A sensitive assay for the evaluation of cytotoxicity and its pharmacologic modulation in human solid tumor-derived cell lines exposed to cancer-therapeutic agents.

PURPOSE: Reliable in vitro cytotoxicity assays are essential for determining the responses of human normal and cancer-derived cells to therapeutic agents and also for the identification and pre-clinical evaluation of new drugs capable of selectively augmenting the susceptibility of cancer cells to conventional therapies. The clonogenic survival assay is considered as the "gold standard" in this regard because it measures the sum of all modes of cell death, encompassing both early and late events such as delayed growth arrest. In this assay, however, the impact of cell-to-cell communication is disregarded because the cells are plated out at very low densities. In addition, here we provide evidence that human breast cancer cell lines cannot be reliably evaluated by clonogenic assays. We developed a novel long-term, High Density Survival (HDS), assay that circumvents the various intrinsic shortcomings of the conventional cytotoxicity assays. METHODS: In the HDS assay, the cells are maintained at a high density for 24 h prior to, and for 24 h after, exposure to a DNA-damaging agent to facilitate intercellular communication. After a carefully scheduled subculturing for approximately 7 days, cultures are assessed for the extent of growth. RESULTS: The degree of radiosensitivity and cisplatin sensitivity evaluated by the HDS assay in human cancer cells was comparable to that measured by the clonogenic assay. Pharmacological inhibitors of CaMKII and/or PI3K signaling elicited a greater degree of radiosensitization when determined by the HDS assay than the clonogenic assay. In all these experiments, there was no relationship between the degree of cytotoxicity measured by the clonogenic survival and viability assays. In the HDS assay, all seven human breast cancer cell lines that we tested exhibited a high degree of radioresistance. CONCLUSIONS: The novel HDS assay appears to be a powerful tool for evaluating cancer cell responses to therapeutic agents under conditions which incorporate some aspects of intercellular communication.