TY - JOUR
T1 - Oligonucleotide conjugated antibodies permit highly multiplexed immunofluorescence for future use in clinical histopathology
AU - Mcmahon, Nathan P.
AU - Jones, Jocelyn A.
AU - Kwon, Sunjong
AU - Chin, Koei
AU - Nederlof, Michel A.
AU - Gray, Joe W.
AU - Gibbs, Summer L.
N1 - Funding Information:
We would like to thank Gemma Kmetz-Gonzalez and Trevor Bingham for experimental assistance. We would like to thank Jenny Eng and Ting Zheng for insightful discussions. We would also like to acknowledge the Oregon Health and Science University Advanced Light Microscopy Core for their assistance in imaging data acquisition. This work was generously funded by NIH/NCI R44CA224994 (Nederlof) and The Prospect Creek Foundation (Gray).
Publisher Copyright:
© The Authors.
PY - 2020/5/1
Y1 - 2020/5/1
N2 - Significance: Advanced genetic characterization has informed cancer heterogeneity and the challenge it poses to effective therapy; however, current methods lack spatial context, which is vital to successful cancer therapy. Conventional immunolabeling, commonplace in the clinic, can provide spatial context to protein expression. However, these techniques are spectrally limited, resulting in inadequate capacity to resolve the heterogenous cell subpopulations within a tumor. Aim: We developed and optimized oligonucleotide conjugated antibodies (Ab-oligo) to facilitate cyclic immunofluorescence (cyCIF), resulting in high-dimensional immunostaining. Approach: We employed a site-specific conjugation strategy to label antibodies with unique oligonucleotide sequences, which were hybridized in situ with their complementary oligonucleotide sequence tagged with a conventional fluorophore. Antibody concentration, imaging strand concentration, and configuration as well as signal removal strategies were optimized to generate maximal staining intensity using our Ab-oligo cyCIF strategy. Results: We successfully generated 14 Ab-oligo conjugates and validated their antigen specificity, which was maintained in single color staining studies. With the validated antibodies, we generated up to 14-color imaging data sets of human breast cancer tissues. Conclusions: Herein, we demonstrated the utility of Ab-oligo cyCIF as a platform for highly multiplexed imaging, its utility to measure tumor heterogeneity, and its potential for future use in clinical histopathology.
AB - Significance: Advanced genetic characterization has informed cancer heterogeneity and the challenge it poses to effective therapy; however, current methods lack spatial context, which is vital to successful cancer therapy. Conventional immunolabeling, commonplace in the clinic, can provide spatial context to protein expression. However, these techniques are spectrally limited, resulting in inadequate capacity to resolve the heterogenous cell subpopulations within a tumor. Aim: We developed and optimized oligonucleotide conjugated antibodies (Ab-oligo) to facilitate cyclic immunofluorescence (cyCIF), resulting in high-dimensional immunostaining. Approach: We employed a site-specific conjugation strategy to label antibodies with unique oligonucleotide sequences, which were hybridized in situ with their complementary oligonucleotide sequence tagged with a conventional fluorophore. Antibody concentration, imaging strand concentration, and configuration as well as signal removal strategies were optimized to generate maximal staining intensity using our Ab-oligo cyCIF strategy. Results: We successfully generated 14 Ab-oligo conjugates and validated their antigen specificity, which was maintained in single color staining studies. With the validated antibodies, we generated up to 14-color imaging data sets of human breast cancer tissues. Conclusions: Herein, we demonstrated the utility of Ab-oligo cyCIF as a platform for highly multiplexed imaging, its utility to measure tumor heterogeneity, and its potential for future use in clinical histopathology.
KW - cancer heterogeneity
KW - cyclic immunostaining
KW - multiplexed immunostaining
KW - oligonucleotide conjugated antibody
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U2 - 10.1117/1.JBO.25.5.056004
DO - 10.1117/1.JBO.25.5.056004
M3 - Article
C2 - 32445299
AN - SCOPUS:85085265939
SN - 1083-3668
VL - 25
JO - Journal of biomedical optics
JF - Journal of biomedical optics
IS - 5
M1 - 056004
ER -