive beyond postnatal week 18.

Article Snippet: An HDAC9 cDNA construct (Origene) was transfected into ECs using Lipofectamine 2000 (Life Technologies) per manufacturer’s recommendations.

Techniques:

Journal: Oncotarget

Article Title: TCGA data and patient-derived orthotopic xenografts highlight pancreatic cancer-associated angiogenesis.

doi: 10.18632/oncotarget.3233

Figure Lengend Snippet: Figure 6: Ruxolitinib suppresses mitogenic cross-talk between endothelial cells and PCCs. (A) 3D co-cultures of ECs (red) and KRC PCCs (green) shows that compared with vehicle (DMSO, left), ruxolitinib ([100 nM], right) suppresses PCC growth. Shown are representative phase contrast and fluorescent images taken on day 8. Scale bars, 200 μm. (B) Fluorescence intensity quantitation shows that compared with 3D cultures in which ECs and PCCs are cultured independently (single culture), culturing ECs and PCCs together in 3D (co-culture) significantly enhances PCC growth, which is blocked by ruxolitinib (open bars). Data are mean ± SEM from three independent experiments. *P < 0.05, and **P < 0.01. (C) Schematic representation of PCC and EC cross-talk. TGF-β activates canonical Smad- dependent signaling in PCCs (top) leading to enhanced production of pro-angiogenic factors, which can be blocked by SB505124. These factors activate JAK/STAT3 signaling in ECs (bottom), which promotes EC proliferation through HDAC9, and ruxolitinib blocks these effects. ECs also produce factors (angiocrine factors) that can exert growth-stimulatory effects on PCCs through JAK/STAT3 signaling, which can also be targeted with ruxolitinib.

Article Snippet: An HDAC9 cDNA construct (Origene) was transfected into ECs using Lipofectamine 2000 (Life Technologies) per manufacturer’s recommendations.

Techniques: Fluorescence, Quantitation Assay, Cell Culture, Co-Culture Assay