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Cancer enhances the risk of venous thromboembolism, but a hypercoagulant microenvironment also promotes cancer progression. Although anticoagulants have been suggested as a potential anticancer treatment, clinical studies on the effect of such modalities on cancer progression have not yet been successful for unknown reasons. In normal physiology, complex formation between the subendothelial-expressed tissue factor (TF) and the blood-borne liver-derived factor VII (FVII) results in induction of the extrinsic coagulation cascade and intracellular signaling via protease-activated receptors (PARs). In cancer, TF is overexpressed and linked to poor prognosis. Here, we report that increased levels of FVII are also observed in breast cancer specimens and are associated with tumor progression and metastasis to the liver. In breast cancer cell lines, tumor-expressed FVII drives changes reminiscent of epithelial-to-mesenchymal transition (EMT), tumor cell invasion, and expression of the prometastatic genes, SNAI2 and SOX9. In vivo, tumor-expressed FVII enhanced tumor growth and liver metastasis. Surprisingly, liver-derived FVII appeared to inhibit metastasis. Finally, tumor-expressed FVII-induced prometastatic gene expression independent of TF but required a functional endothelial protein C receptor, whereas recombinant activated FVII acting via the canonical TF:PAR2 pathway inhibited prometastatic gene expression. Here, we propose that tumor-expressed FVII and liver-derived FVII have opposing effects on EMT and metastasis.

Background: Glioblastoma patients are considered to be at high risk of venous thromboembolism (VTE) and major bleeding (MB), although reliable incidence estimates are lacking. Moreover, the risk of arterial thromboembolism (ATE) in these patients is largely unknown. Our aim was to assess the cumulative incidence, predictors, and prognostic impact of VTE, ATE, and MB on subsequent complications and mortality. Methods: Cohort study of 967 consecutive patients diagnosed with glioblastoma between 2004–2020 in two hospitals. Patients were followed from 6 months before date of histopathological glioblastoma diagnosis up to 2 years after, or until an outcome of interest (VTE, ATE, and MB) or death occurred, depending on the analysis. Cumulative incidences were estimated with death as competing risk. Cox regression was used to identify predictors and the prognostic impact. Results: A total of 101 patients were diagnosed with VTE, 50 with ATE, and 126 with MB during a median follow-up of 15 months (interquartile range 9.0–22). The adjusted 1-year cumulative incidence of VTE was 7.5% (95% confidence interval [CI] 5.9–9.3), of ATE 4.1% (95% CI 3.0–5.6), and of MB 12% (95% CI 9.6–14). Older age, type of surgery, and performance status were predictors of VTE. Incident VTE during follow-up was associated with MB (adjusted HR 4.7, 95% CI 2.5–9.0). MB and VTE were associated with mortality (adjusted HR 1.7, 95% CI 1.3–2.1 and 1.3, 95% CI 1.0–1.7, respectively). Conclusion: We found considerable incidences of VTE and MB in glioblastoma patients, with both complications associated with poorer prognosis. Our observations emphasize the need for prospective studies to determine optimal thromboprophylaxis and VTE treatment strategy in these patients.

Glioblastoma (GBM) patients have one of the highest risks of venous thromboembolism (VTE), which is even further increased upon treatment with chemotherapy. Tissue factor (TF) is the initiator of the extrinsic coagulation pathway and expressed by GBM cells. In this study, we aimed to examine the effect of routinely used chemotherapeutic agents Temozolomide (TMZ) and Lomustine (LOM) on TF procoagulant activity and expression in GBM cells in vitro. Three human GBM cell lines (U-251, U-87, U-118) were exposed to 100 µM TMZ or 30 µM LOM for 72 h. TF procoagulant activity was assessed via an FXa generation assay and TF gene and protein expression through qPCR and Western blotting. The externalization of phosphatidylserine (PS) was studied using Annexin V flow cytometry. Treatment with TMZ and LOM resulted in increased procoagulant activity in all cell lines. Furthermore, both agents induced procoagulant activity in the supernatant and tumor-cell-secreted extracellular vesicles. In line, TF gene and protein expression were increased upon TMZ and LOM treatment. Additionally, PS externalization and induction of inflammatory-associated genes were observed. Overall, the chemotherapeutic modalities TMZ and LOM induced procoagulant activity and increased TF gene and protein expression in all GBM cell lines tested, which may contribute to the increased VTE risk observed in GBM patients undergoing chemotherapy.

Cancer patients have an increased risk of developing venous thromboembolism (VTE) and this combination is reported to result in poorer survival compared to cancer alone. The aim of this study was to investigate the impact of VTE on survival of cancer patients in a general population. The Scandinavian Thrombosis and Cancer (STAC) cohort, a population-based cohort including 144,952 subjects without previous VTE or cancer was used. During follow-up, cancer and VTE incidences were registered. 'Cancer-related VTE' was defined as VTE diagnosed in patients with overt or occult cancer. Survival of subjects without cancer and/or VTE ('disease-free') was compared with survival of subjects with cancer and cancer-related VTE. Cox-regression models with cancer and VTE as time-varying exposures were performed to calculate hazard ratios for death. Sub-analyses were performed across cancer types and stages and VTE type (deep vein thrombosis or pulmonary embolism). During follow-up (mean 11.7 years) 14,621 subjects developed cancer and 2,444 developed VTE, of which 1241 where cancer-related. The mortality rates (per 100 person-years) for disease-free subjects, VTE only, cancer only and cancer-related VTE were 0.63 (95%CI 0.62-0.65), 5.0 (4.6-5.5), 9.2 (9.0-9.5) and 45.3 (41.1-50.0), respectively. Compared with cancer only patients, the risk of death for cancer-related VTE patients was increased 3.4-fold (95%CI 3.1-3.8). Within all cancer types, the occurrence of VTE increased the mortality risk 2.8 to 14.7-fold. In a general population, cancer patients with VTE had a 3.4-fold higher mortality risk than cancer patients without VTE, independent of cancer type. Cancer patients have an increased risk of developing venous thromboembolism (VTE) and this combination is reported to result in poorer survival compared to cancer alone. The aim of this study was to investigate the impact of VTE on survival of cancer patients in a general population. The Scandinavian Thrombosis and Cancer (STAC) cohort, a population-based cohort including 144,952 subjects without previous VTE or cancer was used. During follow-up, cancer and VTE incidences were registered. 'Cancer-related VTE' was defined as VTE diagnosed in patients with overt or occult cancer. Survival of subjects without cancer and/or VTE ('disease-free') was compared with survival of subjects with cancer and cancer-related VTE. Cox-regression models with cancer and VTE as time-varying exposures were performed to calculate hazard ratios for death. Sub-analyses were performed across cancer types and stages and VTE type (deep vein thrombosis or pulmonary embolism). During follow-up (mean 11.7 years) 14,621 subjects developed cancer and 2,444 developed VTE, of which 1241 where cancer-related. The mortality rates (per 100 person-years) for disease-free subjects, VTE only, cancer only and cancer-related VTE were 0.63 (95%CI 0.62-0.65), 5.0 (4.6-5.5), 9.2 (9.0-9.5) and 45.3 (41.1-50.0), respectively. Compared with cancer only patients, the risk of death for cancer-related VTE patients was increased 3.4-fold (95%CI 3.1-3.8). Within all cancer types, the occurrence of VTE increased the mortality risk 2.8 to 14.7-fold. In a general population, cancer patients with VTE had a 3.4-fold higher mortality risk than cancer patients without VTE, independent of cancer type.

