Alternatively spliced Tissue Factor (asTF) is a secreted form of Tissue Factor (TF), the trigger of blood coagulation whose expression levels are heightened in several forms of solid cancer, including pancreatic ductal adenocarcinoma (PDAC). asTF binds to β1 integrins on PDAC cells, whereby it promotes tumor growth, metastatic spread, and monocyte recruitment to the stroma. In this study, we determined if targeting asTF in PDAC would significantly impact tumor progression. We here report that a novel inhibitory anti-asTF monoclonal antibody curtails experimental PDAC progression. Moreover, we show that tumor-derived asTF is able to promote PDAC primary growth and spread during early as well as later stages of the disease. This raises the likelihood that asTF may comprise a viable target in early- and late-stage PDAC. In addition, we show that TF expressed by host cells plays a significant role in PDAC spread. Together, our data demonstrate that targeting asTF in PDAC is a novel strategy to stem PDAC progression and spread.

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BACKGROUND Pro-coagulant full length Tissue Factor (flTF) and its minimally coagulant alternatively spliced isoform (asTF), promote breast cancer (BrCa) progression via different mechanisms. We previously showed that flTF and asTF are expressed by BrCa cells, resulting in autoregulation in a cancer milieu. BrCa cells often express hormone receptors such as the estrogen receptor (ER), leading to the formation of hormone-regulated cell populations. OBJECTIVE To investigate whether TF isoform-specific and ER-dependent pathways interact in BrCa. METHODS TF isoform-regulated gene sets were assessed using ingenuity pathway analysis (IPA). Tissues from a cohort of BrCa patients were divided into ER positive and ER negative groups. Associations between TF isoform levels and tumor characteristics were analyzed in these groups. BrCa cells expressing TF isoforms were assessed for proliferation, migration and in vivo growth in the presence or absence of estradiol. RESULTS IPA analysis pointed to similarities between ER- and TF-induced gene expression profiles. In BrCa tissue specimens, asTF expression associated with grade and stage in ER positive but not in ER negative tumors. flTF only associated with grade in ER positive tumors. In MCF-7 cells, asTF accelerated proliferation in the presence of estradiol in a β1 integrin-dependent manner. No synergy between asTF and the ER pathway was observed in a migration assay. Estradiol accelerated the growth of asTF-expressing tumors but not control tumors in vivo in an orthotopic setting. CONCLUSION TF isoform and estrogen signalling share downstream targets in BrCa; concomitant presence of asTF and estrogen signalling is required to promote BrCa cell proliferation.

Cancer patients have an increased risk of developing venous thromboembolism (VTE), a condition that is associated with increased morbidity and mortality. Although risk assessment tools have been developed, it is still very challenging to predict which cancer patients will suffer from VTE. The scope of this review is to summarize and discuss studies focusing on the link between genetic alterations and risk of cancer‐associated thrombosis (CAT). Thus far, classical risk factors that contribute to VTE have been tried as risk factors of CAT, with low success. In support, hypercoagulant plasma profiles in patients with CAT differ from those with only VTE, indicating other risk factors that contribute to VTE in cancer. As germline mutations do not significantly contribute to elevated risk of VTE, somatic mutations in tumors may significantly associate with and contribute to CAT. As it is very time‐consuming to investigate each and every mutation, an unbiased approach is warranted. In this light we discuss our own recent unbiased proof‐of‐principle study using RNA sequencing in isolated colorectal cancer cells. Our work has uncovered candidate genes that associate with VTE in colorectal cancer, and these gene profiles associated with VTE more significantly than classical parameters such as platelet counts, D‐dimer, and P‐selectin levels. Genes associated with VTE could be linked to pathways being involved in coagulation, inflammation and methionine degradation. We conclude that tumor cell‐specific gene expression profiles and/or mutational status has superior potential as predictors of VTE in cancer patients.

Background Venous thromboembolism (VTE) is a frequent complication in patients with cancer, and is associated with significant morbidity and mortality. However, the mechanisms behind cancer-associated thrombosis are still incompletely understood. Objectives To identify novel genes that are associated with VTE in patients with colorectal cancer (CRC). Methods Twelve CRC patients with VTE were age-matched and sex-matched to 12 CRC patients without VTE. Tumor cells were isolated from surgical samples with laser capture microdissection approaches, and mRNA profiles were measured with next-generation RNA sequencing. Results This approach led to the identification of new genes and pathways that might contribute to VTE in CRC patients. Application of ingenuity pathway analysis indicated significant links with inflammation, the methionine degradation pathway, and increased platelet function, which are all key processes in thrombus formation. Tumor samples of patients with VTE had a proinflammatory status and contained higher levels of fibrin and fibrin degradation products than samples of those without VTE. Conclusion This case-control study provides a proof-of-principle that tumor gene expression can discriminate between cancer patients with low and high risks of VTE. These findings may help to further unravel the pathogenesis of cancer-related VTE. The identified genes could potentially be used as candidate biomarkers to select high-risk CRC patients for thromboprophylaxis.

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Background Full length Tissue factor (flTF) is a key player in hemostasis and also likely contributes to venous thromboembolism (VTE), the third most common cardiovascular disease. flTF and its minimally coagulant isoform, alternatively spliced TF (asTF), have been detected in thrombi, suggesting participation of both isoforms in thrombogenesis, but data on participation of asTF in hemostasis is lacking. Therefore, we assessed the role of asTF in flTF cofactor activity modulation, using a co-expression system. Objective To investigate the interplay between flTF and asTF in hemostasis on endothelial cell surface. Methods Immortalized endothelial (ECRF) cells were adenovirally transduced to express asTF and flTF, after which flTF cofactor activity was measured on cells and microvesicles (MVs). To study co-localization of flTF/asTF proteins, confocal microscopy was performed. Finally, intracellular distribution of flTF was studied in the presence or absence of heightened asTF levels. Results Levels of flTF antigen and cofactor activity were not affected by asTF co-expression. asTF and flTF were found to localize in distinct subcellular compartments. Only upon heightened overexpression of asTF, lower flTF protein levels and cofactor activity were observed. Heightened asTF levels also induced a shift of flTF from non-raft to lipid raft plasma membrane fractions, and triggered the expression of ER stress marker BiP. Proteasome inhibition resulted in increased asTF – but not flTF – protein expression. Conclusion At moderate levels, asTF appears to have negligible impact on flTF cofactor activity on endothelial cells and MVs; however, at supra-physiological levels, asTF is able to reduce the levels of flTF protein and cofactor activity.