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

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.

Oligonucleotide conjugated antibody strategies for cyclic immunostaining

“…All human formalin fixed paraffin embedded (FFPE) tissues were obtained from the OHSU Knight Biolibrary as deidentified human tissue blocks. Ab-oligo conjugates were validated in cells and FFPE tissues as previously described 29 . Ab-oligo conjugate optimization studies were also performed on FFPE MCF7 xenograft tissues grown in mice for unrelated studies.…”

“…The cells were stained with 300 nM 4′,6-diamidino-2-phenylindole (DAPI, MilliporeSigma) for 10 min at RT after which they were washed with PBS or 2X SSC (2 × 5 min) prior to imaging. FFPE tissue sections (4–5 µm thickness) were stained using a similar procedure as previously described 29 , 40 . Stained slides were mounted in Fluoromount G (Southern Biotech, Birmingham, AL) or Prolong Gold Antifade with DAPI (ThermoFisher Scientific) prior to coverslipping.…”

“…Antibody barcoding techniques (e.g., DNA-Exchange Image 25 , Immuno-SABER 26 , Nanostring 24 , 27 and CODEX 28 ) have demonstrated the ability for highly multiplexed immunostaining using non-destructive signal removal techniques. We have optimized a barcoded antibody cyclic IF (cyCIF) technique where a complementary fluorophore-labeled DNA strand is used for in situ detection, facilitating collection of high-dimensional proteomic data 29 . Notably, signal removal of complementary fluorophore-labeled DNA strands can be achieved through a variety of non-destructive methods, making this technique advantageous over fluorophore-labeled antibody-based cyCIF and MSI.…”

Highly multiplexed tissue imaging using repeated oligonucleotide exchange reaction

“…Another approach uses DNA-barcoded antibodies that are visualized by cyclic addition and removal of fluorescently labeled DNA probes. Techniques based on this principle include exchangepoints accumulation in nanoscale topography (PAINT) [22], DNA exchange imaging (DEI) [23], immunostaining with signal amplification by exchange reaction (immuno-SABER) [24], quantumdot SABER [25], barcoded-antibody based cyclic immunofluorescence (cyCIF) [26], and CO-Detection by indEXing (CODEX) [27,28]. These DNA-based systems have the advantages of a single staining procedure, fast run times, and comparably simple chemistries.…”

A framework for multiplex imaging optimization and reproducible analysis

“…Direct immunofluorescence using fluorophore-conjugated primary antibodies and chemical inactivation of fluorescent dyes enables detection of over 50 protein targets in a single tissue section, in cyclic immunofluorescence (CyCIF) [7][8][9] and NeoGenomics' MultiOmyx platform 10 . Furthermore, fluorophore-conjugated DNA barcodes (i.e., oligonucleotides) facilitate multiplexing in co-detection by indexing (Akoya's CODEX) 11 , Ultivue's InSituPlex 12 , and Ab-oligo cyCIF 13 . Imaging mass cytometry 14 and multiplex ion beam imaging 15 can Adopting the method of Lin et al 7 , we stained formalin-fixed, paraffin-embedded (FFPE) tissues using a cyclic process in which four proteins per cycle were labeled using a direct immunofluorescence (IF) protocol, imaged, and quenched of fluorescence signal (Fig.…”