A knowledge gap exists in understanding the factors that control the motility and invasiveness of glioblastoma stem cells (GSCs). Until this knowledge gap is filled, it is unlikely that effective treatments can be devised to effectively impede GSC invasion, and consequently, glioblastoma will remain an incurable cancer. The long-term goal is to elucidate factors and mechanisms that promote GSC motility and invasiveness. The overall objective of this study is to reveal novel means by which the neural recognition molecule L1CAM (L1; CD171) stimulates GSC motility and invasiveness. The central hypothesis is that GSC motility and invasiveness is stimulated by L1 from different sources and in different forms (i.e., soluble autocrine vs. soluble paracrine vs. patterned deposits). The rationale for the proposed work is based on previous findings that L1 is abnormally expressed, proteolyzed, and released (shed) by GBM cells.
Specific Aim 1 of this small, defined, exploratory research project will characterize recently generated patient-derived GSCs for L1 expression, release, and responsiveness. GSCs derived from patient GBM tumors will be grown as adherent cultures and characterized for expression of GSC markers and expression and release of L1ecto.
Specific Aim 2 will measure the differences in responsiveness to stimulation of GSCs by different forms of L1ecto in vitro. To approach autocrine effects, L1ecto will be ectopically expressed or attenuated in GSCs characterized in Aim 1. To address paracrine effects, GSCs will be grown adjacent to cells releasing soluble L1ecto. To address effects of insoluble patterns, GSCs will be grown on immobilized L1ecto. GSC motility will be tracked and analyzed for cell speed and directionality by time-lapse microscopy.
Specific Aim 3 will identify stimulatory effects of L1ecto in a complex tissue microenvironment. Green GFP labeled GSCs will be injected into an in vivo xenograft brain tumor model, either alone or mixed with red mCherry labeled L1ecto(+) or (-) GBM non-stem cells. The effects of autocrine and paracrine L1ecto on GSC invasion will be assessed using confocal 3-D reconstructions, including GSC proximity relationships to L1ecto shedding cells and to tracks of deposited L1ecto. Upon successful completion of the proposed research, its contribution is expected to be the discovery of novel means by which L1 stimulates GSC motility and invasiveness. This contribution will be significant because it is expected to have broad importance in understanding novel mechanisms of how normal neural molecules are abnormally expressed, released, and presented by GBM cells to GSCs to stimulate their invasiveness. This research is innovative by shifting focus to effects of L1 on stimulating GSC invasiveness and to the ability of different sources and different presentations of L1 to stimulate invasiveness differently.
The project is relevant to NIH's mission because it is expected to have broad importance in understanding novel mechanisms by which brain cancer cells abnormally express, release, and deposit a normal brain cell molecule to stimulate their own spreading throughout the brain. This increased understanding is expected to lead to the development of appropriate strategies to block or reduce brain cancer cell spread, which ultimately could result in treatments that prolong brain cancer patient survival.