Endoglin expression by CRC cells, HT29 proliferation and in vivo data HT29 only injections

Characterization of patient-derived NFs and CAFs

Purpose:Cancer-associated fibroblasts (CAF) are a major component of the colorectal cancer tumor microenvironment. CAFs play an important role in tumor progression and metastasis, partly through TGF-β signaling pathway. We investigated whether the TGF-β family coreceptor endoglin is involved in CAF-mediated invasion and metastasis.Experimental Design:CAF-specific endoglin expression was studied in colorectal cancer resection specimens using IHC and related to metastases-free survival. Endoglin-mediated invasion was assessed in vitro by transwell invasion, using primary colorectal cancer–derived CAFs. Effects of CAF-specific endoglin expression on tumor cell invasion were investigated in a colorectal cancer zebrafish model, whereas liver metastases were assessed in a mouse model.Results:CAFs specifically at invasive borders of colorectal cancer express endoglin and increased expression intensity correlated with increased disease stage. Endoglin-expressing CAFs were also detected in lymph node and liver metastases, suggesting a role in colorectal cancer metastasis formation. In stage II colorectal cancer, CAF-specific endoglin expression at invasive borders correlated with poor metastasis-free survival. In vitro experiments revealed that endoglin is indispensable for bone morphogenetic protein (BMP)-9–induced signaling and CAF survival. Targeting endoglin using the neutralizing antibody TRC105 inhibited CAF invasion in vitro. In zebrafish, endoglin-expressing fibroblasts enhanced colorectal tumor cell infiltration into the liver and decreased survival. Finally, CAF-specific endoglin targeting with TRC105 decreased metastatic spread of colorectal cancer cells to the mouse liver.Conclusions:Endoglin-expressing CAFs contribute to colorectal cancer progression and metastasis. TRC105 treatment inhibits CAF invasion and tumor metastasis, indicating an additional target beyond the angiogenic endothelium, possibly contributing to beneficial effects reported during clinical evaluations.See related commentary by Becker and LeBleu, p. 6110.

Background and objectives: Patients with glioblastoma have a high risk of developing venous thromboembolism (VTE). However, the role of underlying genetic risk factors remains largely unknown. Therefore, the aim of this study was to discover whether genetic aberrations in glioblastoma associate with VTE risk. Methods: In this cohort study, all consecutive patients diagnosed with glioblastoma in two Dutch hospitals between February 2017 and August 2020 were included. Targeted DNA next-generation sequencing of all glioblastomas was performed for diagnostic purposes and included mutational status of the genes ATRX, BRAF, CIC, FUBP1, H3F3A, IDH1, IDH2, PIK3CA, PTEN and TP53 and amplification/gain or deletion of BRAF, CDKN2A, EGFR, NOTCH1 and PTEN. The primary outcome was VTE within three months before glioblastoma diagnosis until two years after. Cumulative incidences were determined using competing risk analysis adjusting for mortality. Univariable Cox regression analysis was performed to determine hazard ratios. Results: From 324 patients with glioblastoma, 25 were diagnosed with VTE. Patients with a CDKN2A deletion had a 12-month adjusted cumulative incidence of VTE of 12.5 % (95%CI: 7.3–19.3) compared with 5.4 % (95%CI: 2.6–9.6) in patients with CDKN2A wildtype (p = 0.020), corresponding to a HR of 2.53 (95%CI: 1.12–5.73, p = 0.026). No significant associations were found between any of the other investigated genes and VTE. Conclusion: This study suggests a potential role for CDKN2A deletion in glioblastoma-related VTE. Therefore, once independently validated, CDKN2A mutational status may be a promising predictor to identify glioblastoma patients at high risk for VTE, who may benefit from thromboprophylaxis.

Tissue Factor (TF) is the initiator of blood coagulation but also functions as a signal transduction receptor. TF expression in breast cancer is associated with higher tumor grade, metastasis and poor survival. The role of TF signaling on the early phases of metastasis has never been addressed. Here, we show an association between TF expression and metastasis as well as cancer stemness in 574 breast cancer patients. In preclinical models, blockade of TF signaling inhibited metastasis tenfold independent of primary tumor growth. TF blockade caused a reduction in epithelial-to-mesenchymal-transition, cancer stemness and expression of the pro-metastatic markers Slug and SOX9 in several breast cancer cell lines and in ex vivo cultured tumor cells. Mechanistically, TF forms a complex with beta 1-integrin leading to inactivation of beta 1-integrin. Inhibition of TF signaling induces a shift in TF-binding from alpha 3 beta 1-integrin to alpha 6 beta 4 and dictates FAK recruitment, leading to reduced epithelial-to-mesenchymal-transition and tumor cell differentiation. In conclusion, TF signaling inhibition leads to reduced pro-metastatic transcriptional programs, and a subsequent integrin beta 1 and beta 4-dependent reduction in metastasic dissemination.

