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Mass Culture and Characterization of Tumor Cells From a Naturally Occurring Invertebrate Cancer Model: Applications for Human and Animal Disease and Environmental Health

Department of Molecular, Cellular and Biomedical Sciences, Rudman Hall, University of New Hampshire, Durham, New Hampshire 03824

^* To whom correspondence should be addressed. E-mail: cwwalker{at}christa.unh.edu

On the northeastern coast of the United States and Canada, Mya arenaria, the soft shell clam, develops a diffuse, hemopoetic tumor (a fatal leukemia-like cancer) resulting from inactivation of p53-like family member proteins.These malignant cells provide a model for an unrelated set of human cancer cells that are also characterized by mortalin-based cytoplasmic sequestration of wild-type p53 tumor suppressor protein (mortalin is the mitochondrial Hsp70 protein). Here we describe methods for mass culture and long-term storage of tumor cells from this cancer. These are the first successful efforts at maintaining malignant cells from any marine invertebrate in vitro. Following passage (subculture), these cultures undergo transition from primary cultures to non-immortalized cell lines that continue to proliferate and do not re-differentiate the normal hemocyte phenotype. We also characterize normal clam hemocytes and the pathology of cancerous clam hemocytes in vitro and in vivo using light and electron microscopy, cyto- and immunocytochemistry, molecular biology, and a phagocytosis assay. Our protocols provide biomedical and environmental researchers with ready access to this naturally occurring cancer model. We discuss the clam cancer model regarding (a) human health and disease; (b) animal health, disease, and aquaculture; (c) environmental health monitoring; and (d) future research directions.

Abbreviations: BrdU, 5-bromo-2'-deoxyuridine • CCH, cancerous clam hemocytes • 1E10, cancerous clam hemocyte-specific monoclonal antibody • PAS, periodic acid–Schiff • NCH, normal clam hemocytes