Multicolor Fluorescence-Based Approaches for Imaging Cytokine-Induced Alterations in the Neovascularization, Growth, Metastasis, and Apoptosis of Murine Neuroblastoma Tumors

Neuroblastoma is one of the most common solid tumors in children. The prognosis of patients with advanced neuroblastoma is poor overall despite standard therapeutic modalities and has stimulated substantial interest in the potential role for biologics such as immunotherapeutic and/or antiangiogenic agents for the treatment of neuroblastoma. To facilitate preclinical investigation of the efficacy and mechanisms of action of new biologic agents for the treatment of neuroblastoma, a comprehensive panel of disease-specific fluorescence-based model systems has been developed by our group to image the growth, neovascularization, metastasis, and apoptosis of neuroblastoma tumors. These model systems use fluorescent proteins to monitor cytokine-induced alterations in the growth and metastasis of neuroblastoma and allow for monitoring and/or quantitation of even minimal residual disease that is localized to visceral organ sites such as the liver, lung, and/or bone marrow. Further, based on the differential spectra of red fluorescent protein, green fluorescent protein (GFP), and agents such as 4′-6-diamidino-2-phenylindole (DAPI) (blue) and fluorescein isothiocyanate–dextran (green), multicolor systems have now been established by our group that allow for combined assessment of parameters, including the macroscopic relation of tumors to their associated vasculature and, within tissue sections, simultaneous quantitation of tumor neovascularization and evaluation of therapy-induced apoptosis within the tumor and vascular endothelial compartments. Further, by engineering cells to express specific mediators of apoptosis that have been linked to GFP (ie, BID-EGFP), these systems can also be used to dissect mechanisms by which neuroblastoma cells are induced to undergo apoptosis in vitro as well as in vivo. Collectively, these model systems provide important tools for investigation of the biology of neuroblastoma tumors and evaluation of mechanisms that mediate the regression of these tumors in response to novel therapeutic agents, including cytokines such as interleukin-12.