MALDI‐MSI and label‐free LC‐ESI‐MS/MS shotgun proteomics to investigate protein induction in a murine fibrosarcoma model following treatment with a vascular disrupting agent

Tumour vasculature is notoriously sinusoidal and leaky, and is hence susceptible to vascular disruption. Microtubule destabilising drugs such as the combretastatins form the largest group of tumour vascular disrupting agents (VDAs) and cause selective shutdown of tumour blood flow within minutes to hours, leading to secondary tumour cell death. Targeting the tumour vasculature is a proven anticancer strategy but early treatment response bio-markers are required for personalising treatment planning. Protein induction following treatment with combretastatin A4-phosphate (CA4P) was examined in a mouse fibrosarcoma model (fs 188), where tumour cells express only the matrix-bound isoform of vascular endothelial growth factor A (VEGF188). These tumours are relatively resistant to vascular disruption by CA4P and hence a study of protein induction following treatment could yield insights into resistance mechanisms. The distribution of a number of proteins induced following treatment were visualised by MALDI-MSI. Responses identified were validated by LC-ESI-MS/MS and immunohistochemical (IHC) staining. Significant changes in proteins connected with necrosis, cell structure, cell survival and stress-induced molecular chaperones were identified. Protein-protein interactions were identified using STRING 9.0 proteomic network software. These relationship pathways provided an insight into the activity of the active tumour milieu and a means of linking the identified proteins to their functional partners.