Abstract
.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.
Highlights
Our understanding of cancer has evolved from a view of a collection of cells exhibiting unchecked proliferation to the realization that cancers include heterogenous genetic cellJournal of Biomedical OpticsMay 2020 Vol 25(5)McMahon et al.: Oligonucleotide conjugated antibodies permit highly multiplexed immunofluorescence. . .populations able to evade death through oncogenic dysregulation[1,2] and complex interactions with the tumor microenvironment.[3,4,5] We owe much of our recent understanding to large cancer genome sequencing efforts that uncovered novel cancer genes and genetic diversity using molecular characterization technologies such as generation sequencing and quantitative polymerase chain reaction sequencing
The resulting images were qualitatively and quantitatively assessed, showing increased Signal-to-background ratio (SBR) from 100 to 350 nM imaging strand (IS) concentrations, with little SBR increase with further increases in IS concentration [Fig. 1(d)], resulting in the selection of 350 nM IS as the optimal staining concentration
As a more direct comparison, primary antibody directly conjugated to AF555 was used to stain serial sections and SBR was quantified for comparison
Summary
Populations able to evade death through oncogenic dysregulation[1,2] and complex interactions with the tumor microenvironment.[3,4,5] We owe much of our recent understanding to large cancer genome sequencing efforts that uncovered novel cancer genes and genetic diversity using molecular characterization technologies such as generation sequencing and quantitative polymerase chain reaction sequencing. The translation of complex genomic analyses to gold standard pathological diagnoses remains challenging as conventional immunohistochemical (IHC) and immunofluorescence (IF) staining are limited to approximately two to five antigens per sample.[6] while genomic analyses are a powerful therapeutic tool, they are deployed at the cost of spatial context of biomarker distribution. To further understand the diagnostic and prognostic implications of these relationships, a molecular profiling technology to measure both expression and spatial context of biomarkers, while preserving both, is required
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