Abstract A114: Alternatively spliced tissue factor: A new therapeutic target in pancreatic cancer
Authors: Unruh Dusten , Qi Xiaoyang , Chu Zhengtao , Sturm Robert , Keil Ryan , Ahmad Syed , Versteeg Henri H. , Bogdanov Vladimir Y. 
Published: October 2015

Abstract
Background: Pancreatic ductal adenocarcinoma (PDAC) is marked by high expression of Tissue Factor (TF), the trigger of blood coagulation. Alternatively spliced TF (asTF) is a secreted TF form that signals non-proteolytically via β1/β3 integrins. We recently reported that asTF is abundant in PDAC lesions and, when overexpressed in PDAC cells, it promotes tumor growth, metastatic spread, monocyte recruitment, and elevates procoagulant potential of PDAC cells and cell-derived microparticles. In this study, we sought to determine whether i) delayed-onset overexpression of asTF yields a phenotype distinct from that obtained with constitutive overexpression; ii) asTF mechanistically contributes to PDAC cell migration; and iii) antibody-based targeting of asTF slows PDAC progression and/or asTF release into systemic circulation. Methods: Cohorts of nude mice (n=5/cohort) were orthotopically transplanted with grade III human PDAC cells Pt45P1 harboring a doxycycline (Dox)-inducible asTF transgene system (line Pt45P1/asTFi, 5x105 cells/mouse); mice began receiving Dox (2µg/mL) in sucrose at day 1 of the study (“Dox”), day 25 of the study (“late Dox”), or sucrose alone (“no Dox”), and tumor progression was monitored in vivo via SapC-DOPS imaging over 7 wks. PDAC cell migration toward serum was assessed using laminin-coated transwell inserts seeded with Pt45P1/asTFi cells. Plasma samples from mice bearing PDAC tumors were assayed for asTF using a custom sandwich ELISA. asTF-specific rabbit monoclonal antibody Rb1 was co-implanted with Pt45P1 cells, and tumor progression was monitored as above over 5 wks. Tumor specimens were assessed for vessel density (isolectin B4) and monocyte/macrophage infiltration (anti-F4/80 antibody). Results: “Late Dox” mice developed tumors that were comparable in size and vessel density to those in “Dox” mice, yet less infiltrated with macrophages (Dox: 70±22 cells/LPF, late Dox: 51±17 cells/LPF, p=0.009); interestingly, tumor spread was also significantly less pronounced in “late Dox” mice compared to “Dox” mice ([7.52e+7] vs [1.22e+8]P/s/mm2, p=0.01). “Dox” mice also had high levels of asTF in circulation (~1 ng/mL), which did not differ significantly from those in “late Dox” mice. PDAC cell migration assay revealed a ~4 fold increase in the migration of Pt45P1/asTFi cells treated with Dox compared to untreated cells (p&lt;0.001). When cells were co-treated with Dox and inhibitory anti-β1 antibody, migration was inhibited to the levels observed with untreated cells; anti-α6 antibody had a partial effect. Strikingly, migration of Pt45P1/asTFi cells co-treated with Dox and Rb1 was inhibited to the levels observed with untreated cells and/or Dox/anti-β1 antibody co-treated cells; rabbit isotype control IgG had no effect. Pt45P1 cells, when co-implanted with just 100μg of Rb1, grew significantly smaller tumors that were less vascularized and had ~3.5 fold fewer stromal macrophages compared to the cells co-implanted with vehicle (PBS) and/or isotype control IgG (Rb1: avg. wt. 0.835g, 20±9 cells/LPF; PBS: avg. wt. 1.624g, 73±30 cells/LPF; IgG: avg. wt. 1.665g, 66±47 cells/LPF; p&lt;0.005 Rb1 vs PBS/IgG). Mice in the Pt45P1/Rb1 cohort had ~2 fold decrease in the levels of circulating asTF compared to mice in the Pt45P1/PBS and/or isotype IgG cohorts (0.27 ng/mL vs 0.60/0.54 ng/mL; p&lt;0.001). Conclusion: asTF can promote PDAC progression during earlier as well as later stages of the disease. asTF-β1 integrin interactions render PDAC cells more motile, likely via competing with laminin for β1 integrin binding; Rb1 fully inhibits asTF-potentiated PDAC cell migration. When co-implanted with Rb1, Pt45P1 cells grow much smaller tumors with fewer monocytes and blood vessels that release less asTF in the circulation. Thus, antibody-based targeting of asTF may comprise a novel therapeutic strategy to stem PDAC progression.
Citation Format: Dusten Unruh, Xiaoyang Qi, Zhengtao Chu, Robert Sturm, Ryan Keil, Syed Ahmad, Henri H. Versteeg, Vladimir Y. Bogdanov. Alternatively spliced tissue factor: A new therapeutic target in pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A114.