Immunoelectron microscope observation of exosomes using anti-exosome antibodies

 

Nao Nishida- Aoki Department of Molecular and Cellular Therapy, National Cancer Center Research Institute

Products

• Anti CD9 for Exosome Isolation, Human (Mouse) Unlabeled, 12A12 (Product number: CAC-SHI-EXO-M01)
• Anti CD63 for Exosome Isolation, Human (Mouse) Unlabeled, 8A12 (Product number: CAC-SHI-EXO-M02)

Manufacturer: Cosmo Bio Co., Ltd.

■ Monoclonal antibody for exosome isolation Anti CD9, CD63, CD81

This product is an antibody that specifically recognizes CD9, CD63, and CD81 known as exosome markers, and is an antibody that can isolate exosomes from serum and culture supernatant using immunoprecipitation method.

• Recognize exosome membrane proteins CD9, CD63, CD81 with high specificity
• Exosome surface antigen protein, endogenous RNA (miRNA), useful for protein analysis

Experiment content

Exosomes are vesicles with a lipid bilayer membrane with a diameter of about 100 nm. Cancer cells secrete exosomes to the outside of the cells and adversely affect surrounding cells, promoting cancer invasion and metastasis. If so, could cancer metastasis be suppressed by inhibiting the function of exosomes derived from cancer cells? Therefore, we started experiments with the aim of inhibiting the action of exosomes derived from cancer cells and suppressing cancer metastasis using antibodies that bind to exosomes.

This time, we used mice transplanted with human breast cancer cells as a metastasis model, and used anti-human CD9 antibody (clone 12A12) and anti-human CD63 antibody (clone 8A12) as antibodies to recognize exosomes derived from human breast cancer cells in this mouse model. decided to administer to First, it is necessary to confirm that the antibody binds to the surface of exosomes isolated from breast cancer cells. Since exosomes are vesicles with a diameter of 100 nm, they cannot be observed at normal microscope magnification and must be observed using an electron microscope. There are other ways to confirm that antibodies are bound to exosomes, but seeing is believing. We decided to directly observe by immunoelectron microscopy whether the antibody binds to the exosomes.　

However, immuno-electron microscopy requires examination of various conditions, and it is difficult to try unless an antibody that reliably binds to the target molecule is used. These antibodies had a proven track record of being used for immunoprecipitation and were found to bind to CD9 and CD63 on the surface of exosomes, so it was easy to try them. In fact, CD63 was easily detected, but CD9 was difficult to detect. After changing various conditions, we were finally able to detect it (Fig. 1).

Figure 1. Detection of human CD9 and CD63 proteins on the surface of exosomes by immunoelectron microscopy
(A) Using an anti-human CD9 antibody (clone 12A12), we detected CD9 present on the surface of exosomes derived from human breast cancer cells. (B) Anti-human CD63 antibody (clone 8A12) was used to detect CD63 present on the surface of exosomes derived from human breast cancer cells.

Also, for the purposes of this experiment, species specificity between mice and humans is very important. This is because if an antibody administered to a mouse binds not only to CD9 and CD63 derived from human cancer cells but also to CD9 and CD63 in the body of the mouse, it can be easily predicted that the antibody will be toxic. These antibodies have very high species specificity, and human-derived CD9 and CD63 could be easily detected by Western blotting. could not be detected (Fig. 2). Perhaps for this reason, even when human CD9 and CD63 antibodies were administered to mice, clear toxicity was not observed, and antibody administration experiments could proceed.

Figure 2. Examples of detection of
CD9 and CD63 proteins by Western blot Human breast cancer cell line (MDA -MB-231-D3H2LN) and mouse breast cancer cell line (4T1-luc)-derived cell lysates were detected for CD9 and CD63.

Based on the above results, we were convinced that these antibodies specifically bind to exosomes derived from breast cancer cells. ⁽¹ ) . We believe that the success of this experiment is largely due to the high-quality antibodies. I hope this antibody will help more people in their research.

 

 

 

Product use literature

1. Nishida-Aoki, N., N. Tominaga, F. Takeshita, H. Sonoda, Y. Yoshioka, and T. Ochiya. 2017. 'Disruption of Circulating Extracellular Vesicles as a Novel Therapeutic Strategy against Cancer Metastasis', Mol Ther, 25 : 181-91.