Retention of fluorescently labeled affibodies (black, WT; blue, N23BP; red, Z) in 3D tumor spheroids grown from transfected 4T1 cells either (A) induced with 15 μg/mL cumate or uninduced (B). Additional induced spheroids were irradiated with 365 nm light for 30 min after 3.5 h of incubation and again measured for retention (dashed lines, triangles) and compared to nonirradiated spheroids (solid lines, circles). Concentrations of retained affibodies were calculated by comparing spheroid lysate fluorescence to standard curves prepared for each labeled affibody. (C) Retention of fluorescently labeled affibodies (black, WT; blue, N23BP) with (dotted line, diamonds) or without (dashed line, triangles) addition of EGF.