Radiation treatment did not notably alter the increased expression of vimentin in TTA with CSC grown in hypoxia. TTA − CSC and TTA + CSC maintained in hypoxia at 5 days after 5 Gy irradiation were incubated with rabbit anti-vimentin polyclonal antibody followed by anti-rabbit secondary Alexa Fluor 633 (red, panel A) and monoclonal mouse anti-vimentin (Dako, Inc.) followed by subsequent DAB staining (panel B). Treatment regimens are as follows (left to right for panels A and B): i. TTA − CSC control; ii. TTA − CSC 5 Gy radiation; iii. TTA + CSC control; iv. TTA + CSC 5 Gy radiation. Panel C: Intensity analysis of antibody staining for vimentin in the respective groups shown in panel A. The average pixel intensity for vimentin in the different treatment groups was analyzed using ImageJ software. Mean ratio values are shown. Abbreviations are as follows: −CSC (A549, endothelial, fibroblast), +CSC (A549, endothelial, fibroblast, CSC). (n = 4 TTA were used per treatment assess spheroid size.) **P < 0.009 and *P < 0.01 indicate a statistically significant difference between the treatments.