Investigation of protein induction in vascular-targeted strategies.

The aims of the study reported in this thesis were to develop and utilise mass spectrometry imaging techniques (MALDI-MSI), in combination with conventional proteomic methodologies, to investigate protein induction in vascular-targeted strategies. Proteins thought to be involved in tumourigenesis and drug treatment resistance were observed along with the responses from proteins identified via the techniques used, in this global analysis study. MALDI-MSI, LC-ESI-MS/MS, LC-MALDI-MS/MS with iTRAQ labelling and immunohistochemistry intended to provide cross validation of the effects post administration of vascular disrupting agent CA-4-P. Two mouse fibrosarcoma models (expressing VEGF120/ VEGF188 isoforms only) following treatment with the tubulin-binding tumour vascular disrupting agent, combretastatin A-4-phosphate (CA-4-P) have been studied. The gross haemorrhagic pharmacological response elicited by CA-4-P was visible by MALDI-MSI throughout the fibrosarcoma 120 time course. The latter encouraged the prospect that other proteins could potentially be observed induced via a dose response relationship. The haemoglobin time course using the resistant 188 tumour model gave quite different results to those previously seen in the MALDI-MSI of the fibrosarcoma 120 data set. The first indication of the 'switch back to tissue viability' concept was revealed. The experimental work using LC-ESI-MS/MS revealed many proteins connected with necrosis, apoptosis, cell structural reorganisation, polymerisation, tumour survival and stress induced molecular chaperones. The inverse correlation of structural proteins, haemoglobin and heat shock molecular chaperones gave the required validation and identification to relate these responses to those seen in MALDI-MSI. The relationship pathways generated by using STRING 9.0 proteomic network software gave an invaluable insight into the activity of the active tumour milieu and provided a means of linking the identified proteins to their functional partners. Protein-protein interactions could be observed to help interpretation of the MALDI-MSI, LC-ESI-MS/MS and iTRAQ LC-ESI-MS/MS response graphs. Overall, the dose relationships observed in the iTRAQ data by the proteins involved in haemorrhaging, structural remodelling, were in good agreement with the other techniques employed here.It could be said that MALDI-MSI could potentially forge a place in the workflow of clinical diagnostics. Targeted approaches for the observation of disease biomarkers could be visualised using MALDI-MSI and serve as a complimentary technique to standard clinical imaging. A novel method reported here using a multi-peptide recombinant standard could prove an important diagnostic tool for the analysis of patient biopsies and tissue micro-arrays. The exciting prospect is the diversity of a multi-peptide recombinant standard, an artificial construct that can be engineered to include any prospective biomarkers for both research and diagnostic screening applications.