Characterization of hypoxic cells in solid tumors

The authors utilized (a) centrifugal elutriation in conjunction with flow cytometric (FCM) analysis as well as (b) fluorescence activated cell sorting (FACS) based on the diffusion properties of the fluorochrome Hoechst 33342 to characterize hypoxic cells in KHT sarcomas. In the elutriation studies, tumors were irradiated with a range of doses in situ either in dead or air-breathing animals prior to separating the cells into various phases of the cell cycle. Clonogenic cell survival in these phases then was determined and the results showed that hypoxic cells in KHT sarcomas existed primarily in the G/sub 1/ phase. For comparison, KHT sarcoma-bearing mice were injected i.v. with Hoechst 33342 and the cells derived from the tumors sorted on the basis of their Hoechst 33342 staining intensity. The results indicated that the oxic (bright) and hypoxic (dim) cell subpopulations were not equally distributed about the cell cycle. When comparing the 10% most bright and 10% most dim populations they observed (i) a greater proportion of cycling (S) cells in the bright subpopulation than in the total cell population, and (ii) --90% of the dim cells to be in the G/sub 1/ phase. The latter results were consistent with the elutriation studies of hypoxic cells. These investigations suggest that cell separation will aid understanding of the nature of hypoxic cells and interpretation of tumor responses to drugs and radiation