Dataset Information

Development of a CD63 Aptamer for Efficient Cancer Immunochemistry and Immunoaffinity-Based Exosome Isolation.

ABSTRACT: CD63, a member of transmembrane-4-superfamily of tetraspanin proteins and a highly N-glycosylated type III lysosomal membrane protein, is known to regulate malignancy of various types of cancers such as melanoma and breast cancer and serves as a potential marker for cancer detection. Recently, its important role as a classic exosome marker was also emphasized. In this work, via using a magnetic bead-based competitive SELEX (systematic evolution of ligands by exponential enrichment) procedure and introducing a 0.5M NaCl as elution buffer, we identified two DNA aptamers (CD63-1 and CD63-2) with high affinity and specificity to CD63 protein (K_d = 38.71nM and 78.43, respectively). Furthermore, CD63-1 was found to be efficient in binding CD63 positive cells, including breast cancer MDA-MB-231 cells and CD63-overexpressed HEK293T cells, with a medium binding affinity (K_d~ 100 nM) as assessed by flow cytometry. When immunostaining assay was performed using clinical breast cancer biopsy, the CD63-1 aptamer demonstrated a comparable diagnostic efficacy for CD63 positive breast cancer with commercial antibodies. After developing a magnetic bead-based exosome immunoaffinity separation system using CD63-1 aptamer, it was found that this bead-based system could effectively isolate exosomes from both MDA-MB-231 and HT29 cell culture medium. Importantly, the introduction of the NaCl elution in this work enabled the isolation of native exosomes via a simple 0.5M NaCl incubation step. Based on these results, we firmly believe that the developed aptamers could be useful towards efficient isolation of native state exosomes from clinical samples and various theranostic applications for CD63-positive cancers.

SUBMITTER: Song Z

PROVIDER: S-EPMC7730289 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Development of a CD63 Aptamer for Efficient Cancer Immunochemistry and Immunoaffinity-Based Exosome Isolation.

Song Zhenguo Z Mao Jun J Barrero Roberto A RA Wang Peng P Zhang Fengqiu F Wang Tao T

Molecules (Basel, Switzerland) 20201127 23

CD63, a member of transmembrane-4-superfamily of tetraspanin proteins and a highly N-glycosylated type III lysosomal membrane protein, is known to regulate malignancy of various types of cancers such as melanoma and breast cancer and serves as a potential marker for cancer detection. Recently, its important role as a classic exosome marker was also emphasized. In this work, via using a magnetic bead-based competitive SELEX (systematic evolution of ligands by exponential enrichment) procedure and ...[more]

PMID: 33261145

Similar Datasets

Project description:Exosomes are defined as submicron (30-150 nm), lipid bilayer-enclosed extracellular vesicles (EVs), specifically generated by the late endosomal compartment through fusion of multivesicular bodies with the plasma membrane. Produced by almost all cells, exosomes were originally considered to represent just a mechanism for jettisoning unwanted cellular moieties. Although this may be a major function in most cells, evolution has recruited the endosomal membrane-sorting pathway to duties beyond mere garbage disposal, one of the most notable examples being its cooption by retroviruses for the generation of Trojan virions. It is, therefore, tempting to speculate that certain cell types have evolved an exosome subclass active in intracellular communication. We term this EV subclass "signalosomes" and define them as exosomes that are produced by the "signaling" cells upon specific physiological or environmental cues and harbor cargo capable of modulating the programming of recipient cells. Our recent studies have established that signalosomes released by mesenchymal stem/stromal cells (MSCs) represent the main vector of MSC immunomodulation and therapeutic action in animal models of lung disease. The efficacy of MSC-exosome treatments in a number of preclinical models of cardiovascular and pulmonary disease supports the promise of application of exosome-based therapeutics across a wide range of pathologies within the near future. However, the full realization of exosome therapeutic potential has been hampered by the absence of standardization in EV isolation, and procedures for purification of signalosomes from the main exosome population. This is mainly due to immature methodologies for exosome isolation and characterization and our incomplete understanding of the specific characteristics and molecular composition of signalosomes. In addition, difficulties in defining metrics for potency of exosome preparations and the challenges of industrial scale-up and good manufacturing practice compliance have complicated smooth and timely transition to clinical development. In this manuscript, we focus on cell culture conditions, exosome harvesting, dosage, and exosome potency, providing some empirical guidance and perspectives on the challenges in bringing exosome-based therapies to clinic.

Dataset Information

Development of a CD63 Aptamer for Efficient Cancer Immunochemistry and Immunoaffinity-Based Exosome Isolation.

Publications

Development of a CD63 Aptamer for Efficient Cancer Immunochemistry and Immunoaffinity-Based Exosome Isolation.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets