Figure 3. Characterization of 3E10 cellular penetration, mechanism of uptake, and tumor targeting.

(A) Representative confocal immunofluorescence images and of HeLa cells treated with 750 nM humanized AlexaFluor 680-labeled 3E10 variants for 24 h. (B) Quantification of (A). n ≥ 250 cells per treatment group. (C, D) Quantification of antibody uptake in K562 cells treated with inhibitors of ENT2 (C) and of canonical cellular uptake pathways (D) as assessed by flow cytometry. Cells were treated with 750 nM AlexaFluor 680-labeled antibody for 2 h. Labeled IgG1 isotype was used as a negative control. Al680: AlexaFluor 680; EIPA: 5-(N-ethyl-N-isopropyl)-amiloride; NBMPR: S-(4-nitrobenzyl)-6-thioinosine. Data are mean ± SEM, n = 3 replicates. (E) Quantification and representative IVIS images of AlexaFluor 680 fluorescence in EMT6 mouse tumors isolated from tumor-bearing mice. Mice (n = 2 per group) were treated intravenously with AlexaFluor 680-labeled antibody (100 μg) and tumors were harvested 24 h after treatment. (F) Representative IVIS images showing fluorescence signal in mice injected intravenously with AlexaFluor680-labeled antibodies. One representative image of all major organs plus EMT6 tumors is shown per treatment group.