Analysis of apoptosis in solid tumors by laser-scanning cytometry

Apoptosis (programmed cell death) plays an important role in tissue physiology and pathology. First, tumor development and progression is often associated with defective regulation of apoptotic pathways, and measurement of apoptosis might be equally important for the analysis of cell proliferation. Second, tumor cells die by apoptosis during chemotherapy or radiotherapy, so monitoring the level of apoptosis might prove useful in modulating treatment or in predicting the biologic behavior of the tumor. Flow cytometry (FC), in conjunction with DNA strand-break labeling assays, is commonly used to assess apoptosis, but unless combined with cell sorting, FC results cannot correlate with morphology. Also, analysis by FC is associated with cell loss during preparation of the specimen. In this study, we identified apoptotic cells by in situ DNA strand-break labeling in solid tumors and then analyzed those cells by laser-scanning cytometry (LSC). LSC provides data comparable to those obtained by FC, but because the cells are prepared and measured on a slide, there is no cell loss. Also, with LSC, each measured cell can be relocated and examined visually by conventional or fluorescence microscopy to confirm its identity. With use of this approach, we analyzed apoptosis in 30 primary solid tumors of different types. The number of cells with DNA strand breaks varied from tumor to tumor and ranged from 0.5 to 28.1% (average, 7.0%). FC on the same tumors gave similar results. More than 95% of the relocated cells with DNA strand breaks either had the typical appearance of late apoptosis or showed strong green fluorescence within or at the periphery of the nucleus. Our results suggest that different morphologic variants of apoptosis might be common in different tumor types and that cytometric quantification might provide biologically useful information not otherwise easy to obtain.