Glioblastoma Multiforme Invasion Assay Optimization and Analysis

A promising category of GBM therapeutic agents are activators of cyclic mediated adenosine monophosphate (cAMP) production. cAMP, a type of second messenger, has been shown to have lower expression levels in many different types of cancer but the reason and mechanism of its downregulation is unknown. The compartmentalized functions of the cAMP pathway and the signaling that occurs in distinct microdomains make it complicated to study. Here, we describe the effects of cAMP activation on the growth and invasion of GBM neurospheres. The pharmaceutical treatment forskolin and the photoactivatable bPAC were used as methods of cAMP production activation in MMK1 cells. Forskolin significantly decreased the growth and invasion of neurospheres in the presence and absence of bPAC, supporting the hypothesis that cAMP production activation decreases GBM invasion. However, cells treated with bPAC as a means for activating cAMP production did not show significant differences in growth or invasion. Future studies should continue to optimize the use of bPAC site-specifically, in order to localize the activation of cAMP production.Glioblastoma Multiforme (GBM) is a type of highly invasive brain tumor. The visualization of invasive cellular structures, known as invadapodia, has been an important tool for measuring GBM cancer cell invasion. One assay for measuring invasion relies on imaging dark areas created by the invadapodia as they degrade a fluorescent gelatin matrix. Here, the optimization of this assay is described, along with its use in a preliminary study investigating the role of the cofilin protein in invasion. Additionally, an automated invasion analysis pipeline was developed using the Cell Profiler software. The high-throughput approach can save time and remove user bias from the image analysis process. Along with the assay, the pipeline provides a new method to quantify GBM cancer cell invasion. This method can be used to elucidate which proteins are important to invadopodia formation and subsequently build more targeted therapeutics for GBM.Bachelor of Scienc