Study of the thrombogenicity induced by the cytotoxic treatment of malignant disease

Hall, Jessica

Abstract

Cancer and its treatment are frequently complicated by the development of venous thromboembolism (VTE). Interestingly, VTE incidence rates vary according to tumour type and the chemotherapy regimen administered. Yet, the precise mechanisms responsible for the increase in VTE in cancer patients remain unclear. Tissue factor (TF), the primary cellular initiator of the coagulation cascade, is over-expressed in many solid malignancies, particularly pancreatic cancer, and the number of circulating TF+ microparticles (MP) are increased in cancer patients. It is hypothesised that the increased risk of VTE in cancer patients generated by cytotoxic treatment may be partly attributed to the apoptotic process, involving the exposure of procoagulant phosphatidylserine (PS) on tumour cells or other chemotherapy damaged cells and increased release of TF+ MP into the circulation. The aim of this study was to explore how coagulation can be initiated in cancer cells and be potentiated by chemotherapy, with respects to the specific expression of TF and PS, and generation of MP.

Flow cytometry was used to evaluate expression of cell surface TF and level of apoptosis in untreated/doxorubicin (Dox)-treated cancer cell lines, and number of MP in platelet-free plasma (PFP) from untreated pancreatic cancer patients, myeloproliferative disorder (MPD) patients, and multiple myeloma (MM) patients before, during, and after chemotherapy. The cell proliferation of untreated/Dox-treated cancer cells was assessed by an MTS assay. Procoagulant activity (PCA) of untreated/Dox-treated cancer cells and their isolated cell-free supernatants containing MP, and also MM and MPD patient PFP was measured using a prothrombin time assay. Enzyme-linked immunosorbent assay quantified levels of TF in unfiltered/0.1 μm filtered cell-free supernatants isolated from cancer cell lines, and serum levels of soluble cell adhesion molecules CD106 and CD54 from MM patients.

Pancreatic (AsPC-1, CFPAC-1, MIA-PaCa-2), ovarian (A270, ES2, SKOV-3), colorectal (CaCo-2, LoVo), breast (MCF-7, MDA-MB-231, T47D), and haematological (JJN3, U937) cancer cells were found to support coagulation in a cell number-dependent manner, defined by a logarithmic relationship that was consistent across all cell lines. Furthermore, single cell clotting time (CT) was determined for each cancer cell line from the slope and y axis intercept of a logarithmically transformed data plot. A near linear relationship was observed between TF expression and single cell CT where a higher expression of TF results in a proportionally faster CT (P = 0.01). In addition, tumour cell-derived MP were shown to be procoagulant and the majority of procoagulant potential could be removed by passing isolated cell-free supernatants through a 0.1 μm filter. A dose-dependent CT was observed with AsPC-1, CFPAC-1, ES2, SKOV-3, LoVo, and MDA-MB-231 cell-free supernatants.

The cytotoxic chemotherapeutic agent Dox was found to decrease the number of viable ovarian (ES2), breast (MDA-MB-231, T47D), and haematological (MM.1S, U937) cancer cells in a time- and dose-dependent manner, and cell death was shown to be induced by apoptosis and subsequently necrosis at higher drug concentrations. Cell surface expression of PS was found to increase following Dox treatment, while TF was not upregulated. Furthermore, Dox was shown to dose-dependently increase PCA in all cancer cells examined, although the effect of cell-free supernatants on PCA was less consistent; ES2, MDA-MB-231, and U937 cell-free supernatants isolated from Dox-treated cells demonstrated increased PCA at 0.01 and 0.1 μM concentrations. The Dox-induced increase in PCA of cancer cells and cell-free supernatants were found to correlate with tumour cell viability (r = 0.79 to 0.97, P < 0.01).

Numbers of TF+ MP were significantly higher in 35 untreated pancreatic cancer patients in comparison with 15 MM patients prior to chemotherapy (P < 0.0005). Furthermore, numbers of endothelial cell-derived MP (EMP), monocyte-derived MP, plasma cell-derived MP, and PS+ MP, were significantly higher in 6 MPD patients in comparison with 15 MM patients prior to chemotherapy (P < 0.005), but not levels of platelet-derived MP (PMP) or TF+ MP. Markers of endothelial dysfunction, including EMP and soluble cell adhesion molecules CD106 and CD54, were elevated after thalidomide (Thal)- or lenalidomide (Len)-based therapies in MM patients. Furthermore, PCA was significantly increased in MM patients after treatment (P = 0.007), and also levels of PMP, plasma cell-derived MP, and PS+ MP (P < 0.05), but not monocyte-derived MP (P = 0.33) and TF+ MP (P = 0.41).

In summary, this study shows that across a range of tumour sites a consistent relationship is seen between procoagulant potential and both cell number and TF cell surface expression. Dox can increase PCA of cancer cells through reduced cell viability that leads to PS exposure. Importantly, this Dox-induced procoagulant mechanism was not found to involve an upregulation of surface TF antigen on ovarian, breast, or haematological cancer cells. In MM patients treated with Thal- or Len-based therapies increased PCA was observed, which may be mediated by endothelial dysfunction and increased generation of MP.

Citation

Hall, J. Study of the thrombogenicity induced by the cytotoxic treatment of malignant disease. (Thesis). Hull York Medical School, the University of Hull and the University of York. https://hull-repository.worktribe.com/output/4216550

• Thesis Type: Thesis
• Deposit Date: May 22, 2015
• Publicly Available Date: Feb 23, 2023
• Keywords: Medicine
• Public URL: https://hull-repository.worktribe.com/output/4216550
• Additional Information: Hull York Medical School, The University of Hull and the University of York
• Award Date: Sep 1, 2014