Immunoelectron microscopy of exosomes using anti-exosome antibodies

 

User Report

Nao Nishida-Aoki

National Cancer Center Research Institute of Molecular and Cellular Therapy

Products

  • ●Anti CD9 for Exosome Isolation, Human (Mouse) Unlabeled, 12A12（Cat.No. SHI-EXO-M01）
  • ●Anti CD63 for Exosome Isolation, Human (Mouse) Unlabeled, 8A12（Cat.No. SHI-EXO-M02）
Products Page

Experimental findings

Exosomes are lipid bilayer-delimited vesicles of about 100 nm in diameter. Cancer cells adversely affect surrounding cells by secreting exosomes that promote cancer invasion and metastasis. We wondered whether inhibiting the function of exosomes derived from cancer cells would suppress cancer metastasis. Toward this end, we used antibodies that bind to exosomes to inhibit the action of exosomes derived from cancer cells.

Antibodies that recognize exosomes from human breast cancer cells were sought for use in a mouse xenotransplantation model with human breast cancer cells. First, we wanted to check whether anti-human CD9 and anti-human CD63 would bind to the surface of exosomes isolated from breast cancer cell conditioned media. The small size of exosomes prevented the use of conventional microscopy. Thus, it was necessary to employ immunoelectron microscopy. However, immunoelectron microscopy is challenging and requires antibodies that bind well to their targets.

Anti-human CD9 (clone 12A12) and anti-human CD63 (clone 8A12) have proven track records in immunoprecipitation and were thus promising candidates to test in immunoelectron microscopy. These reagents easily detected CD63. The detection of CD9, however, required substantial protocol modifications before we achieved successful staining (Figure 1). In addition, for the purposes of this experiment, it was important that the chosen antibodies have exquisite specificity for human and not mouse to prevent toxicity and off-target effects. The antibodies were found to be very human-specific in western blot experiments, readily detecting CD9 and CD63 derived from human, but not mice (Figure 2).

Having established detection protocols and the human specificity of both antibodies, it was possible to advance to in vivo studies. Anti-human CD9 (clone 12A12) and anti-human CD63 (clone 8A12) were administered in the murine xenograft model of breast cancer to determine whether it was possible to suppress metastasis. We were able to successfully inhibit cancer metastasis in this model (Reference 1). We consider the success of this experiment to be solely due to the high quality of the antibodies we used. I hope that these antibodies are able to help more people in their studies.

Figures

Figure 1. Immunoelectron microscopic detection of human CD9 and CD63 protein on exosome surfaces

(A) Anti-human CD9 antibody (clone 12A12) detected CD9 on the surface of exosomes from human breast cancer cells. (B) Anti-human CD63 antibody (clone 8A12) also detected CD63 on the surface of exosomes from human breast cancer cells.

Figure 2. Western blot for human and mouse CD9 and CD63 protein

Anti-human CD9 antibody (clone 12A12), anti-human CD63 antibody (clone 8A12), anti-mouse CD9 antibody and anti-mouse CD63 antibody were used to probe blots containing lysates from human breast cancer cell lines (MDA-MB-231-lucD3H2LN) and murine breast cancer cell line (4T1-luc).

References

  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.