Research ArticleBasic Science Investigation

Recording Intracellular Molecular Events from the Outside: Glycosylphosphatidylinositol-Anchored Avidin as a Reporter Protein for In Vivo Imaging

Steffi Lehmann, Elisa Garcia Garayoa, Alain Blanc, Ruth Keist, Roger Schibli and Markus Rudin
Journal of Nuclear Medicine March 2011, 52 (3) 445-452; DOI: https://doi.org/10.2967/jnumed.110.082412

Article Figures & Data

Figures

  • FIGURE 1.
    FIGURE 1.

    Characterization of the avidin–GPI reporter construct (pH3SVG). (A) A schematic drawing of the pH3SVG reporter construct driving the expression of avidin-GPI from a minimal SV40 promoter, further regulated by 3 hypoxia-response elements (HREs). (B) Immunofluorescence staining of a C51 clone, stably expressing pH3SVG. After treatment of cells with dimethyloxalyglycine (top), there was increased expression of avidin–GPI on the cell surface. The first column displays an immunofluorescence staining of avidin, and the second column shows cell nuclei stained with 4′,6-diamidino-2-phenylindole (DAPI);. The third column represents the overlay of the first 2 columns. The scale bar indicates 25 μm. (C) pH3SVG-positive cells treated with dimethyloxalyglycine were simultaneously stained with a FITC-coupled anti-avidin antibody (column 1) and alexa-594-biocytin (column 2). The overlay of the 2 images (column 3) shows colocalization of the 2 stainings. The scale bar is 10 μm.

  • FIGURE 2.
    FIGURE 2.

    FACS analysis to assess HIF-dependent regulation of reporter expression. C51 control cells (A), a C51 clone stably expressing pH3SVG (B), or avidin–GPI pcDNA3.1 (C) were subjected to FACS analysis after treatment with dimethyloxalyglycine. A shift in fluorescence intensity in response to dimethyloxalyglycine treatment was observed only for the pH3SVG cells. (D) Geometric means of the fluorescence intensity histograms (A–C). The geometric mean of pH3SVG cells was significantly increased after dimethyloxalyglycine treatment (P = 0.00002). Values are shown as mean ± SD. Av-GPI = avidin–GPI; DMOG = dimethyloxalyglycine.

  • FIGURE 3.
    FIGURE 3.

    In vivo assessment of avidin–GPI reporter activity. (A) In vivo assessment of tumor fluorescence after intravenous injection of either alexa-594-biocytin or alexa-594-cadaverine in pH3SVG tumor animals. Images only show the spectrally unmixed alexa-594 fluorescence component. (B) Original fluorescence, spectrally unmixed, and white light images for pH3SVG animals treated with biocytin or cadaverine and a C51 control animal injected with the biotinylated compound at 24 h after injection. (C) Tumor-to-background ratios calculated on spectrally unmixed fluorescence images. Whereas this ratio did not change for control animals, there was increased tumor fluorescence in pH3SVG tumors at 24 h after injection of the dye. The high variability between animals may reflect different degrees of hypoxia in the analyzed tumors. Values are shown as mean ± SD.

  • FIGURE 4.
    FIGURE 4.

    Analysis of ex vivo tumor sections showing distribution of alexa-594-biocytin in tumor sections extracted from animals carrying pH3SVG tumors treated with fluorescent biocytin or cadaverine and animals carrying C51 tumors injected with alexa-594-biocytin. Red fluorescence in tumor sections was observed only for pH3SVG animals injected with the biotinylated compound. When analyzing the distribution patterns of the dye and pimonidazole, a marker of hypoxia, we found a good overlap, implying that the reporter activity is increased in hypoxic areas. However, the staining pattern of alexa-594-biocytin extended regions positive for pimonidazole. The scale bar indicates 40 μm.

  • FIGURE 5.
    FIGURE 5.

    In vivo SPECT imaging of avidin–GPI expression. (A) A cell-binding assay demonstrated specific binding of 67Ga-DOTA-biotin to pH3SVG-transfected tumor cells. Dimethyloxalyglycine (DMOG) treatment further potentiated probe binding to these cells. (B) Biodistribution experiments after intravenous injection of 67Ga-DOTA-biotin showed higher tracer uptake in tumors expressing avidin–GPI than in C51 control tumors. This difference in uptake is present at both 3 and 24 h after probe injection. The uptake is displayed as percentage of injected dose normalized to weight of tissue (%ID/g). Values are shown as mean + SD. (C) In vivo SPECT/CT scans confirmed the utility of 67Ga-DOTA-biotin in targeting tumor cells expressing avidin–GPI in vivo. Whereas high activity was detected in positive tumors, the signal arising from negative control tumors of similar size was weak. The tumor surfaces are visualized on an isosurface CT image and marked with arrowheads.

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