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

摘要

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 molecularcharacterization technologies such as next generation sequencing have helped unravel this network of interactions andhave vastly affected how cancer is diagnosed and treated. However, the translation of complex genomic analyses topathological diagnosis remains challenging using conventional immunofluorescence (IF) staining, which is typicallylimited to 2-5 antigens. Numerous strategies to increase distinct antigen detection on a single sample have beeninvestigated, but all have deleterious effects on the tissue limiting the maximum number of biomarkers that can beimaged on a single sample and none can be seamlessly integrated into routine clinical workflows. To facilitate readyintegration into clinical histopathology, we have developed a novel cyclic IF (cycIF) technology based on antibodyconjugated oligonucleotides (Ab-oligos). In situ hybridization of complementary oligonucleotides (oligos) facilitatesbiomarker labeling for imaging on any conventional fluorescent microscope. We have validated a variety of oligoconfigurations and their respective signal removal strategies capable of diminishing fluorescent signal to levels ofautofluorescence before subsequent staining cycles. Robust signal removal is performed without the employment ofharsh conditions or reagents, maintaining tissue integrity and antigenicity for higher dimensionality immunostaining of asingle sample. Our platform Ab-oligo cycIF technology uses conventional fluorophores and microscopes, allowing fordissemination to a broad audience and congruent integration into clinical histopathology workflows.