1. Volume 25, Issue 1, Pages 181-191 (January 2017)
Disruption of Circulating Extracellular Vesicles as a Novel Therapeutic Strategy against Cancer Metastasis 
Nao Nishida-Aoki, Naoomi Tominaga, Fumitaka Takeshita, Hikaru Sonoda, Yusuke Yoshioka, Takahiro Ochiya 
Molecular Therapy 
Volume 25, Issue 1, Pages (January 2017)
DOI: /j.ymthe
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

2. Molecular Therapy 2017 25, 181-191DOI: (10.1016/j.ymthe.2016.10.009)
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

3. Figure 1
Specific Recognition of Anti-CD9 and Anti-CD63 Antibodies on the Surfaces of Cancer-Derived EVs
(A) A representative image of EVs isolated from MDA-MB-231-luc-D3H2LN cells under transmission electron microscopy. Scale bar, 100 nm. (B) The particle size distributions and concentrations of EVs from MDA-MB-231-luc-D3H2LN cells were measured using a NanoSight nanoparticle tracking system. (C) Immunoelectron microscopy images of MDA-MB-231-luc-D3H2LN EVs labeled by anti-human CD9 and anti-human CD63 antibodies. Scale bar, 100 nm. (D) Human-specific recognitions of anti-human CD9 and anti-human CD63 antibodies. The EVs and cell lysates from a human breast cancer cell line (MDA-MB-231-luc-D3H2LN; depicted as MM231) and a mouse breast cancer cell line (4T1-luc; depicted as 4T1) were analyzed by western blot using anti-human CD9, anti-mouse CD9, anti-human CD63, and anti-mouse CD63 antibodies. Hsp70 was used as the loading control.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

4. Figure 2
Uptake of Cancer-Derived EVs by Macrophages Was Stimulated by the Anti-CD9 and Anti-CD63 Antibodies
(A) Representative observations of the uptake of the breast-cancer-derived EVs by mouse macrophage RAW264.7 cells incubated with cytochalasin D and EIPA. The EVs from MDA-MB-231-luc-D3H2LN were stained with the green fluorescent dye PKH67. The cells were incubated with inhibitors for 30 min prior to the addition of the stained EVs. Uptake was observed under a confocal microscope. Scale bar, 50 μm. (B) Observation of the uptakes by the breast cancer-derived EVs incubated with or without antibodies under confocal microscopy. The EVs were stained by PKH67 and incubated with the same protein amount of anti-human CD9, anti-human CD63, control IgG, or without any antibody. PBS without EVs stained by PKH67 was added for the controls. In the lower panel, the cells were incubated with cytochalasin D (cytoD) for 30 min prior to the addition of stained EVs. The images were collected 6 hr after supplementing the cells with EVs. Scale bar, 50 μm. (C) The amount of EVs that was incorporated into the macrophages, as evaluated by the fluorescence intensities of the images in Figure S1C. The cells were incubated with EVs from MDA-MB-231-luc-D3H2LN cells with anti-human CD9, anti-human CD63, control IgG, or PBS. After 15 hr, the cells were washed, fixed, and observed using fluorescent microscopy. The averages of the mean brightness of five observed fields from independent wells are shown. The error bars represent the ± SDs. **p < 0.01, *p < 0.05, ANOVA with Dunnett’s test. (D) The numbers of EVs remaining in the culture medium after 15 hr of incubation. The numbers of remaining EVs were evaluated after 15 hr by measuring the fluorescences of the culture media using a plate reader. The graph represents the means ± SDs of five independent well measurements. RFU, relative fluorescence units. ***p < 0.001, ANOVA with Dunnett’s test.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

5. Figure 3
Treatments with the Anti-CD9 and Anti-CD63 Antibodies Did Not Alter Primary Tumor Growth or Metastatic Ability of Cancer Cells
(A) A timetable of the tumor implantation, antibody injection, in vivo imaging, and dissection. (B) Mouse weight shift. The average weights of each treatment group at each time point are shown. The graph represents the mean ± SD. CD9 n = 16, CD63 n = 13, IgG n = 18, PBS n = 9. (C) Primary tumor size as evaluated by the photon radiance of the cancer cell bioluminescence at each indicated area. Representative images from mice in each treatment group are shown (left panel). The regions of interest are circled in red (right panel). The primary tumor sizes were quantified using photon radiance. The data are shown as the mean ± SD. CD9 n = 16, CD63 n = 13, IgG n = 18, PBS n = 9. (D) Representative images of the gross tumors dissected from tumor-bearing mice (left panel). Scale bar, 1 cm. The average approximate tumor volumes from each treatment group were calculated based on the longest and shortest diameters (right panel). CD9 n = 5, CD63 n = 13, IgG n = 7. Error bars represent ± SD.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

6. Figure 4
Anti-CD9 and Anti-CD63 Antibodies Suppressed the Pulmonary Metastasis of Breast Cancer Cells
(A) Metastatic cancer cells in the areas of the axillary lymph nodes and thoracic cavity at day 35 were evaluated using the photon radiance of the cancer cell bioluminescence after dissection. Representative images from the mice of each group are shown below. CD9 n = 16, CD63 n = 13, IgG n = 18, PBS n = 9, and the bars represent the lower quartile, median, and upper quartile. The regions of interest are depicted in red. **p < 0.01 by the Steel-Dwass method, followed by a Kruskal-Wallis rank test. (B) Metastatic cancer cells in the lungs at day 35, as evaluated by the photon radiances of the cancer cell bioluminescence from the whole lungs. Representative in vivo images of the mice from each group are shown in the right panel. The regions of interest are depicted in red. *p < 0.05 and **p < 0.01 by the Steel-Dwass method, followed by a Kruskal-Wallis rank test. CD9 n = 11, CD63 n = 13, IgG n = 13, PBS n = 5. The bars represent the lower quartile, median, and upper quartile. (C) Immunohistochemistry of the metastatic lungs. Metastatic cancer cells were labeled with anti-human vimentin antibodies. The black arrows indicate the metastatic foci. The lower panel depicts a magnification of the area in the black frame of the upper panel. Scale bars, 500 μm (upper panel) and 100 μm (lower panel). (D) Quantifications of the metastatic foci in the lungs of each treatment group. The numbers of metastatic foci per area (cm2) were calculated. CD9 n = 5, CD63 n = 13, IgG n = 7. *p < 0.05 and **p < 0.01 by the Steel-Dwass method, followed by a Kruskal-Wallis rank test. The bars represent the lower quartile, median, and upper quartile.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

7. Figure 5
Macrophage-Dependent Stimulation of EV Clearance by Anti-CD9 and Anti-CD63 Antibodies
The localizations of the EVs with anti-CD9 or anti-CD63 antibodies in the whole organs were evaluated using the DiR-stained EVs. The results from mice without treatment (macrophage +, the left panel) and with depletion of the macrophages with clodronate liposomes (macrophage −, the right panel) are shown. Representative images are shown in the lower panels. The DiR-stained EVs were incubated with antibodies and intravenously injected into mice via the tail vein. PBS without EVs stained by DiR was injected for the controls. The relative amounts of EV were evaluated according to the radiant efficiency in the areas of the organs at 24 hr after injection. The graph represents the mean ± SD. Macrophage +: EVs only n = 11, CD9 n = 9, CD63 n = 9, IgG n = 5. Macrophage −: EVs only n = 6, CD9 n = 4, CD63 n = 4, IgG n = 4. ***p < 0.001, ANOVA with Dunnett’s test.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions