The goal of this renewal proposal is to characterize inhibition of antitumor immune responses by tumor gangliosides in vivo. The general hypothesis underlying this work is that gangliosides are shed by tumor cells, act as intercellular signaling molecules, block the cellular immune response, and protect tumor cells from host immune destruction. Findings of significant shedding and of potent immunosuppressive activity of human neuroblastoma gangliosides, and new preliminary data showing inhibition of murine cellular immune response to syngeneic tumor cells by these molecules, lay the foundation to directly test the hypothesis that tumor gangliosides downregulate host cellular immune responses to syngeneic tumor cell both in vitro and in vivo. Two approaches will be used to obtain neuroblastoma tumor gangliosides, homogeneous in both carbohydrate and ceramide structure, in large quantities: isolation from human neuroblastoma tumors (natural) and chemical synthesis (synthetic). All gangliosides will be further purified by HPLC to remove potential traces of contaminants prior to biological study. Molecular structures of the gangliosides will be confirmed by mass spectrometry and chemical methods. The delineation of inhibitory effects of tumor gangliosides upon both the afferent (priming) and efferent (effector function) phases of the cellular immune response in murine tumor models in vivo will be complemented by determination of the effects of these same gangliosides on the human cellular immune response in vitro. The in vivo studies will be performed in two murine syngeneic tumor models, FBL erythroleukemia and B78H1 melanoma. The proposed studies will conclusively demonstrate downregulation of syngeneic antitumor immune response by neuroblastoma tumor gangliosides in vivo. They will provide the foundation for future investigations (longer term aims) to elucidate the molecular mechanism(s) of inhibition by tumor gangliosides and to develop strategies to eliminate these immunosuppressive molecules (such as removal of gangliosides from the circulation or modulation of ganglioside biosynthesis and shedding by pharmacologic agents), with the ultimate goal of achieving improved therapeutic approaches to neuroblastoma and other neuroectodermal tumors such as melanoma.
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