A Method for the Selective Measurement of the Radiosensitivity of Quiescent Cells in Solid Tumors: Combination of Immunofluorescence Staining to BrdU and Micronucleus Assay

Abstract

C3H/He mice bearing the SCC VII tumor were irradiated after being given 10 injections of 5-bromo-2'-deoxyuridine (BrdU) to label all proliferating cells in the tumors, and the tumors were then excised and trypsinized. The tumor cell suspensions were incubated with cytochalasin-B (which blocks cytokinesis), and the micronucleus frequency in unlabeled cells was determined using immunofluorescence staining to BrdU. The micronucleus frequency was then used to calculate the surviving fraction of the unlabeled cells, using the regression line relating the micronucleus frequency to the surviving fraction determined separately for the total tumor cell population. Using this technique, a cell survival curve could be determined for the unlabeled cells, which were regarded as the quiescent cells. Assays performed both immediately after and 24 h after irradiation of normally-aerated tumors showed that unlabeled cells were more radioresistant and had a greater capacity for repair of potentially lethal damage than the tumor cell population as a whole. Moreover, when the assay was performed immediately after the irradiation of both normally-aerated and hypoxic tumors, it was found that unlabeled cells had a much higher hypoxic fraction than the tumor cell population as a whole. This appears to be a useful method for determining the responses of quiescent cells in solid tumors to various treatments.