Characterization of radiation resistant hypoxic cell subpopulations in KHT sarcomas. (ii) Cell sorting

Abstract

Summary Hypoxic cells in KHT sarcomas were characterized using fluorescence activated cell sorting based on the diffusion properties of the fluorochrome Hoechst 33342. Tumour-bearing female C3H/HeJ mice were injected i.v. with lOjgg-g Hoechst 33342 and the cells derived from the tumours sorted on the basis of their staining intensities. For each sorted fraction the DNA histogram was evaluated using FCM analysis. The results indicated that the bright and dim cells were not equally distributed about the cell cycle. For example, a greater proportion of S phase cells were in the bright subpopulations whereas the dim subpopulations contained an increased proportion of cells in GV. When the tumours were irradiated with a single dose of radiation prior to cell sorting, the dim cells survived preferentially. Dose response curves for the 20% most dim and 20% most bright cells, sorted on the basis of fluorescence intensity, then were determined. The survival curves of the dim and bright cells were found to have slopes similar to those of KHT cells irradiated in situ in dead animals or in vitro under fully oxic conditions, respectively. In addition, when KHT sarcomabearing mice were given a 2.5mmolkg-1 dose of misonidazole (MISO) prior to irradiation and cell sorting, the dim subpopulation was sensitized whereas the bright subpopulation was not. These findings suggest that (i) compared to well-oxygenated areas, hypoxic regions of KHT tumours contain a smaller percentage of cells actively proliferating and (ii) Hoechst 33342 sorting may allow the detailed in situ evaluation of agents acting directly against hypoxic cells in solid tumours.

Cell cycle distribution of hypoxia and progression of hypoxic tumour cells in vivo

“…Cells are thought to arrest in GI as they move away from blood vessels, becoming hypoxic and nutritionally deprived (Tannock, 1968;Hirst and Denekamp, 1979) (Sutherland et al, 1986). However, our observation of significant numbers of hypoxic S and G/M cells is also supported by other investigators (Pallavicini et al, 1979;Siemann and Keng, 1988). Within each cell cycle phase (defined by DNA content) in SaF, CaNT and Rh tumours, the highest proportion of hypoxic cells was located within G2 ( Figure 4 and Table 3).…”

Intermittent blood flow in the KHT sarcoma--flow cytometry studies using Hoechst 33342

“…We interpret these results as indicating that the blood flow to at least some regions of the tumour is intermittent resulting in a breakdown in the relationship between sort fraction and oxygen status. This conclusion is in contrast to that made by Siemann and Keng (1988) who suggested on the basis of experiments using the injection of stain 20 minutes prior to irradiation, that chronic hypoxia was the dominant form of hypoxia within the KHT/Ro sarcoma. Several explanations for these divergent conclusions can be suggested.…”

“…Although a comparison between the two sites has not been made for either of these versions of the KHT tumour, Brown (1979) documented differences in the radiosensitivity of the RIF-1 tumour when implanted in these two sites. Therefore, it is possible that histological differences both inherent within the tumour and related to the site of implantation, may contribute to the differences observed by Siemann and Keng (1988) and those found in this study. However, we feel it is important when assessing the presence of acute hypoxia to use two different staining protocols, one where the stain is administered simultaneously with the radiation treatment and another where the staining is carried out temporally divorced from the radiation period.…”

“…Several explanations for these divergent conclusions can be suggested. Siemann and Keng (1988) used a different variant of the tumour used in this study and implanted it intramuscularly rather than subcutaneously. Although a comparison between the two sites has not been made for either of these versions of the KHT tumour, Brown (1979) documented differences in the radiosensitivity of the RIF-1 tumour when implanted in these two sites.…”

Radiation sensitivity of tumour cells stained in vitro or in vivo with the bisbenzimide fluorochrome Hoechst 33342

“…In addition, techniques involving diffusion-limited staining of tumour cell populations with H33342 have been developed in an effort to isolate cells from solid tumours as a function of their distance from the vasculature Olive et al, 1985;Loeffler et al, 1987;Siemann & Keng, 1988) or depth in multi-cellular spheroids (Durand, 1982). These procedures have been used to study tumour cells which reside in hypoxic regions, so that the efficacy of treatments directed at this resistant subpopulation can be specifically evaluated.…”