FIGURE 1 from Mechanistic Characterization of Cancer-associated Fibroblast Depletion via an Antibody–Drug Conjugate Targeting Fibroblast Activation Protein

HuB12 internalizes into FAP-expressing cells. A, Confocal microscopy images of hPrCSC-44 cells (top) or PC3 cells (bottom) after incubation with Alexa Fluor-647 (AF647)-labeled huB12 (50 nmol/L) for 1 hour. Single-channel images of huB12-AF647 localization, fluorescein-dextran labeled endosomes, and CellBrite Orange-labeled plasma membrane are shown. Composite images depicting whole-cell and enlarged regions of interest are shown as colored fluorescence overlays. B, Quantification of the organellar distribution of huB12-AF647, assessed using MCC with either fluorescein dextran (endosome marker) or CellBrite Orange (membrane marker). Values represent average ± SD of n ≥ 6 images, *, P <0.05 using two-tailed Student’s t-test. C, Representative images of CWR-R1FAP or parental CWR-R1 cells after 16 hours incubation with the indicated antibody (40 nmol/L). A panel of phase-contrast images overlaid with an outline of individual cells (yellow) and pHrodoRed fluorescence (red) is shown. D, Time course of pHrodoRed fluorescence intensity in CWR-R1FAP cells incubated with the indicated concentration of huB12-pHrodoRed, data shown from a representative experiment. Dose–response curves of peak pHrodoRed fluorescence values in CWR-R1 (gray) or CWR-R1FAP (red) cells after 24 hours incubation with increasing concentrations of huB12-pHrodoRed (E) or isotyped control IgG-pHrodoRed (F). Values represent average ± SEM from n = 3 replicates.