It is unknown how lower-leg injury and knee arthroscopy, both associated with venous thromboembolism (VTE), affect coagulation. To study the effect of (1) lower-leg trauma and (2) knee arthroscopy on coagulation, plasma samples of the Prevention of Thrombosis following CAST immobilization (POT-CAST, #NCT01542762) and Prevention of Thrombosis following Knee Arthroscopy (POT-KAST, #NCT01542723) trials were used, which were collected shortly after lower-leg trauma and before/after (

Background Colorectal cancer patients have an increased risk of developing venous thromboembolism (VTE), resulting in increased morbidity and mortality. Because the exact mechanism is yet unknown, risk prediction is still challenging; therefore, new biomarkers are needed. MicroRNAs (miRNAs) are small, relatively stable RNAs, that regulate a variety of cellular processes, and are easily measured in body fluids. Objective The aim of this study was to identify novel tumor-expressed miRNAs associated with VTE. Methods In a cohort of 418 colorectal cancer patients diagnosed between 2001 and 2015 at the Leiden University Medical Center, 23 patients (5.5%) developed VTE 1 year before or after cancer diagnosis. Based on availability of frozen tumor material, tumor cells of 17 patients with VTE and 18 patients without VTE were isolated using laser capture microdissection and subsequently analyzed on the Illumina sequencing platform NovaSeq600 using 150-bp paired-end sequencing. Cases and controls were matched on age, sex, tumor stage, and grade. Differential miRNA expression was analyzed using edgeR. Results A total of 547 miRNAs were detected. Applying a 1.5-fold difference and false discovery rate of

Cancer patients have a four- to sevenfold increased risk of developing cancer-associated thrombosis (CAT), which is associated with a strong increase in morbidity and mortality. Not all cancer patients receive thromboprophylaxis as this may lead to adverse events in a cancer population that is already at increased risk for major bleedings. Different risk prediction models have been developed to identify cancer patients at high risk of developing CAT that may be selected for thromboprophylaxis. However, risk models using the currently established biomarkers and clinical parameters perform poorly, particularly when validated in independent cohorts. Discovery of new and better biomarkers are therefore urgently needed. This review describes how aberrations in the genetic profile of the tumor and host influence a hypercoagulable state, and explores how these can be used as novel biomarker to improve CAT risk prediction.

Glioblastoma (GBM) is a devastating cancer of the brain with an extremely poor prognosis. For this reason, besides clinical and preclinical studies, novel in vitro models for the assessment of cancer response to drugs and radiation are being developed. In such context, three-dimensional (3D)engineered cellular microenvironments, compared to unrealistic two-dimensional (2D) monolayer cell culture, provide a model closer to the in vivo configuration. Concerning cancer treatment, while X-ray radiotherapy and chemotherapy remain the current standard, proton beam therapy is an appealing alternative as protons can be efficiently targeted to destroy cancer cells while sparing the surrounding healthy tissue. However, despite the treatment's compelling biological and medical rationale, little is known about the effects of protons on GBM at the cellular level. In this work, we designed novel 3D-engineered scaffolds inspired by the geometry of brain blood vessels, which cover a vital role in the colonization mechanisms of GBM cells. The architectures were fabricated by two-photon polymerization (2PP), cultured with U-251 GBM cells and integrated for the first time in the context of proton radiation experiments to assess their response to treatment. We employed Gamma H2A.X as a fluorescent biomarker to identify the DNA damage induced in the cells by proton beams. The results show a higher DNA doublestrand breakage in 2D cell monolayers as compared to cells cultured in 3D. The discrepancy in terms of proton radiation response could indicate a difference in the radioresistance of the GBM cells or in the rate of repair kinetics between 2D cell monolayers and 3D cell networks. Thus, these biomimetic-engineered 3D scaffolds pave the way for the realization of a benchmark tool that can be used to routinely assess the effects of proton therapy on 3D GBM cell networks and other types of cancer cells. KEYWORDS: engineered cell microenvironments, two-photon polymerization, cancer, glioblastoma, proton therapy, DNA damage

Renal cell carcinoma (RCC) can be complicated by a venous tumor thrombus (TT), of which the optimal management is unknown.This study sought to assess the prevalence of TT in RCC, its current management, and its association with venous thromboembolism (VTE), arterial thromboembolism (ATE), major bleeding (MB), and mortality.Patients diagnosed with RCC between 2010 and 2019 in our hospital were included and followed from RCC diagnosis until 2 years after, or until an outcome of interest (VTE, ATE, and MB) or death occurred, depending on the analysis. Cumulative incidences were estimated with death as a competing risk. Cause-specific hazard models were used to identify predictors and the prognostic impact.Of the 647 patients, 86 had a TT (prevalence 13.3%) at RCC diagnosis, of which 34 were limited to the renal vein, 37 were limited to the inferior vena cava below the diaphragm, and 15 extended above the diaphragm; 20 patients started therapeutic anticoagulation and 45 underwent thrombectomy with/without anticoagulation. During follow-up (median 24.0 [IQR: 7.0-24.0] months), 17 TT patients developed a VTE, 0 developed an ATE, and 11 developed MB. TT patients were more often diagnosed with VTE (adjusted HR: 6.61; 95% CI: 3.18-13.73) than non-TT patients, with increasing VTE risks in more proximal TT levels. TT patients receiving anticoagulation still developed VTE (HR: 0.56; 95% CI: 0.13-2.48), at the cost of more MB events (HR: 3.44; 95% CI: 0.95-12.42) compared with those without anticoagulation.Patients with RCC-associated TT were at high risk of developing VTE. Future studies should establish which of these patients benefit from anticoagulation therapy.

Background Atrial fibrillation (AF) can lead to the loss of microvascular integrity thereby enhancing AF progression. Mechanistically, the pro-coagulant state that drives the risk of stroke in patients with AF may also play a causal role in microvascular loss. Direct oral anticoagulants (DOACs), the preferred anticoagulants for AF, can target factors upstream (factor Xa [FXa]) or downstream (thrombin) in the coagulation cascade and mediate differential vascular effects through interaction with protease-activated receptors (PARs). Objective To investigate the potential effect of different DOACs on vascular integrity. Methods To model the impact of DOACs on vascular integrity, we utilized platelet-free plasma in thrombin generation assays and endothelial barrier assays under identical experimental conditions. These multifactorial systems provide all coagulation factors and their respective natural inhibitors in physiological ratios in combination with the pro-coagulant endothelial surface on which coagulation is initiated. Furthermore, the system provides pro- and anti-barrier factors and monitoring both assays simultaneously permits coupling of thrombin kinetics to endothelial barrier dynamics. Results We provide evidence that the anti-FXa DOAC rivaroxaban and the anti-thrombin DOAC dabigatran are efficient in blocking their target proteases. However, while rivaroxaban could preserve endothelial barrier function, dabigatran failed to protect endothelial integrity over time, which could be prevented in the presence of a custom-made peptide that blocks thrombin's exosite-I. Conclusions Proteolytically inactive thrombin in complex with dabigatran evokes loss of barrier function that can be prevented by a protease-activated receptor-1 mimicking peptide blocking thrombin's exosite-I.

