Proliferation and
hypoxia affect the efficacy of
radiotherapy, but radiation by itself also affects the tumor microenvironment. The purpose of this study was to analyze temporal and spatial changes in
hypoxia, proliferation and apoptosis after irradiation (20 Gy) in cells of a murine
adenocarcinoma tumor line (C38). The
hypoxia marker
pimonidazole was injected 1 h before irradiation to label cells that were hypoxic at the time of irradiation. The second
hypoxia marker,
CCI-103F, and the proliferation marker BrdUrd were given at 4, 8 and 28 h after irradiation. Apoptosis was detected by means of activated
caspase 3 staining. After immunohistochemical staining, the
tumor sections were scanned and analyzed with a semiautomatic image analysis system. The hypoxic fraction decreased from 22% in unirradiated
tumors to 8% at both 8 h and 28 h
after treatment (P < 0.01). Radiation did not significantly affect the fraction of perfused vessels, which was 95% in unirradiated
tumors and 90%
after treatment. At 8 h after irradiation, minimum values for the BrdUrd labeling index (LI) and maximum levels of apoptosis were detected. At 28 h
after treatment, the BrdUrd labeling and density of apoptotic cells had returned to pretreatment levels. At this time, the cell density had decreased to 55% of the initial value and a proportion of the cells that were hypoxic at the time of irradiation (
pimonidazole-stained) were proliferating (BrdUrd-labeled). These data indicate an increase in
tumor oxygenation after irradiation. In addition, a decreased
tumor cell density without a significant change in
tumor blood perfusion (Hoechst labeling) was observed. Therefore, it is likely that in this
tumor model the decrease in tumor cell hypoxia was caused by reduced oxygen consumption.