Novel Laser Capture Microdissection-Proteomic Analysis Identifies Spatially Distinct Extracellular Matrix Signatures in the Core and Infiltrating Edge of Human Glioblastoma

Abstract

AbstractBackgroundGlioblastoma is the most common and aggressive primary brain tumour in adults. Hallmarks of glioblastoma include its intra-tumoural heterogeneity and extensive invasion of the surrounding brain. Glioblastoma is known to remodel the extracellular matrix (ECM) of the brain, resulting in altered mechanical properties and the establishment of a tumour-promoting microenvironment. How changes in the expression and spatial distribution of ECM constituents within glioblastoma contribute to invasion and disease progression is still unclear.MethodsHere we report on a protocol for laser-capture microdissection coupled with mass spectrometry (LCM-proteomics) that allowed a spatially resolved and unbiased analysis of the regional ECM proteome (matrisome) in formalin-fixed and paraffin-embedded (FFPE) samples of human glioblastoma. We investigated five molecularly characterised hemispheric adult glioblastomas where the brain/tumour interface and tumour epicentre were represented in the surgical specimens and snap-frozen tissue was available. LCM-proteomic analysis was validated with immunohistochemistry.ResultsLCM-proteomics identified 53 matrisome proteins in FFPE tissue, demonstrating comparable performance with conventional analysis of snap-frozen tissue. The analysis revealed distinct matrisome components in the brain/tumour interface versus the tumour epicentre. Guided by data from LCM-proteomic analysis, immunostaining for tenascin-R confirmed greater staining in the brain/tumour interface, whilst expression of fibronectin was higher in the tumour epicentre.ConclusionThe protocol described in this work allowed for accurate, spatially resolved analysis of ECM in FFPE tissues, with performance comparable to analysis of snap-frozen tissue. While the focus for this work was on the regional ECM composition of glioblastoma, we found that the LCM-proteomics protocol is also applicable to the study of the wider proteome, and represents a valuable tool for investigating tumour/tissue heterogeneity. This protocol opens the possibility to apply LCM-proteomics to retrospective studies with the advantage of accessing clinical history and follow-up information, providing a valuable resource for translational research in glioblastoma.