Date of Award

2014

Document type

Thesis

Degree Name

PhD (Doctor of Philosophy)

First Supervisor

Professor Niamh Moran

Second Supervisor

Professor David Williams

Funder/Sponsor

Health Research Board

Keywords

Neoplasms, therapy, Blood Platelets, Platelet Activation

Abstract

Platelets play a role in cancer progression by acting as a protective shield and dynamic reservoir of effectors which facilitate tumour vascularisation, growth and metastasis. However, little information is available about the mechanism of tumour cell-induced platelet secretion (TCIPS) or the molecular machinery by which soluble mediators are released from platelets.

Here we demonstrate that tumour cells directly induce platelet secretion.

Platelet secretion was assessed by employing a luminometric assay capable of detecting ATP/ADP released by platelet dense granules following activation. Preincubation of platelets with increasing doses (0.1-100 X 103 cells/mL) of human colon cancer cells (Caco-2), prostate cancer cells (PC3M-luc) or breast cancer cells (MDA-MB-231; MCF-7) resulted in a marked dose-dependent secretion of dense granules. Importantly, TCIPS preceded aggregation which displayed a lag-time characteristic for each cell type. Secretion could not be induced in Boyden chambers where a permeable barrier separated the tumour cells from the platelets, strongly suggesting that direct tumour cell contact with platelet surface proteins was required.

Using a pharmacological approach, we next attempted to identify the nature of the platelet receptor(s) and signalling pathways involved in PC3M cell-induced platelet secretion and aggregation (TCIPS/A). The most potent inhibitors of TCIPS/A were the pharmacological antagonists of Syk Kinase, Phospholipase C (PLC) and Protein Kinase C (PKC), downstream mediators of the immunoreceptor tyrosine-based activation motif (ITAM) cascade in platelets. Supporting this, we demonstrated a central role for the immune Fey Receptor lia, (FcyRlla) in mediating platelet-tumour cell cross-talk.

There is increasing evidence in the literature suggesting that platelet secretion can be differentially tuned according to the stimuli. Evidence is provided that shows that the composition of platelet secretóme can vary in response to diverse cancer cell lines. By using an initial human protein array and a complementary ELISA analysis, a differential secretion from platelets was demonstrated according to the nature of the stimulus (pro-thrombotic or cellular) or the potency of the stimulus (Caco2>PC3M~=TRAP>> PC3). The potential for this bespoke release to be used for therapeutic benefit was discussed.

In conclusion, we show that cancer cells can differentially promote both dense and a-granule secretion from platelets. ADP, derived from dense granules, is required to augment platelet aggregation. In addition, a critical primary interactor for PC3M cells is the platelet surface immune receptor, FcyRlla, providing a potential target for the development of novel anti-metastatic therapies. Finally, we confirm that platelet secretion can be dictated by the nature of stimuli, with different cancer cell lines inducing diverse responses depending on their aggressiveness, opening the opportunity to develop personalised treatment strategies for cancer patients by tailoring platelet secretion to particular goals.

Creative Commons License

Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-Share Alike 4.0 License.

File Size

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Comments

A thesis submitted for the degree of Doctor of Philosophy from the Royal College of Surgeons in Ireland in 2014.

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