Glioblastoma multiforme (GBM) is the most aggressive and prevalent malignant brain tumour in adults. GBM is diffusely infiltrative which makes complete surgical resection difficult. Tumour invasion of neighbouring tissues is facilitated by cell migration and degradation of the extracellular matrix (ECM). Invadopodia are actin-rich organelles that protrude from the ventral side of the plasma membrane in direct contact with the ECM and play and important role 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 and perform comparative membrane proteomic analyses of highly invasive vs less-invasive cells.
Nine 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 h at 37oC. Fluorescence microscopy revealed areas devoid of Cy3-gelatin indicating gelatin degradation by GBM cell invasion.The membrane proteomes from GBM cells with varying invasive potentials were enriched by differential ultracentrifugation and compared using isobaric tags for relative and absolute quantitation (iTRAQ) coupled with multi-dimensional liquid chromatography and tandem mass spectrometry (2D LC-MS/MS). Fluorescence microscopy was used to show the co-localisation of membrane proteins and invadopodia.
All 9 GBM lines produced invadopodia and degraded Cy3-gelatin. There was a significant difference between the most invasive (U87MG) and least invasive (LN229) cells (65%, percentage of total cell area; p=0.0001). Overall, 1667 proteins were identified from duplicate runs, of which 76% mapped to membrane structures using the David bioinformatics database (http://david.abcc.ncifcrf.gov). The differential abundance of 38 proteins significantly correlated to the degree of invasion (r2 > 0.45 or r 2 <- 0.45; n ≥ 9; p < 0.05). Cell surface proteins were involved in cell morphology and cellular movement.
Differentially abundant proteins identified by iTRAQ LC-MS/MSanalysis are involved in cellular movement, cell-cell and interactions. Such, invadopodia-associated membrane proteins could be novel targets for anti-invasive therapies.