Fig. 4. Targeting the tumor vasculature with selected cystine-knot miniproteins.
a Specific binding of MC-FN-010 to tissue sections derived from the human U-87 MG glioblastoma cell line grown as mouse xenograft tumor. Representative immunofluorescence staining of U-87 MG tumor tissues and normal mouse brain with EDB ligand MC-FN-010 and negative control MC-FN-0115. Tissue sections (6 µm) were stained with tetramerized cystine-knot miniprotein-biotin/strepatividin-Cy3 complex (red) and an anti-CD31 antibody to visualize vasculature (green). Scale bars, 100 µm. b In vivo and ex vivo imaging of U-87 MG bearing mice. Tumors derived from human U-87 MG cells injected s.c. in flanks of mice were imaged after i.v. application of 3.34 nmol AF680-(MC-FN-010)3, AF680-(MC-FN-016)3 (EDB binder), and control AF680-(MC-FN-0115)3. Mice (n = 3) were stratified according to their tumor sizes. c Kinetics of fluorescence signals in different organs quantified and normalized to the respective organ/tumor weight. Each of the two cystine-knot miniproteins was compared to the control (AF680-(MC-FN-010)3 vs. AF680-(MC-FN-0115)3 and AF680-(MC-FN-016)3 vs. AF680-(MC-FN-0115)3, respectively) in a given organ at a given time point. d Kinetics of tumor-to-lung, tumor-to-liver, and tumor-to-kidney ratio. Ratios resulting from ex vivo imaging were derived from normalized fluorescence signals after i.v. injection of 3.34 nmol AF680-(MC-FN-010)3, AF680-(MC-FN-016)3, and AF680-(MC-FN-0115)3 at 1, 2, and 6 h. e Specific tumor targeting of AF680-(MC-FN-010)3 in a competitive experiment. Tumor-to-lung, tumor-to-liver, and tumor-to-kidney ratio of ex vivo imaging are calculated on the basis of the normalized fluorescence signals to the respective tumor weight at 1, 2, and 6 h (Supplementary Fig. 6). All data are shown as mean ± SD of triplicates. Statistical significance was calculated with two-way ANOVA (*p < 0.05; **p < 0.01; ***p < 0.001). Source data are provided as a Source Data file.