Alternatively Spliced Tissue Factor As a New Therapeutic Target in Pancreatic Cancer
Authors: Unruh Dusten , Qi Xiaoyang , Chu Zhengtao , Sturm Robert , Keil Ryan , Ahmad Syed A , Sovershaev Timofey , Rak Janusz W , Mackman Nigel , Versteeg Henri H , Bogdanov Vladimir Y 
Published: October 2019

Abstract
Pancreatic ductal adenocarcinoma (PDAC) is marked by high expression of Tissue Factor (TF). Alternatively spliced TF (asTF) is a secreted TF form that signals non-proteolytically via β1/β3 integrins. asTF is abundant in PDAC lesions and its constitutive overexpression promotes tumor growth, metastatic spread, monocyte recruitment, and elevated procoagulant potential of PDAC cells and cell-derived microparticles. In this study, we determined if 1) delayed-onset overexpression of asTF yields a phenotype distinct from that obtained with constitutive overexpression; 2) asTF contributes to PDAC cell migration; and 3) monoclonal antibody-based targeting of tumor-derived asTF slows PDAC progression and/or asTF release into circulation in an orthotopic setting.
Nude mice were orthotopically transplanted with human PDAC cells harboring a doxycycline (Dox)-inducible asTF transgene system (line Pt45P1/asTFi); mice received Dox (2µg/mL) in sucrose at day 1 (“Dox”), day 25 (“late Dox”), or sucrose alone (“no Dox”), and tumor progression was monitored in vivo over 7 weeks via SapC-DOPS imaging. Migration of Pt45P1/asTFi cells toward serum was assessed using laminin-coated transwell inserts. Plasma of PDAC tumor-bearing mice was assayed for asTF using a custom ELISA. Pt45P1 cells were resuspended in PBS containing anti-asTF rabbit monoclonal antibody Rb1 and implanted in a) nude mice; b) SCID mice, and c) SCID mice with TF levels reduced by 99% (SCID/TF-low) and tumor progression was monitored as above over 5 weeks. Paraffin-embedded tumor specimens were assessed for vessel density (isolectin B4 and/or anti-CD31 staining) and monocyte/macrophage infiltration (anti-F4/80 and/or anti-CD206 staining).
“Late Dox” mice developed tumors 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); tumor spread was significantly reduced 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. The PDAC cell migration assay revealed a ~4 fold increase in the migration of Pt45P1/asTFi cells treated with Dox compared to untreated cells (p<0.001). When cells were co-treated with Dox and inhibitory anti-β1 antibody, migration was inhibited to the levels observed with untreated cells. Migration of Pt45P1/asTFi cells co-treated with Dox and Rb1 was inhibited to the levels observed with Dox/anti-β1 antibody co-treated cells; rabbit isotype control IgG had no effect. Pt45P1 cells, when implanted together with just 100 μg of Rb1, grew significantly smaller tumors that were less vascularized and had ~3.5 fold fewer stromal macrophages compared to the cells implanted together with vehicle (PBS) and/or isotype control IgG (Rb1: avg. wt. 0.835g, 20±9 cells/low power field (LPF); PBS: avg. wt. 1.624g, 73±30 cells/LPF; IgG: avg. wt. 1.665g, 66±47 cells/LPF; p<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<0.001). While Pt45P1 tumors grown in SCID and SCID/TF-low mice were comparable in size, tumor spread was significantly diminished in SCID/TF-low mice (7.74e+8 P/s/mm2 vs 1.09e+9 P/s/mm2, p=0.007); tumors in SCID/TF-low mice also had fewer M2 polarized macrophages (p=0.011) and lower vessel density (4,576µm2/LPF vs 1,834µm2/LPF, p=0.017). In the presence of Rb1, Pt45P1 cells implanted in SCID mice grew tumors significantly smaller compared to IgG control (p=0.005); the effects of Rb1 on primary tumor growth in SCID/low-TF mice were similar to those in SCID mice.
We here report that asTF can promote PDAC progression during early and late stages of the disease. asTF-β1 integrin interaction renders PDAC cells more motile, while Rb1 fully inhibits asTF-potentiated PDAC cell migration. Host-derived TF has no impact on the size of primary PDAC tumor, yet it contributes to tumor vascularization and, consequently, metastatic spread. In the presence of Rb1 in nude and/or SCID mice, Pt45P1 cells grow significantly 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 strategy to stem PDAC progression.

Disclosures
No relevant conflicts of interest to declare.