Journal: Scientific Reports

Article Title: In vivo imaging of long-term accumulation of cancer-derived exosomes using a BRET-based reporter

doi: 10.1038/s41598-020-73580-5

Figure Lengend Snippet: CD63-Antares2 expression enables the detection of exosomes at long wavelength. ( a ) Schematic diagram of Antares2 and Antares2-fused CD63 (CD63-Antares2). ( b ) Western blot analysis of control (Mock), CD63-Antares2-, and Antares2-expressing PC3 cells. Total cell lysates were immunoblotted with antibodies against the indicated proteins. ( c ) BRET signal of culture media containing PC3/CD63-Antares2 cells that were treated with FRZ or DTZ (500, 50, 5, and 0.5 μM) and imaged using the in vivo imaging system (IVIS). The data are representative of at least three independent experiments. ( d ) Emission spectrum of CD63-Antares2 in culture medium containing PC3/CD63-Antares2 cells. ( e ) BRET signal intensities in culture medium before and after ultracentrifugation (UC). The upper image shows BRET signals visualized using IVIS. ( f ) Western blot analysis of CD63-Antares2 and Antares2 expression in exosomes secreted from the cells indicated in ( b ). TSG101 and ALIX were used as exosome marker proteins. ( g ) Size distribution of isolated exosome particles derived from the cells indicated in ( b ) by NTA analysis. ( h ) Effect of ectopic CD63-Antares2 or Antares2 expression on exosome production. Exosome number per cell from the data ( g ) are expressed as means ± SD. All data are representative of at least 3 independent experiments each. *** P < 0.001, by ANOVA with Dunnett's post hoc analysis. Uncropped gel images for panels d and f are shown in Supplementary Fig. .

Article Snippet: A mixture of EGFP-labeled PC3/CD63-Antares2 cells (1 × 10 6 cells/100 μL) and Matrigel (100 μL) was subcutaneously implanted into 5-week-old BALB/c-nu/nu male mice (Japan SLC Inc., Shizuoka, Japan).

Techniques: Expressing, Western Blot, In Vivo Imaging, Marker, Isolation, Derivative Assay

Journal: Scientific Reports

Article Title: In vivo imaging of long-term accumulation of cancer-derived exosomes using a BRET-based reporter

doi: 10.1038/s41598-020-73580-5

Figure Lengend Snippet: PC3/CD63-Antares2 xenograft mice model enables continuous exosome tracking analysis in vivo. ( a ) Timeline of in vivo exosome tracking analysis in a xenograft mouse model established using PC3/CD63-Antares2- and EGFP-expressing (PC3/CD63-Antares2/EGFP) cells. Whole body imaging analyses using IVIS and BRET intensity measurement in blood samples were performed every 5 days after subcutaneous inoculation of control (PC3/Mock) or PC3/CD63-Antares2/EGFP cells into the mice. ( b ) Tumor growth in control (PC3/Mock) and PC3/CD63-Antares2/EGFP xenograft mice. ( c ) Time course of BRET signal in the blood of control (PC3/Mock) and PC3/CD63-Antares2/EGFP xenograft mice. ( d ) Sequential bioluminescence images of the whole body of control (PC3/Mock) and PC3/CD63-Antares2/EGFP xenograft mice. BRET emission derived from Antares2 was imaged using IVIS. ( e ) Bioluminescence images of organs harvested from control (PC3/Mock) and PC3/CD63-Antares2/EGFP tumor-bearing mice 5 weeks after inoculation. BRET emission derived from Antares2 and EGFP fluorescence were imaged using IVIS. ( f ) Immunohistochemical analysis of Nluc (Antares2) in the lungs, spleen, mesenteric nodes, and adipose tissues from mice bearing PC3/CD63-Antares2/EGFP cells (40 ×). Boxed with dotted line images are enlarged inset of the panels. Scale bar = 100 μm. Representative images from three mice are shown in (d, e, and f). Means ± SDs of tumor volume and luminescence intensity of BRET were obtained from three mice (b and c).

Article Snippet: A mixture of EGFP-labeled PC3/CD63-Antares2 cells (1 × 10 6 cells/100 μL) and Matrigel (100 μL) was subcutaneously implanted into 5-week-old BALB/c-nu/nu male mice (Japan SLC Inc., Shizuoka, Japan).

Techniques: In Vivo, Expressing, Imaging, Derivative Assay, Fluorescence, Immunohistochemical staining

Journal: Scientific Reports

Article Title: In vivo imaging of long-term accumulation of cancer-derived exosomes using a BRET-based reporter

doi: 10.1038/s41598-020-73580-5

Figure Lengend Snippet: The PC3/CD63-Antares2 xenograft mouse model is useful for evaluating the effects of exosome inhibitors in vivo. ( a ) Numbers of exosomes produced by PC3/CD63-Antares2 cells treated with DMSO or dasatinib (50 or 100 nM). ( b ) Timeline of in vivo exosome tracking analysis using the PC3/CD63-Antares2 xenograft mouse model. Dasatinib was administrated intraperitoneally once every 3 days. Whole body imaging using IVIS and BRET intensity measurement using blood samples were performed every 5 days after subcutaneous inoculation of PC3/CD63-Antares2 cells into the mice. ( c ) PC3/CD63-Antares2 cells were inoculated subcutaneously into the mice. When the tumor volume reached approximately 100 mm 3 , saline (Control) or dasatinib (2.5 mg/kg or 5 mg/kg) was administrated. Mean ± SD values of tumor volume (mm 3 ) obtained from 3 mice are plotted against days after inoculation. ( d ) Sequential BRET signal in the blood samples from control (saline administrated) or dasatinib-administrated (2.5 mg or 5 mg/kg) PC3/CD63-Antares2 xenograft mice. Means ± SDs of BRET signal obtained from three mice are plotted against days after inoculation. ( e ) Bioluminescence images of organs excised from control (saline-administrated) and dasatinib-administrated PC3/CD63-Antares2 xenograft mice 5 weeks after inoculation. (f) Immunohistochemical analysis of Nluc (Antares2) in the lungs and spleen from control (saline administrated) and dasatinib-administrated PC3/CD63-Antares2 xenograft mice (40 ×). Boxed in dotted line images are enlarged inset of the panels. Scale bar = 100 μm. * P < 0.05, ** P < 0.01, by ANOVA with Dunnett's post hoc analysis.

Article Snippet: A mixture of EGFP-labeled PC3/CD63-Antares2 cells (1 × 10 6 cells/100 μL) and Matrigel (100 μL) was subcutaneously implanted into 5-week-old BALB/c-nu/nu male mice (Japan SLC Inc., Shizuoka, Japan).

Techniques: In Vivo, Produced, Imaging, Immunohistochemical staining