Signal removal methods for highly multiplexed immunofluorescent staining using antibody conjugated oligonucleotides

Nathan McMahon, Jocelyn Jones, Jennifer Eng, Sunjong Kwon, Young Hwan Chang, Guillaume Thibault, Koei Chin, Michel Nederlof, Joe Gray, Summer L. Gibbs

Research output: Chapter in Book/Report/Conference proceedingConference contribution

Abstract

Successful cancer treatment continues to elude modern medicine and its arsenal of therapeutic strategies. Therapy resistance is driven by significant tumor heterogeneity, complex interactions between malignant, microenvironmental and immune cells and cross talk between signaling pathways. Advances in molecular characterization technologies such as next generation sequencing have helped unravel this network of interactions and have vastly affected how cancer is diagnosed and treated. However, the translation of complex genomic analyses to pathological diagnosis remains challenging using conventional immunofluorescence (IF) staining, which is typically limited to 2-5 antigens. Numerous strategies to increase distinct antigen detection on a single sample have been investigated, but all have deleterious effects on the tissue limiting the maximum number of biomarkers that can be imaged on a single sample and none can be seamlessly integrated into routine clinical workflows. To facilitate ready integration into clinical histopathology, we have developed a novel cyclic IF (cycIF) technology based on antibody conjugated oligonucleotides (Ab-oligos). In situ hybridization of complementary oligonucleotides (oligos) facilitates biomarker labeling for imaging on any conventional fluorescent microscope. We have validated a variety of oligo configurations and their respective signal removal strategies capable of diminishing fluorescent signal to levels of autofluorescence before subsequent staining cycles. Robust signal removal is performed without the employment of harsh conditions or reagents, maintaining tissue integrity and antigenicity for higher dimensionality immunostaining of a single sample. Our platform Ab-oligo cycIF technology uses conventional fluorophores and microscopes, allowing for dissemination to a broad audience and congruent integration into clinical histopathology workflows.

Original languageEnglish (US)
Title of host publicationImaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues XVII
EditorsJames F. Leary, Attila Tarnok, Daniel L. Farkas
PublisherSPIE
ISBN (Electronic)9781510624047
DOIs
StatePublished - Jan 1 2019
EventImaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues XVII 2019 - San Francisco, United States
Duration: Feb 4 2019Feb 6 2019

Publication series

NameProgress in Biomedical Optics and Imaging - Proceedings of SPIE
Volume10881
ISSN (Print)1605-7422

Conference

ConferenceImaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues XVII 2019
CountryUnited States
CitySan Francisco
Period2/4/192/6/19

Keywords

  • Antibody conjugation
  • Biomarker distribution
  • Cyclic immunofluorescence
  • Fluorescence microscopy
  • Oligonucleotide
  • Photocleavable linker
  • Restriction enzyme
  • Tumor heterogeneity

ASJC Scopus subject areas

  • Electronic, Optical and Magnetic Materials
  • Biomaterials
  • Atomic and Molecular Physics, and Optics
  • Radiology Nuclear Medicine and imaging

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  • Cite this

    McMahon, N., Jones, J., Eng, J., Kwon, S., Chang, Y. H., Thibault, G., Chin, K., Nederlof, M., Gray, J., & Gibbs, S. L. (2019). Signal removal methods for highly multiplexed immunofluorescent staining using antibody conjugated oligonucleotides. In J. F. Leary, A. Tarnok, & D. L. Farkas (Eds.), Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues XVII [108810X] (Progress in Biomedical Optics and Imaging - Proceedings of SPIE; Vol. 10881). SPIE. https://doi.org/10.1117/12.2510573