Introduction Pancreatic cancer is associated with a high risk of venous thromboembolism (VTE). However, comprehensive data on incidence, timing and relevant determinants of VTE in this particular population are scarce. Current study assesses incidence, timing and predictors of VTE in pancreatic cancer through different phases of disease. Methods All pancreatic cancer patients treated in our tertiary referral center between 2013 through 2017 were studied. Occurrence of VTE was evaluated from diagnosis through end of follow-up or death. Relevant determinants of VTE were identified in logistic regression models. Hazard ratios were calculated to evaluate impact of VTE on overall survival. Results In total, 361 patients were followed for a median period of 43 months; 64 were diagnosed with VTE (18%). Most were tumor related thrombosis (59%), incidental (75%) and occurred after anti-cancer treatment had been stopped (80%), only 1.6% occurred during remission phase. Stage IV pancreatic cancer was a predictor for VTE (hazard ratio (HR) 2.46, 95% confidence interval (CI) 0.9–6.8). Biliary drainage (HR 0.52, 95%CI 0.28–0.98) and tumor resection (HR 0.45, 95%CI 0.45–1.83) were protective factors. VTE was not associated with worse survival (HR 1.3; 95% CI 0.97–1.74). Conclusions VTE in pancreatic cancer is disease-stage dependent, with 80% occurring in advanced phases of disease when patients no longer receive active treatment. We speculate that this is the main reason for the absence of a survival effect of VTE in our cohort. These practice-based findings should be taken into account when considering wide-spread introduction of primary thromboprophylaxis in patients with pancreatic cancer.

Simple Summary The relationship between cancer and blood clotting has been well established. The activation of blood coagulation proteins regulates the fate of cells and is known to be used by cancer cells to enhance survival and proliferation. Cells strictly regulate the initiation of coagulation through controlling the action of the protein “tissue factor (TF)”. In addition to initiating clotting, TF also acts as a deciding factor to determine the extent of damage and instructs cells to proliferate and repair or, when severely damaged, to die. Therefore, normal cells keep TF in a dormant state, achieved through mechanisms called “TF encryption”. Understanding the mechanisms by which the cells control the activity of TF is crucial, especially since cancer cells bypass these regulatory mechanisms, ensuring survival and tumour growth. This study has elucidated essential molecular mechanisms by which cells regulate TF clotting activity, and also the cellular signals arising from these. Abstract In this study, the role of de-palmitoylation of tissue factor (TF) in the decryption of its activity was explored. TF-tGFP constructs were prepared by mutagenesis-substitution at Cys245 to prevent or mimic palmitolyation. Additionally, to reduce TF de-palmitoylation, the expression of palmitoyl-protein thioesterases (PPT) was suppressed. Other TF mutants were prepared with altered flexibility, hydrophobicity or length of the transmembrane domain. The outcome of these alterations on fXa-generation, fVIIa binding, Ser253 phosphorylation and TF-microvesicle release were assessed in endothelial cells, and the influence on endothelial and MCF-7 cell proliferation and apoptosis was analysed. Preventing TF palmitoylation (TFSer245-tGFP), increasing the hydrophobicity (TFPhe241-tGFP) or lengthening (TFLongTM-tGFP) of the transmembrane domain enhanced fXa-generation in resting cells compared to cells expressing TFWt-tGFP, but fXa-generation was not further increased following PAR2 activation. Extending the available length of the transmembrane domain enhanced the TF-tGFP release within microvesicles and Ser253 phosphorylation and increased cell proliferation. Moreover, prevention of PKCα-mediated Ser253 phosphorylation with Gö6976 did not preclude fXa-generation. Conversely, reducing the hydrophobicity (TFSer242-tGFP), shortening (TFShortTM-tGFP) or reducing the flexibility (TFVal225-tGFP) of the transmembrane domain suppressed fXa-generation, fVIIa-HRP binding and Ser253 phosphorylation following PAR2 activation. PPT knock-down or mimicking palmitoylation (TFPhe245-tGFP) reduced fXa-generation without affecting fVIIa binding. This study has for the first time shown that TF procoagulant activity is regulated through de-palmitoylation, which alters the orientation of its transmembrane domain and is independent of TF phosphorylation. However, Ser253 phosphorylation is facilitated by changes in the orientation of the transmembrane domain and can induce TF-cellular signalling that influences cellular proliferation/apoptosis.

In 2021, pancreatic ductal adenocarcinoma (PDAC) is the 3rd leading cause of cancer deaths in the United States. This is largely due to a lack of symptoms and limited treatment options, which extend survival by only a few weeks. There is thus an urgent need to develop new therapies effective against PDAC. Previously, we have shown that the growth of PDAC cells is suppressed when they are co-implanted with RabMab1, a rabbit monoclonal antibody specific for human alternatively spliced tissue factor (asTF). Here, we report on humanization of RabMab1, evaluation of its binding characteristics, and assessment of its in vivo properties. hRabMab1 binds asTF with a KD in the picomolar range; suppresses the migration of high-grade Pt45.P1 cells in Boyden chamber assays; has a long half-life in circulation (~ 5 weeks); and significantly slows the growth of pre-formed orthotopic Pt45.P1 tumors in athymic nude mice when administered intravenously. Immunohistochemical analysis of tumor tissue demonstrates the suppression of i) PDAC cell proliferation, ii) macrophage infiltration, and iii) neovascularization, whereas RNAseq analysis of tumor tissue reveals the suppression of pathways that promote cell division and focal adhesion. This is the first proof-of-concept study whereby a novel biologic targeting asTF has been investigated as a systemically administered single agent, with encouraging results. Given that hRabMab1 has a favorable PK profile and is able to suppress the growth of human PDAC cells in vivo, it comprises a promising candidate for further clinical development.

