Oligonucleotide conjugated antibodies permit highly multiplexed immunofluorescence for future use in clinical histopathology.

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.