Development of the highly sensitive chemosensitivity testing using ATP bioluminescence assay

5329 Objective: Chemosensitivity testing is thought to be a useful method for selecting susceptive agents or eliminating resistant agents prior to treatment. However, one unsolved problem is that these assays require a relatively large amount of tumor tissue, which hampers wide clinical application. For example, conventional MTT assay usually requires 50,000 cells /well for determining chemosensitivity. In order to minimize the sample size for chemosensitivity testing, we have developed a highly sensitive assay using ATP bioluminescence methods. Materials and Methods: Human gastric cancer cell line MKN-45 was used for the basic experiment. Cells were plated onto 96-well microplates at a concentration of 2,000, 1,000, 500, 250 cells / well and then anti-cancer drugs were added at various concentrations, and were cultured for 72 hrs. Cell viability was determined by ATP bioluminescence assay using highly sensitive synthetic firefly luciferase and compared with that in MTT assay. In clinical samples, obtained from 64 patients with various kinds of cancers, cells were plated at 20,000, 10,000, 5,000cells /well, and drug sensitivity was determined by the ATP assay in a similar manner. Results: In the basic examination using gastric cancer cell line, intracellular ATP levels showed significant linear correlation with the number of cells ranging from 10 to 1,000,000 cells/ml. The ATP assay could detect drug sensitivity regardless number of cells applied, while MTT assay failed to do so. Minimal number of cells for MTT assay were considered to be >50,000 cells /well. In clinical samples, chemosensitivity testing was evaluable at any concentration of cells applied in 47 of 64 samples (73%), and the results were always consistent regardless of cell concentration. Consideration: ATP bioluminescence assay enables reproducible chemosensitivity testing for a much smaller size of cancer cells as compared to MTT assay, suggesting a wide clinical application for chemosensitivity testing with small sample such as biopsy specimen.