HOST-CELL CYTOTOXICITY, CELLULAR REPOPULATION DYNAMICS, AND PHASE-SPECIFIC CELL-SURVIVAL IN X-IRRADIATED RAT RHABDOMYOSARCOMA TUMORS

Postirradiation tumor volume response, cellular repopulation dynamics, cell-cycle perturbations, and phase-specific cell survival were characterized in rat rhabdomyosarcoma R-1 tumors (the R2C5 subline) following an in situ 10-Gy dose of 225-kVp X rays. This X-ray dose produced a 7.5-day delay in tumor growth to twice the volume measured at the time of irradiation, and reduced the initial surviving fraction of R2C5 cells to 0.17 as measured by the excision assay procedure. The surviving fraction of R2C5 cells returned to unity by the 16th day after tumor irradiation. On the basis of flow cytometry measurements of DNA content in tumor cells stained with a noncytotoxic concentration of Hoechst 33342 (5 μM, 2 h, 37°C), a transient G2 block was observed 1 day after irradiation. Flow cytometry measurements also demonstrated that the tetraploid R2C5 cells constituted only 30% of the total tumor cell population, with the remainder being diploid host cells comprised of macrophages, monocytes, lymphocytes, and granulocytes. Large numbers of host cells infiltrated the irradiated tumors, leading to an increase in the percentage of diploid cells by Day 2 and reaching a level of more than 80% of the total tumor cell population by 4 to 8 days after irradiation. The influx of host cells into irradiated tumors was correlated temporally with a significant 12-fold decrease in the surviving fraction of R2C5 cells that occurred between Days 2 and 4 postirradiation. When the diploid host cell population was removed by cell sorting procedures, the surviving fraction of R2C5 cells at Day 4 was substantially greater than that in the presence of the host cells. Experiments involving the mixing of 4/1 and 12/1 ratios of diploid host cells and tetraploid tumor cells isolated from irradiated and unirradiated tumors demonstrated that the cytotoxic effect of the host cells was specific for the irradiated tumor cells. The significant toxic effect of host cells on irradiated tumor cells was observed only at 2 to 4 days after irradiation, and not earlier or later times. The data obtained in these experiments indicate that the immunogenicity of R2C5 cells is increased significantly by irradiation, and a resultant cell-mediated host immune response produces a delayed decrease in tumor cell survival that is most pronounced 4 days after irradiation. The cell survival characteristics of R2C5 cells in different cell-cycle phases were found to be similar throughout the 16-day postirradiation interval that was studied. No differences were observed in the clonogenicity or susceptibility of R2C5 cells to the toxic effect of the host cells in the G1, S, or G2 + M phases of the cell cycle at early or late times following an in situ 10-Gy dose of X rays.