The coagulation protein tissue factor (TF) regulates inflammation and angiogenesis via its cytoplasmic domain in infection, cancer and diabetes. While TF is highly abundant in the heart and is implicated in cardiac pathology, the contribution of its cytoplasmic domain to post-infarct myocardial injury and adverse left ventricular (LV) remodeling remains unknown.Methods: Myocardial infarction was induced in wild-type mice or mice lacking the TF cytoplasmic domain (TF increment CT) by occlusion of the left anterior descending coronary artery. Heart function was monitored with echocardiography. Heart tissue was collected at different time-points for histological, molecular and flow cytometry analysis.Results: Compared with wild-type mice, TF increment CT had a higher survival rate during a 28-day follow-up after myocardial infarction. Among surviving mice, TF increment CT mice had better cardiac function and less LV remodeling than wild-type mice. The overall improvement of post-infarct cardiac performance in TF increment CT mice, as revealed by speckle-tracking strain analysis, was attributed to reduced myocardial deformation in the peri-infarct region. Histological analysis demonstrated that TF increment CT hearts had in the infarct area greater proliferation of myofibroblasts and better scar formation. Compared with wild-type hearts, infarcted TF increment CT hearts showed less infiltration of proinflammatory cells with concomitant lower expression of protease-activated receptor-1 (PAR1) -Rac1 axis. In particular, infarcted TF increment CT hearts displayed markedly lower ratios of inflammatory M1 macrophages and reparative M2 macrophages (M1/M2). In vitro experiment with primary macrophages demonstrated that deletion of the TF cytoplasmic domain inhibited macrophage polarization toward the M1 phenotype. Furthermore, infarcted TF increment CT hearts presented markedly higher peri-infarct vessel density associated with enhanced endothelial cell proliferation and higher expression of PAR2 and PAR2-associated pro-angiogenic pathway factors. Finally, the overall cardioprotective effects observed in TF increment CT mice could be abolished by subcutaneously infusing a cocktail of PAR1-activating peptide and PAR2-inhibiting peptide via osmotic minipumps.Conclusions: Our findings demonstrate that the TF cytoplasmic domain exacerbates post-infarct cardiac injury and adverse LV remodeling via differential regulation of inflammation and angiogenesis. Targeted inhibition of the TF cytoplasmic domain-mediated intracellular signaling may ameliorate post-infarct LV remodeling without perturbing coagulation.

Background Genome wide association studies (GWAS) identified SLC44A2 as a novel susceptibility gene for venous thrombosis (VT) and previous work established that SLC44A2 contributed to clot formation upon vascular injury.Objective To further investigate the role of SLC44A2 in VT by utilizing SLC44A2 deficient mice (Slc44a2(-)(/)(-)) in two representative disease models.Methods Mice were included in a hypercoagulability model driven by siRNA-mediated hepatic gene silencing of anticoagulants Serpinc1 (antithrombin) and Proc (protein C) and a flow restriction (stenosis) model induced by partial ligation of the inferior vena cava.Results In the hypercoagulability model, no effect in onset was observed in Slc44a2(-)(/)(-) animals; however, a drop in plasma fibrinogen and von Willebrand factor coinciding with an increase in blood neutrophils was recorded. In the neutrophil dependent stenosis model after 48 hours, Slc44a2(-)(/)(-) mice had significantly smaller thrombi both in length and weight with less platelet accumulation as a percentage of the total thrombus area. During the initiation of thrombosis at 6 hours post-stenosis, Slc44a2(-)(/)(-) mice also had smaller thrombi both in length and weight, with circulating platelets remaining elevated in Slc44a2(-)(/)(-) animals. Platelet activation and aggregation under both static- and venous and arterial shear conditions were normal for blood from Slc44a2(-)(/)(-) mice.Conclusions These studies corroborate the original GWAS findings and establish a contributing role for SLC44A2 during the initiation of VT, with indications that this may be related to platelet-neutrophil interaction. The precise mechanism however remains elusive and warrants further investigation.

Item does not contain fulltext An increasing body of evidence suggests an association between cancer and atrial fibrillation (AF). The exact magnitude and underlying mechanism of this association are however unclear. Cancer-related inflammation, anti-cancer treatment and other cancer-related comorbidities are proposed to affect atrial remodelling, increasing the susceptibility of cancer patients for developing AF. Moreover, cancer is assumed to modify the risk of thromboembolisms and bleeding. A thorough and adequate understanding of these risks is however lacking, as current literature is scarce and show ambiguous results in AF patients. The standardized risk-models that normally aid the clinician in the decision of initiating anticoagulant therapy do not take the presence of malignancy into account. Other factors that complicate risk assessment in AF patients with cancer include drug-drug interactions and other cancer-related comorbidities such as renal impairment. In this review, we highlight the available literature regarding epidemiological association, risk assessment and anticoagulation therapy in AF patients with cancer.

BACKGROUND Patients with glioblastoma have a high risk of developing venous thromboembolism (VTE). However, the underlying genetic risk factors remain largely unknown. Therefore, the aim of our study was to discover whether genetic aberrations in glioblastoma associate with VTE. MATERIAL AND METHODS In this retrospective cohort study, all patients diagnosed with glioblastoma between February 2017 and August 2020 in two hospitals (Leiden University Medical Center and Haaglanden Medical Center) were included (n=341). Targeted DNA next-generation sequencing (NGS) had been performed of all glioblastomas for diagnostic purposes and included tumor mutational status of the genes ATRX, BRAF, CIC, FUBP1, H3F3A, IDH1, IDH2, PIK3CA, PTEN and TP53 and amplification/gain or deletion of BRAF, CDKN2A, EGFR, NOTCH1 and PTEN. Through extensive chart review, we collected data on VTE events (deep vein thrombosis and/or pulmonary embolism) three months before until two years after glioblastoma diagnosis, which were adjudicated by an independent investigator. Patients with cerebral vein thrombosis (n=3) and low quality NGS data (n=1; minimum reads/samples: RESULTS Of the 337 patients, 215 had died, 26 were diagnosed with VTE and 37 were lost to follow-up. CDKN2A deletion was found to associate most significantly with VTE (HR: 2.65, 95%CI: 1.18–5.94, p=0.018). Competing risk analysis confirmed this finding, demonstrating a 12-month adjusted cumulative incidence of 12.7% (95%CI: 7.5–19.3) compared to 5.2% (95%CI: 2.5–9.2) in patients with CDKN2A wild-type (p=0.013). No significant association was found between any of the investigated genes, including CDKN2A deletion, and death. CONCLUSION This study demonstrates that CDKN2A deletion is associated with VTE in glioblastoma patients. Therefore, CDKN2A mutational status may be a promising predictor to identify patients with glioblastoma at high risk of VTE, who may benefit from thromboprophylaxis.

Metastasis results from a complex set of traits acquired by tumor cells, distinct from those necessary for tumorigenesis. Here, we investigate the contribution of enhancer elements to the metastatic phenotype of osteosarcoma. Through epigenomic profiling, we identify substantial differences in enhancer activity between primary and metastatic human tumors and between near isogenic pairs of highly lung metastatic and nonmetastatic osteosarcoma cell lines. We term these regions metastatic variant enhancer loci (Met-VELs). Met-VELs drive coordinated waves of gene expression during metastatic colonization of the lung. Met-VELs cluster nonrandomly in the genome, indicating that activity of these enhancers and expression of their associated gene targets are positively selected. As evidence of this causal association, osteosarcoma lung metastasis is inhibited by global interruptions of Met-VEL-associated gene expression via pharmacologic BET inhibition, by knockdown of AP-1 transcription factors that occupy Met-VELs, and by knockdown or functional inhibition of individual genes activated by Met-VELs, such as that encoding coagulation factor III/tissue factor (F3). We further show that genetic deletion of a single Met-VEL at the F3 locus blocks metastatic cell outgrowth in the lung. These findings indicate that Met-VELs and the genes they regulate play a functional role in metastasis and may be suitable targets for antimetastatic therapies.

