Effect of FGFR and FAK Inhibitors on Glioma Cell Migration
University of Delaware
Glioblastoma multiforme (GBM), the most lethal of brain tumors, spreads rapidly in the brain. The mechanisms that promote extensive GBM cell migration are not completely known. The Galileo lab has shown that the neural recognition protein L1CAM (L1) is a key factor. It acts by being abnormally expressed by GBM cells, proteolyzed to release a large ectodomain fragment, and autocrine signaling through integrin receptors and fibroblast growth factor receptors. While autocrine stimulation by L1 has been shown to increase glioma cell migration and proliferation, there may be other extrinsic influences at play that promote glioma aggressiveness. Brain cells, or more specifically astrocytes, the most abundant cell in the human brain, also may stimulate GBM cells. In order to determine relative contributions of mixed brain cells or purified astrocytes to glioma cell motility, time-lapse experiments of co-cultured GFP-expressing T98G human glioma cells and chick embryo brain cells were performed. Results showed that chick brain cells may further stimulate glioma cells. Astrocytes also were isolated from primary rat brain tissue and plated as a monolayer for co-cultured glioma cells to interact with. The effect of cell-to-cell contact between astrocytes and glioma cells was inconclusive due to astrocyte sensitivity to the applied methods. L1-FGFR interaction was seen through double immunostaining. Results showed that L1 and FGFR do in fact act as binding partners but also separately to other molecules and receptors. Furthermore, T98G-dFGFR cells were injected into chick optic tectum to observe the effect of FGFR in vivo. Results showed that glioma cells lacking FGFR were minimally invasive compared to the control (T98G-2605) that showed invasive character. In a separate experiment, media was extracted from astrocyte and L1- expressing tumor cell line (U118-L1LE) and placed in cell scratches of cells lacking L1 (T98G-shL1), cells lacking FGFR (T98G-FGFR), and cells expressing L1 (T98G). The cell velocities were recorded using time-lapse microscopy. In addition, cell proliferation rates were determined through cell cycle analysis using propidium iodide staining and FACS. Results showed that paracrine stimulation by astrocytes can stimulate glioma cell motility and proliferation to near autocrine stimulation levels in the absence of L1. This suggests that Astrocytes, via paracrine stimulation, may have other factors or molecules that stimulate glioma cell motility through mechanisms other than FGFR receptors. It was also found that L1 autocrine signaling is the biggest source of stimulation for normal glioma cells.