Differential Staining Cytotoxicity Assay: A Review

Differential Staining Cytotoxicity (DiSC) assay is the prototype for a closely related family of assays based on the concept of total cell kill, or, in other words, cell death occurring in the entire population of tumor cells. It is probably the most versatile of the cell-death end points, in that it (1) can be applied to both solid and hematologic neoplasms, (2) can be applied to specimens in which it is not possible to obtain a pure population of highly enriched tumor cells, and (3) can be applied to a wide variety of drugs, ranging from traditional cytotoxic agents to biological response modifiers with activity mediated through tumor-infiltrating effector cells, to “targeted” kinase inhibitors, and to antivascular agents, such as bevacizumab and pazopanib. The basic principles of the assay are to culture three-dimensional fresh tumor cell clusters in anchorage-independent conditions. At the conclusion of the culture period, Fast Green dye is added to the microwells, the contents of which are then sedimented onto permanent Cytospin centrifuge slides and then counterstained with hematoxylin–eosin or Wright–Giemsa. “Living” cells stain with the cytologic stain in question and can be identified as either normal or neoplastic, based on standard morphologic criteria. “Dead” cells stain blue-green. Nonviable endothelial cells appear as strikingly hyperchromatic, blue-black, and often refractile objects, which may be readily distinguished from other types of dead cells. This assay has been biologically and clinically validated in a number of ways, as described in this chapter.