Introduction: Recent genome wide association studies (GWAS) identified a novel susceptibility locus for thrombosis, harbouring the SLC44A2 gene which encodes the Solute Carrier Family 44 Member 2 protein (SLC44A2). Thus far, SLC44A2 has not been studied in the context of thrombosis, and may be a unique contributor to thrombotic disease. Here we utilize mice lacking SLC44A2 (Slc44a2(-/-)) to evaluate a possible role of SLC44A2 in hemostasis.Methods: Slc44a2(-/-) mice were evaluated in key aspects of normal hemostasis including a challenge of vascular damage by applying laser induced injury to the cremaster muscle arteriole.Results: Slc44a2(-/-) mice had comparable levels of thrombin generation and gene expression of coagulation related genes, as compared to littermate wild type controls. Lower levels of circulating plasma Von Willebrand factor (VWF) were measured in Slc44a2(-/-) mice, while no difference in VWF multimerization or vascular localization was detected. Upon in vivo laser injury of the cremaster arterioles, we detected an impairment of clot formation for Slc44a2(-/-) mice.Conclusions: Although mice lacking SLC44A2 are normal for several hemostasis parameters, we do observe a reduction of plasma VWF levels and an altered response upon vascular damage, which suggests that SLC44A2 contributes to hemostasis upon injury. These findings are in line with the reported GWAS data and support further research on SLC44A2 in thrombosis.

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.

The plasma compartment of the blood holds important information on the risk to develop cardiovascular diseases such as venous thrombosis (VT). Mass spectrometry-based targeted proteomics with internal standards quantifies proteins in multiplex allowing generation of signatures associated with a disease or a condition. Here, to demonstrate the method, we investigate the plasma protein signatures in mice following the onset of VT, which was induced by RNA interference targeting the natural anticoagulants antithrombin and protein C. We then study mice lacking Slc44a2, which was recently characterized as a VT-susceptibility gene in human genome-wide association studies. We use a recently developed panel of 375 multiplexed mouse protein assays measured by mass spectrometry. A strong plasma protein siganture was observed when VT was induced. Discriminators included acute phase response proteins, and proteins related to erythrocyte function. In mice lacking Slc44a2, protein signature was primarily overruled by the difference between sexes and not by the absent gene. Upon separate analyses for males and females, we were able to establish a signature for Slc44a2 deficiency, in which glycosylation-dependent cell adhesion molecule-1 and thrombospondin-1 were shared by both sexes. The minimal impact of Slc44a2 deficiency on the measured plasma proteins suggests that the main effect of Slc44a2 on VT does not lay ultimately in the plasma compartment. This suggests further investigation into the role of this VT-susceptibility gene should perhaps also question the possible involvement in cellular mechanisms.

Background Direct oral anticoagulants (DOACs) are now the first choice thromboprophylaxis in cancer patients who do not have a high risk of bleeding. In addition to the anticoagulant effects, potential anti-tumor effects of DOACs have also been studied in animal cancer models. In this study, we summarize the effects of DOACs on cancer growth and metastasis in animal models through a systematic review with a qualitative analysis. Methods PubMed, EMBASE and Web of Science were systematically searched for original studies that describe animal models of cancer in which one of the experimental groups received DOAC monotherapy, and which reported quantitatively on primary tumor or metastases. Results Nine studies - reporting a total of 19 animal experiments - met the inclusion criteria. These 19 experiments included spontaneous cancer (n = 2), carcinogenicity (n = 2), xenograft (n = 7) and syngeneic (n = 8) models, encompassing orthotopic (n = 7), subcutaneous (n = 5), intraperitoneal (n = 1) and intravenous (n = 2) injection of cancer cells and included treatments with the DOACs ximelagatran (n = 4), dabigatran etexilate (n = 6) and/or rivaroxaban (n = 11). DOAC treatment decreased tumor growth at implanted and metastatic site in 18.8% (3/16) and 20.0% (3/15) of the experiments, respectively. Conversely, DOACs increased tumor growth at implanted and metastatic site in 6.3% (1/16) and 20.0% (3/15) of the experiments, respectively. Conclusion DOAC monotherapy resulted in neoplastic changes in a rat carcinogenicity study, showed a lack of effect in mouse xenograft models, while the effect on cancer growth and metastasis in mouse syngeneic models depended on the timing of DOAC treatment and type of cancer model used.

Background: Home treatment of cancer-associated venous thromboembolism (VTE) is challenging due to thehigh risk of adverse events. While home treatment is quite agreeable to cancer patients, studies evaluating thesafety of VTE home treatment in this setting are largely unavailable.Methods: This was an observational study in patients with cancer-associated VTE. The main outcomes were theproportion of patients treated at home (hospital discharge < 24 h after diagnosis) and the 3-month incidence ofVTE-related adverse events (major bleeding, recurrent VTE and/or suspected VTE-related mortality) in patientsmanaged in hospital versus at home.Results: A total of 183 outpatients were diagnosed with cancer-associated VTE: 69 had deep vein thrombosis(DVT) and 114 had pulmonary embolism (PE ± DVT). Of those, 120 (66%) were treated at home; this was 83%for patients with DVT and 55% for patients with PE ( ± DVT). The 3-month incidence of any VTE-related adverseevent was 13% in those treated at home versus 19% in the hospitalized patients (HR 0.48; 95%CI 0.22–1.1),independent of initial presentation as PE or DVT. All-cause 3-month mortality occurred in 33 patients treated asinpatient (54%) compared to 29 patients treated at home (24%; crude HR 3.1 95%CI 1.9–5.0).Conclusions: Two-third of patients with cancer-associated VTE - including PE - were selected to start antic-oagulant treatment at home. Cancer-associated VTE is associated with high rates of VTE-related adverse eventsindependent of initial in hospital or home treatment. However, home treatment may be a good option forselected patients with cancer-associated DVT or PE.

