Investigation of the Procoagulant Tumour Microenvironment in Breast Cancer

Abstract

Background: Breast cancer patients have a four-fold increased risk of developing a venous thromboembolism (VTE) and those that do have a significantly increased risk of mortality despite adjusting for cancer stage. Tissue Factor (TF) is produced by tumour cells and cancer-associated fibroblasts within the tumour microenvironment. TF and its downstream signalling can induce invasion, angiogenesis and metastasis and therefore presents an innovative therapeutic target. Rivaroxaban, a licensed oral anticoagulant, inhibits this TF-Factor VIIa-Factor Xa complex. The aim of this thesis is to establish: i) if a procoagulant microenvironment, produced using exogenous clotting factors or TF fibroblasts promotes breast cancer cell line proliferation, migration, and stem cell activity and; ii) if these promoting effects are abrogated by inhibitors of the coagulation cascade. Methods: Recombinant exogenous coagulation factors, lentivirally transduced TF expressing fibroblasts (TFF) and their control (CF) or conditioned media (TF FCM and CFCM), were cultured with breast cancer cell lines +/- Rivaroxaban or anti-TF antibody 10H10. Proliferation (sulforhodamine-B/EdU assay), migration (scratch/transwell assay) and stem cell activity (mammosphere assay) were assessed. The mechanisms underlying these functional effects were investigated with RNA Sequencing, qRT-PCR, western blotting, cytokine arrays and mass spectrometry. TFF and CF were co-injected, in vivo NSG mice, with breast cancer cells in the presence/absence of Rivaroxaban. Tumour growth and ex vivo stem cell activity were analysed. Results: Exogenous recombinant TF:FVIIa:FXa promoted proliferation and migration in the oestrogen receptor positive (ER+) MCF-7 breast cancer line, and migration only in the triple negative MDA-MB-231 cell line, with effects abrogated by Rivaroxaban. Thrombin promoted proliferation in the ER+ HER2+ BT474 breast cancer cell line, and migration in both MDA-MB-231 and MCF-7 cell lines, with effects abrogated by Dabigatran. TF FCM promoted proliferation, migration and stem cell activity in MCF-7 cells as compared to CFCM. This was confirmed on 3D co-culture of MCF-7 cells with TFF, with an increase in migratory ability and stem cell activity of the MCF-7 cells cultured with TFF as compared to CF. All 3D co-culture and conditioned media promoting effects in MCF-7 cells were inhibited by 10H10 and Rivaroxaban. In vivo, TF fibroblast co-injection with MCF-7 breast cancer cells promoted primary tumour growth in vivo and stem cell activity ex vivo, with these promoting effects being abrogated by Rivaroxaban. Conclusion: This thesis establishes a role for TF expressing fibroblasts in the promotion of ER+ breast cancer migration and stem cell activity and proposes that targeting the procoagulant tumour microenvironment with Rivaroxaban to reduce metastasis and disease recurrence maybe a valuable therapeutic strategy in ER+ breast cancer patients.

Date of Award	22 Feb 2022
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Cliona Kirwan (Main Supervisor), Robert Clarke (Co Supervisor) & Angelica Santiago-Gomez (Co Supervisor)