Glioblastoma multiforme (GBM) is the most aggressive and prevalent malignant brain tumour in adults. GBM is diffusely infiltrative and its poor demarcation from surrounding brain parenchyma makes complete surgical resection difficult and tumour recurrence inevitable. Tumour invasion of neighbouring tissues is facilitated by cell migration and degradation of the extracellular matrix (ECM). Invadopodia are discrete ECM-degrading structures recently described in GBM and play important roles in tumour invasion. The aim of the current study is to characterize the ‘invasive potential’ of a panel of established GBM cell lines using an invadopodia assay to form the basis of comparative proteomic analyses of highly invasive vs non-invasive cells.
9 GBM cell lines were characterized based on their ability to produce invadopodia using a QCM gelatin invadopodia assay (Millipore). Glass slides were coated with Cy3-gelatin before seeding GBM cells for 24 hr at 37oC (14,000 cells/cm2). Fluorescent microscopy revealed areas devoid of Cy3-gelatin indicating gelatin degradation by GBM cell invasion. The membrane and exosome proteomes from GBM cells with different invasive potentials were enriched and compared using isobaric tags for relative and absolute quantitation (iTRAQ) coupled with multi-dimensional liquid chromatography and tandem mass spectrometry (LC-MS/MS).
All 9 GBM lines produced invadopodia and degraded Cy3-gelatin, with a 65% difference between the most invasive (U87MG) and least invasive (LN229) presented as a percentage of total cell area. SK-MEL-28, a non-invasive melanoma cell line with known low Cy3-gelatin degradation was used as a negative control.
Differentially abundant proteins identified in the quantitative LC-MS/MS analysis may provide insight into the pathways implicated in more invasive GBM phenotypes. Invadopodia-associated and/or exosomal proteins could be targeted to produce novel anti-invasive therapies.