Background The intestinal microbiome plays a versatile role in the etiology of arterial thrombosis. In venous thrombosis, driven chiefly by plasma coagulation, no such role has yet been established. We hypothesized that the intestinal microbiome composition affects coagulation in humans. Methods We used healthy donor fecal microbiota transplant (FMT) to experimentally change the microbiome composition in metabolic syndrome patients. Thirty‐five subjects were randomized in a blinded fashion to healthy donor FMT or autologous FMT as a control in a 2:1 ratio. We measured thrombin generation at baseline and after 6 weeks using automated calibrated thrombinography, and we determined plasma abundance of 32 coagulation related proteins using a targeted mass spectrometry‐based quantitative proteomics assay with heavy labeled internal standards. Results Healthy donor FMT prolonged the thrombinography lag time (median delta 0.0 versus 0.25 minutes, P = .039). The other thrombinography parameters showed no significant difference. Unsupervised cluster analysis suggested overall downregulation of coagulation related plasma proteins in subject clusters containing predominantly subjects that had a metabolic response to healthy donor FMT. FMT treatment status itself showed no clear clustering pattern with up‐ or downregulation, however, and proteins did not cluster according to an apparent biological grouping. Discussion A single healthy donor FMT tends to modestly suppress the onset thrombin generation in metabolic syndrome patients, representing initial proof‐of‐principle that the intestinal microbiota composition might affect the coagulation system in humans. The findings merit external validation as a role for intestinal microbiota in coagulation can have clinically important implications.

Tissue Factor (TF) is the initiator of blood coagulation but also functions as a signal transduction receptor. TF expression in breast cancer is associated with higher tumor grade, metastasis and poor survival. The role of TF signaling on the early phases of metastasis has never been addressed. Here, we show an association between TF expression and metastasis as well as cancer stemness in 574 breast cancer patients. In preclinical models, blockade of TF signaling inhibited metastasis tenfold independent of primary tumor growth. TF blockade caused a reduction in epithelial-to-mesenchymal-transition, cancer stemness and expression of the pro-metastatic markers Slug and SOX9 in several breast cancer cell lines and in ex vivo cultured tumor cells. Mechanistically, TF forms a complex with β1-integrin leading to inactivation of β1-integrin. Inhibition of TF signaling induces a shift in TF-binding from α3β1-integrin to α6β4 and dictates FAK recruitment, leading to reduced epithelial-to-mesenchymal-transition and tumor cell differentiation. In conclusion, TF signaling inhibition leads to reduced pro-metastatic transcriptional programs, and a subsequent integrin β1 and β4-dependent reduction in metastasic dissemination.

Essentials Factor Xa (FXa)-targeting direct oral anticoagulants (DOACs) reduce venous thromboembolism (VTE) The effects of FXa-targeting DOACs on cancer progression remain to be studied In xenograft models, a FXa-targeting DOAC did not inhibit breast cancer growth and metastasis A thrombin-targeting DOAC, dabigatran, also did not inhibit breast cancer growth and metastasis ABSTRACT: Background Factor Xa-targeting DOACs were recently found to reduce recurrent VTE efficiently in cancer patients when compared to the standard treatment with low-molecular-weight heparins (LMWHs). While the anticancer effects of LMWHs have been extensively studied in preclinical cancer models, the effects of FXa-targeting DOACs on cancer progression remain to be studied. Objective We investigated whether the FXa-targeting DOAC rivaroxaban and the thrombin-targeting DOAC dabigatran etexilate (DE) affected human breast cancer growth and metastasis in orthotopic xenograft models. Methods/results Mice that were put on a custom-made chow diet supplemented with rivaroxaban (0.4 or 1.0 mg/g diet) or dabigatran etexilate (DE) (10 mg/g diet) showed prolonged ex vivo coagulation times (prothrombin time [PT] and activated partial thromboplastin time [aPTT] assay, respectively). However, rivaroxaban and DE did not inhibit MDA-MB-231 tumor growth and metastasis formation in lungs or livers of 7-week-old fully immunodeficient NOD/SCID/ƴC -/- (NSG) mice. Comparable data were obtained for rivaroxaban-treated mice when using NOD-SCID mice. Rivaroxaban and DE treatment also did not significantly inhibit tumor growth and metastasis formation when using another human triple negative breast cancer (TNBC) cell line (HCC1806) in NOD-SCID mice. The FXa and thrombin-induced gene expression of the downstream target CXCL8 in both cell lines, but FXa and thrombin, did not significantly stimulate migration, proliferation, or stemness in vitro. Conclusion Although effectively inhibiting coagulation, the DOACs rivaroxaban and DE did not inhibit orthotopic growth and metastasis of human TNBC. It remains to be investigated whether DOACs exert antitumorigenic effects in other types of cancer.

It has been long-established that cancer and thrombosis are linked, but the exact underlying pathological mechanism remains to be unraveled. As the initiator of the coagulation cascade, the transmembrane glycoprotein tissue factor (TF) has been intensely investigated for its role in cancer-associated thrombosis and cancer progression. TF expression is regulated by both specific oncogenes and environmental factors, and it is shown to regulate primary growth and metastasis formation in a variety of cancer models. In clinical studies, TF has been shown to be overexpressed in most cancer types and is strongly associated with disease progression. While TF clearly associates with cancer progression, a prominent role for TF in the development of cancer-associated thrombosis is less clear. The current concept is that cancer-associated thrombosis is associated with the secretion of tumor-derived TF-positive extracellular vesicles in certain tumor types. To date, many therapeutic strategies to target TF—both in preclinical and clinical phase—are being pursued, including targeting TF or the TF:FVIIa complex by itself or by exploiting TF as a docking molecule to deliver cytotoxic compounds to the tumor. In this review, the authors summarize the current understanding of the role of TF in both cancer progression and cancer-associated thrombosis, and discuss novel insights on TF as a therapeutic target as well as a biomarker for cancer progression and VTE.

Tissue factor, coagulation factor XII, platelets, and neutrophils are implicated as important players in the pathophysiology of (experimental) venous thrombosis (VT). Their role became evident in mouse models in which surgical handlings were required to provoke VT. Combined inhibition of the natural anticoagulants antithrombin (Serpinc1) and protein C (Proc) using small interfering RNA without additional triggers also results in a venous thrombotic phenotype in mice, most notably with vessel occlusion in large veins of the head. VT is fatal but is fully rescued by thrombin inhibition. In the present study, we used this VT mouse model to investigate the involvement of tissue factor, coagulation factor XII, platelets, and neutrophils. Antibody-mediated inhibition of tissue factor reduced the clinical features of VT, the coagulopathy in the head, and fibrin deposition in the liver. In contrast, genetic deficiency in, and small interfering RNA–mediated depletion of, coagulation factor XII did not alter VT onset, severity, or thrombus morphology. Antibody-mediated depletion of platelets fully abrogated coagulopathy in the head and liver fibrin deposition. Although neutrophils were abundant in thrombotic lesions, depletion of circulating Ly6G-positive neutrophils did not affect onset, severity, thrombus morphology, or liver fibrin deposition. In conclusion, VT after inhibition of antithrombin and protein C is dependent on the presence of tissue factor and platelets but not on coagulation factor XII and circulating neutrophils. This study shows that distinct procoagulant pathways operate in mouse VT, dependent on the triggering stimulus.

