Low dose-rate irradiation with [³H]-labelled valine to selectively target hypoxic cells in a human colorectal cancer xenograft model

Figures & data

Figure 1. HT29 colorectal cancer cells grown in conditions of intermittent hypoxia (4% oxygen in the atmosphere) and irradiated continuously by low dose-rate [³H]-valine with a dose-rate of 15 or 2.5 mGy/h. Panel A shows the pericellular oxygen concentration of cells irradiated with 15 mGy/h and panel B shows the plating efficiency of cells irradiated with 15 mGy/h, 2.5 mGy/h or an unirradiated control, all seeded on the same day. At confluence, five parallel flasks were seeded for colony formation (marked with arrows in panel A). Standard errors are shown with bars. The cells were recultured four times during the experiment (black stars in panel A) and medium was changed once between recultivations (white stars). In panel C, nuclei DNA histograms of cells grown under conditions of intermittent hypoxia (4% oxygen in the atmosphere) and irradiated with 15 or 2.5 mGy/h for 29 days are shown together with an unirradiated control grown in the same conditions for 29 days.

Figure 2. [³H]-valine turnover in tumor tissue and serum of a PC-3 prostate cancer xenograft model. Turnover of radioactivity was assessed after an initial injection of 41.7 μCi [³H]-valine. Every 4–5 days three mice were sacrificed and tumor activity was measured by scintillation counting. Error bars represent standard error, n = 3.

Figure 3. Tumor volume (panel A) and mouse weight (panel B) of HT29 colorectal cancer xenografts grown in athymic mice, that either were given continuous low dose-rate irradiation with [³H]-valine (eight mice, 16 tumors) or were untreated (control, seven mice, 14 tumors). The dose-rate was maintained constant by repeated i.v. injections of [³H]-valine. Tumor volume was measured with a caliper. Each point represents the mean of minimum 14 tumors and error bars represents standard error.

Figure 4. Hypoxia distribution within HT29 colorectal cancer xenografts after 25 days of treatment with ultra-low dose-rate [³H]-valine irradiation. The tumor volumes were approximately 600 mm³. An example is shown in panel A (HE stained slide) and panel B (pimonidazole stained slide visualizing hypoxia in brown). The tumors were widely hypoxic with areas of necrosis throughout the tumors. Hypoxic tumor regions were identified computationally and are shown in red in panels C and D, and in the excerpt in panels E and F. Necrotic regions were manually delineated by visual inspection of both pimonidazole and HE stained slides, as shown in yellow in panels C–F. The remaining tumor tissue is shown in black (panels C–F).

Figure 5. The upper panel shows an excerpt of a pimonidazole stained slide in a tumor that was given continuous low dose-rate irradiation for 25 days. The lower panel shows the same excerpt of the neighboring slide where tritium autoradiography was performed. Direct comparison between the panels was possible, since specific regions and landmarks were recognized in both panels. Aerobic (A), necrotic (N) and hypoxic (H) regions were delineated in the pimonidazole slides and transferred to the autography slides, as indicated. Black spots show uptake of [³H]-valine, thereby visualizing [³H]-valine incorporation into cellular protein. There was no incorporation of [³H]-valine in hypoxic or necrotic regions, as shown in the lower panel. [³H]-valine was well incorporated in the aerobic regions.