Abstract P5-06-09: Rivaroxaban targets the procoagulant tumour microenvironment in vitro and thereby inhibits breast cancer progression
Authors: Blower Emma L , Castle John , Santiago-Gomez Angelica , Clarke Robert , Kirwan Cliona C 
Published: March 2022

Abstract
Introduction: Breast cancer patients have a four-fold increased risk of developing a venous thromboembolism and those that do have a significantly increased risk of mortality despite adjusting for cancer stage. Tissue Factor (TF) is expressed by breast cancer-associated fibroblasts as well as breast cancer epithelial cells, and at significantly higher levels than by normal breast fibroblasts. TF signals via PAR-1 and PAR-2 to induce proliferation, invasion, angiogenesis and metastasis. Rivaroxaban is a licensed oral anticoagulant that inhibits the TF-Factor VIIa-Factor Xa complex and could therefore be repurposed to target the procoagulant tumour microenvironment in breast cancer. We hypothesise that a procoagulant microenvironment will induce breast cancer progression in vitro and that these promoting effects will be inhibited by anticoagulants, including Rivaroxaban. Methods: Lentivirally transduced TF over-expressing fibroblasts (TFF) and their control (CF) or conditioned media (TFFCM and CFCM), were cultured with oestrogen receptor positive (MCF-7), triple negative (MDA-MB-231) and HER2 positive (BT474) breast cancer cells, in the presence or absence of Rivaroxaban or anti-TF antibody 10H10. Proliferation (sulforhodamine-B/EdU assay), migration (scratch/transwell assay) and stem cell activity (mammosphere forming efficiency (MFE) assay) were assessed. The underlying mechanism was analysed with western blotting and quantitative PCR. The TFFCM and CFCM were analysed with cytokine arrays, enzyme-linked immunosorbent assays (ELISA) and mass spectrometry whilst the TFF and CF were analysed using RNA sequencing. Results: 3D co-culture of MCF-7s with TFF as compared to CF promoted cancer cell migration (p=0.04) and stem cell activity (MFE: p&lt;0.0001), with these effects abrogated by Rivaroxaban (migration: p= 0.0341, MFE: p=0.0003) and 10H10 (migration: p=0.01, MFE: p=0.0028). TFFCM promoted proliferation, migration and stem cell activity in MCF-7 cells (p&lt;0.05) compared to CFCM, with these effects abrogated by Rivaroxaban (migration, MFE: p&lt;0.05) and 10H10 (proliferation, migration, MFE: p&lt;0.05). The cancer-promoting effects of TFFCM in MCF-7 cells was associated with a decrease in CXCL8 (p&lt;0.0001) and an increase in VEGFA (p&lt;0.05) when compared to CFCM; changes which were reversed by Rivaroxaban (VEGFA: p&lt;0.03) and 10H10 (CXCL8, VEGFA: p&lt;0.05). TFFCM also promoted stem cell activity in both MDA-MB-231 (p&lt;0.006) and BT474 (p&lt;0.002) cell lines compared to CFCM. This increased MFE in BT474 was abrogated by Rivaroxaban (p=0.02) and 10H10 (p&lt;0.005) and associated with an increase in VEGFA (p&lt;0.001) and CXCL8 (p&lt;0.0001). Cytokine array and ELISA analysis of TFFCM versus CFCM indicated increased levels of IL-10 (p&lt;0.006), GRO-α (p&lt;0.0001) and IL1-α (p&lt;0.02). GRO-α and IL1-α production was inhibited by fibroblast treatment with Rivaroxaban (GRO-α, p&lt;0.0001 and IL1-α, p&lt;0.001) and 10H10 (p&lt;0.001). TFFCM versus CFCM had elevated levels of plasminogen activator inhibitor 1, integrin linked kinase, thrombospondin 4 and α -fetoprotein on mass spectrometry. RNA sequencing analysis of the TFF as compared to the CF revealed upregulation of AEBP1 (adipocyte enhancer binding protein 1) (p&lt;0.01), NKX2-5 (homeobox protein NKX-2.5) (p&lt;0.0001), TRIM58 (tripartite motif containing 58) (p&lt;0.0001) and KRT81 (keratin 81) (p&lt;0.0001), all of which were decreased by fibroblast treatment with Rivaroxaban (NKX2-5: p&lt;0.01, KRT81: p&lt;0.0001). Conclusion: A procoagulant microenvironment promotes proliferation, migration and stem cell activity in breast cancer in vitro, with these effects abrogated by Rivaroxaban. Targeting the procoagulant tumour microenvironment is a promising future cancer treatment.
Citation Format: Emma L Blower, John Castle, Angelica Santiago-Gomez, Robert Clarke, Cliona C Kirwan. Rivaroxaban targets the procoagulant tumour microenvironment in vitro and thereby inhibits breast cancer progression [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-06-09.