Tissue Factor, the initiator of the extrinsic pathway of coagulation, and its isoform alternatively spliced Tissue Factor (asTF) are often overexpressed in cancer cells. Previously, we have shown that RabMab1, a rabbit monoclonal antibody specific for human asTF, disrupts the binding of asTF to beta-integrins and thereby inhibits the growth of breast cancer and pancreatic ductal adenocarcinoma (PDAC) cell lines in vitro and in vivo. Here, we report on humanization of RabMab1, assessment of its binding characteristics, and determination of its in vivo properties. The variable regions of the heavy and light chains of hybridoma derived RabMab1 were cloned and used to generate a rabbit/human chimera (cRabMab1). Substitutions of species-specific residues were then carried out to produce humanized RabMab1 (hRabMab1; IgG1 isotype). Antigen binding was assessed via anti-human IgG Fc capture biosensor assays. cRabMab1 was found to have a KD of 4.24 nM whereas hRabMab1 was found to have a KD in the low picomolar range, which could not be precisely measured due to an extremely slow off-rate. The in vivo half-lives of each were determined to be 280 and 908 hours, respectively. Orthotopic co-implantation of cRabMab1 with high-grade, human PDAC cell line Pt45.P1 in athymic nude mice resulted in tumors that were 82% and 92% smaller than tumors in the vehicle and IgG control group groups, respectively, with no statistical difference between vehicle and IgG isotype control groups. Intravenous administration of hRabMab1 slowed the growth of pre-formed orthotopic Pt45.P1 tumors in athymic nude mice by 64% and 61% compared to vehicle and isotype control groups, respectively; again, there was no difference between vehicle and IgG isotype control groups. Immunohistochemical analysis of tumor tissue revealed a statistically significant 68% reduction in neovascularization (CD31), a 58% reduction in M2-polarized macrophages (CD206), and a 24% reduction in proliferating cells (Ki67) in the hRabMab1 cohort. RNAseq analysis of tumor tissue revealed that components of the focal adhesion system were the most affected by hRabMab1 treatment. In addition, a significant suppression of pathways that promote mitosis and the cell cycle was seen in hRabMab1-treated tumors. By qRT-PCR, we validated a greater than 82% reduction in the expression of HBEGF, a gene encoding a cell surface mitogen, and a greater than 68% reduction in the expression of STN1, a gene encoding a component of the CST-complex, a telomere replication and maintenance structure. This is the first proof-of-concept study whereby a novel biologic that inhibits asTF has been used as a systemically administered single agent, with encouraging results. Because hRabMab1 has a favorable pharmacokinetic profile and is able to suppress PDAC tumor cell growth in vivo, it is an attractive candidate for further clinical development. Citation Format: Clayton S. Lewis, Aniruddha Karve, Kateryna Matiash, Timothy Stone, Jingxing Li, Jordan Wang, Henri Versteeg, Bruce Aronow, Syed Ahmad, Pankaj Desai, Vladimir Bogdanov. Preclinical in vivo characterization of a first-in-class humanized antibody targeting alternatively spliced tissue factor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 908.

Venous thromboembolism (VTE) and cancer are strongly associated, and present a major challenge in cancer patient treatment. Cancer patients have a higher risk of developing VTE, although the risk differs widely between tumour types. VTE prophylaxis is routinely given to cancer patients, in the form of vitamin K antagonists (VKA) or low molecular weight heparin (LMWH). Several studies have reported that cancer patients receiving anticoagulants show prolonged survival and this effect was more pronounced in patients with a good prognosis, although the mechanism is poorly understood. Tissue Factor (TF) is the initiator of extrinsic coagulation, but its non-haemostatic signalling via protease-activated receptors (PARs) is a potent driver of tumour angiogenesis. Furthermore, coagulation activation is strongly implicated in tumour cell migration and metastasis. This review discusses the effects of anticoagulants on cancer progression in patients, tumour cell behaviour, angiogenesis, and metastasis in in vitro and in vivo models. Inhibition of TF signalling shows great promise in curbing angiogenesis and in vivo tumour growth, but whether this translates to patients is not yet known. Furthermore, non-haemostatic properties of coagulation factors in cancer progression are discussed, which provide exciting opportunities on limiting oncologic processes without affecting blood coagulation.

Essentials Tissue factor (TF) isoforms are expressed in pancreatic neuroendocrine tumors (pNET). TF knockdown inhibits proliferation of human pNET cells in vitro. mTOR kinase inhibitor sapanisertib/MLN0128 suppresses TF expression in human pNET cells. Sapanisertib suppresses TF expression and activity and reduces the growth of pNET tumors in vivo. SUMMARY: Background Full-length tissue factor (flTF) and alternatively spliced TF (asTF) contribute to growth and spread of pancreatic ductal adenocarcinoma. It is unknown, however, if flTF and/or asTF contribute to the pathobiology of pancreatic neuroendocrine tumors (pNETs). Objective To assess TF expression in pNETs and the effects of mTOR complex 1/2 (mTORC1/2) inhibition on pNET growth. Methods Human pNET specimens were immunostained for TF. Human pNET cell lines QGP1 and BON were evaluated for TF expression and responsiveness to mTOR inhibition. shRNA were used to knock down TF in BON. TF cofactor activity was assessed using a two-step FXa generation assay. TF promoter activity was assessed using transient transfection of human TF promoter-driven reporter constructs into cells. Mice bearing orthotopic BON tumors were treated with the mTORC1/2 ATP site competitive inhibitor sapanisertib/MLN0128 (3 mg kg-1 , oral gavage) for 34 days. Results Immunostaining of pNET tissue revealed flTF and asTF expression. BON and QGP1 expressed both TF isoforms, with BON exhibiting higher levels. shRNA directed against TF suppressed BON proliferation in vitro. Treatment of BON with sapanisertib inhibited mTOR signaling and suppressed TF levels. BON tumors grown in mice treated with sapanisertib had significantly less TF protein and cofactor activity, and were smaller compared with tumors grown in control mice. Conclusions TF isoforms are expressed in pNETs. Sapanisertib suppresses TF mRNA and protein expression as well as TF cofactor activity in vitro and in vivo. Thus, further studies are warranted to evaluate the clinical utility of TF-suppressing mTORC1/2 inhibitor sapanisertib in pNET management.