Proliferation and micromilieu during fractionated irradiation of human FaDu squamous cell carcinoma in nude mice

Purpose : Previous functional radiobiological experiments demonstrated a significant acceleration of repopulation after 3 weeks and reoxygenation after 12 days of radiotherapy of FaDu tumours. Owing to the temporal coincidence between repopulation and reoxygenation, it was hypothesized that the improved oxygenation status during fractionated irradiation might be the preceding stimulus for increased proliferation. The study investigated whether these changes in repopulation and re-oxygenation are reflected by histological parameters of proliferation and the tumour micromilieu. Materials and methods : Human FaDu squamous cell carcinomas in nude mice were irradiated with three to 18 fractions of 3 Gy daily or every second day under normal blood flow and clamp hypoxia. At different time points, tumours were excised and stained for Ki67, BrdUrd, epidermal growth factor receptor (EGFR) and markers of the micromilieu (HOECHST 33342, pimonidazole, ER-MP12). Results : On average, Ki67 and BrdUrd labelling indices decreased initially and increased again at later times during the course of fractionated radiotherapy. A similar kinetic pattern was found for the staining intensity of the EGFR. The vascular density in the viable tumour area remained constant during the whole course of irradiation, while the perfused fraction of vessels decreased within the first week of irradiation and returned to baseline values after 2 weeks. There was a corresponding increase in perfusion and a decrease in cellular hypoxia. Conclusions : The histological results were in surprisingly good agreement with the kinetics of clonogen repopulation and re-oxygenation determined previously using functional assays. The results support that the kinetics of repopulation of FaDu squamous cell carcinoma in response to fractionated irradiation are determined not only by intracellular processes, but also by a complex interaction of proliferation parameters with a changing microenvironment.