Flexible Cyclic Immunofluorescence (cyCIF) Using Oligonucleotide Barcoded Antibodies

Abstract

Advances in our understanding of the complex, multifaceted interactions between tumor epithelia, immune infiltrate, and tumor microenvironmental cells have been driven by highly multiplexed imaging technologies. These techniques are capable of labeling many more biomarkers than conventional immunostaining methods. However, multiplexed imaging techniques suffer from low detection sensitivity, cell loss—particularly in fragile samples—, and challenges with antibody labeling. Herein, we developed and optimized an oligonucleotide antibody barcoding strategy for cyclic immunofluorescence (cyCIF) that can be amplified to increase the detection efficiency of low-abundance antigens. Stained fluorescence signals can be readily removed using ultraviolet light treatment, preserving tissue and fragile cell sample integrity. We also extended the oligonucleotide barcoding strategy to secondary antibodies to enable the inclusion of difficult-to-label primary antibodies in a cyCIF panel. Using both the amplification oligonucleotides to label DNA barcoded antibodies and in situ hybridization of multiple fluorescently labeled oligonucleotides resulted in signal amplification and increased signal-to-background ratios. This procedure was optimized through the examination of staining parameters including staining oligonucleotide concentration, staining temperature, and oligonucleotide sequence design, resulting in a robust amplification technique. As a proof-of-concept, we demonstrate the flexibility of our cyCIF strategy by simultaneously imaging with the original oligonucleotide conjugated antibody (Ab-oligo) cyCIF strategy, the novel Ab-oligo cyCIF amplification strategy, as well as direct and indirect immunofluorescence to generate highly multiplexed images.

In situ single-cell therapeutic response imaging facilitated by the TRIPODD fluorescence imaging platform

“…A 10-antibody panel of oligonucleotide (oligo) and fluorophore conjugated antibodies was developed and staining patterns were validated for the Ab-oligo cyCIF workflow (Table 1 ). All Ab-oligos, including their oligonucleotide sequences, were generated using the previously reported methods 73 , 74 . In brief, antibodies to human E-Cadherin (E-Cad), cytokeratin 8 (CK8), EGFR, Akt, pAkt, pMEK, cleaved caspase-3 (CC3) and Ki-67 were purchased from AbCam (Cambridge, UK) and Cell Signaling Technology ([CST], Danvers, MA).…”

“…In our Ab-oligo cyCIF, a complementary, fluorescently labeled oligonucleotide sequence is used for in situ detection 72 . We have also demonstrated an Ab-oligo cyCIF signal amplification strategy as well as the flexibility of integrating Ab-oligo cyCIF with both conventional indirect and direct IF reagents for enhanced spatial proteomics assessment 73 , 74 .…”

Analysis of uveal melanoma scRNA sequencing data identifies neoplastic-immune hybrid cells that exhibit metastatic potential

“…Tissue sections were incubated with antibodies ( Supplemental Table 1 ) in blocking buffer (PBS with 2.0% bovine serum albumin (BSA, bioWORLD, Dublin, OH), 0.05 mg/mL% salmon sperm DNA (Thermo Fisher Scientific, Waltham, MA), and 0.5% dextran sulfate (Sigma-Aldrich, St. Louis, MO)) overnight at 4 °C in a humid chamber, followed by 3 x 5 minute washes in 2X SSC (BD Biosciences, Franklin Lakes, NJ, pH 7.0). Detection methods varied based on antibody type and round of staining ( Supplemental Table 1 ), using either Ab-oligo antibodies + IS [60, 61, 63](MITF, TYR, MLANA, CD45) or directly conjugated fluorescent antibodies (HTR2B, GP100, CD25 and CD203c). [9] Fluorescent signal removal between rounds was performed by exposing slides to UV light for 15 min.…”

“…Formalin-fixed paraffin-embedded (FFPE) tissue sections (5 µm) from enucleated globes were stained for melanocytic and immune markers using a flexible cyclic immunofluorescence method with oligonucleotide conjugated antibodies as previously described [60][61][62][63] (n=3). Briefly, tissue sections were deparaffinized with xylene and rehydrated with graded ethanol baths.…”

“…Supplemental Table 1: Antibody information for hybrid cell identification in UM FFPE sections using cyclic immunofluorescence. [9,[60][61][62][63] Supplemental Table 2: Hybrid cell counts from each patient sample within each original cluster identity as defined by Durante et al [13] Supplemental File 1: All differentially expressed genes & Reactome [31] pathway analysis for UMM063 hybrid cluster 3 vs hybrid cluster 12.…”