Figure 1
Post-radiation survivor selection model yields tumor initiating cells. (A) Outline of technique used for the early isolation of medulloblastoma sub-populations enriched for the most radiation tolerant cells. The method uses Propidium Iodide (PI) and Annexin V to exclude sensitive cells via Fluorescence Activated Cell Sorting (FACS). (B) Radiation dose response curves for UW228, Daoy and R001M, generated by Clonogenic Colony Forming Assay (which only includes actively proliferating cells). (C) Radiation dose response curves for Daoy, R001M and R026M, generated by resazurin based viability assay (measures all cells that have not yet died). (D) Profile of apoptosis initiation over time after irradiation of Daoy cells, detected by Immunofluorescence using Annexin V (240 to 1700 cells assessed per treatment, over 3 to 7 wells). Error bars in panels B to D represent +/− 1 SD. “**” = p-value < 0.01, “***” = p-value < 0.001, for control compared with irradiated cells. (E) Tumorsphere culture of Daoy cells surviving 10 Gy radiation. After an initial lag, independent survivor sub-lines (SLs) yield proliferative sphere cultures, which like non-irradiated (control) SLs, are able to be proliferated long term (spheres were passaged weekly). (F) Medulloblastoma cells shown in panel E, that survived 10 Gy, initiated large tumors when implanted into the cerebellum of immuno-compromised mice. Whole brain shown (olfactory bulbs removed) with typical tumor indicated by arrow.