We will utilize two of the cell lines, one MYCN not-amplified and one MYCN amplified cell line that can be grown in a xenograft model. Among other assays we will use the Real-Time Cell Electronic Sensing System (RT-CES system; Acea Biosciences, CA, USA) as well as the BD Pathway 96-well confocal microscope. The RT-CES is an electronic device that allows a label-free, cell based monitoring of cell number and size in real time without the requirement for manipulating the cells. The data is collected from living cells in real-time in a 96-well format, over the course of the entire experiment. Each well incorporates a gold circle-on-line microelectrode array, where the cells attach and the impendence of each well is measured (Cell Index) which is proportional to the cell number. The BD Pathway microscope allows for high content screening of siRNA gene silencing and monitors cell number, apoptosis, and cell cycle parameters. We have tested several drugs and siRNAs with these methods and find it a robust measure of cell number. The most promising targets and the appropriate siRNA will be further evaluated in the two xenograft animal models as outlined above. All positive hits will be further screened in a wider panel of NB Xenografts which will be part of a panel of the Pediatric Preclinical Testing Program (PPTP) which is currently being utilized as a pipeline to screen new drugs and make recommendation for human pediatric phase 1/2 trials (http://ctep.cancer.gov/resources/child.html. We have successfully optimized the high through put siRNA screening using a set of approximately 400 apoptosis related genes and the findings are being prepared for publication.
