TY - JOUR
T1 - Development of a cd63 aptamer for efficient cancer immunochemistry and immunoaffinity-based exosome isolation
AU - Song, Zhenguo
AU - Mao, Jun
AU - Barrero, Roberto A.
AU - Wang, Peng
AU - Zhang, Fengqiu
AU - Wang, Tao
N1 - Funding Information:
F.Z. and J.M. acknowledges the funding from the National Natural Science Foundation of China (No.81773175 and No.11704343) and the China Postdoctoral Science Foundation (No. 2018M630839). Program for tackling key problems of of Henan Department of Science and Technolody, No.202102310033.
Funding Information:
Funding: F.Z. and J.M. acknowledges the funding from the National Natural Science Foundation of China (No.81773175 and No.11704343) and the China Postdoctoral Science Foundation (No. 2018M630839). Program for tackling key problems of of Henan Department of Science and Technolody, No.202102310033.
Publisher Copyright:
© 2020 by the authors. Licensee MDPI, Basel, Switzerland.
PY - 2020/12/1
Y1 - 2020/12/1
N2 - 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 0.5 M NaCl as elution buffer, we identified two DNA aptamers (CD63-1 and CD63-2) with high affinity and specificity to CD63 protein (Kd = 38.71 nM 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 (Kd ~ 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 0.5 M 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.
AB - 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 0.5 M NaCl as elution buffer, we identified two DNA aptamers (CD63-1 and CD63-2) with high affinity and specificity to CD63 protein (Kd = 38.71 nM 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 (Kd ~ 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 0.5 M 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.
KW - Aptamer
KW - Breast cancer
KW - Cancer
KW - CD63
KW - Exosome
KW - SELEX
UR - http://www.scopus.com/inward/record.url?scp=85097035999&partnerID=8YFLogxK
U2 - 10.3390/molecules25235585
DO - 10.3390/molecules25235585
M3 - Article
C2 - 33261145
SN - 1420-3049
VL - 25
JO - Molecules
JF - Molecules
IS - 23
M1 - 5585
ER -