Abstract
A number of highly multiplexed immunostaining and imaging methods have advanced spatial proteomics of cancer for improved treatment strategies. While a variety of methods have been developed, the most widely used methods are limited by harmful signal removal techniques, difficulties with reagent production and antigen sensitivity. Multiplexed immunostaining employing oligonucleotide (oligos)-barcoded antibodies is an alternative approach that is growing in popularity. However, challenges remain in consistent conjugation of oligos to antibodies with maintained antigenicity as well as non-destructive, robust and cost-effective signal removal methods. Herein, a variety of oligo conjugation and signal removal methods were evaluated in the development of a robust oligo conjugated antibody cyclic immunofluorescence (Ab-oligo cyCIF) methodology. Both non- and site-specific conjugation strategies were assessed to label antibodies, where site-specific conjugation resulted in higher retained binding affinity and antigen-specific staining. A variety of fluorescence signal removal methods were also evaluated, where incorporation of a photocleavable link (PCL) resulted in full fluorescence signal removal with minimal tissue disruption. In summary, this work resulted in an optimized Ab-oligo cyCIF platform capable of generating high dimensional images to characterize the spatial proteomics of the hallmarks of cancer.
Highlights
A number of highly multiplexed immunostaining and imaging methods have advanced spatial proteomics of cancer for improved treatment strategies
Oligos containing an alkyne group in the form of a DBCO triethylene glycol (TEG) spacer modification on the 5′ end permitted Cu-free click chemistry-mediated covalent attachment (Fig. 1D). While both the Solulink and SiteClick kits resulted in Ab-oligo conjugates with maintained affinity and specificity, retention of binding affinity was substantially higher for the site-specific SiteClick kit compared to the non-specific Solulink kit (~ 90% vs. ~ 50%, respectively)
Over the past few decades, immunofluorescence imaging tools have advanced dramatically yet have remained limited by both the dimensionality and spectral detection space on a single sample, both of which prevent high dimensional imaging of biomarkers expressed in tissues
Summary
A number of highly multiplexed immunostaining and imaging methods have advanced spatial proteomics of cancer for improved treatment strategies. A variety of oligo conjugation and signal removal methods were evaluated in the development of a robust oligo conjugated antibody cyclic immunofluorescence (Ab-oligo cyCIF) methodology Both non- and site-specific conjugation strategies were assessed to label antibodies, where site-specific conjugation resulted in higher retained binding affinity and antigen-specific staining. The TME must be characterized to understand the multiple evolving obstacles that hamper effective drug delivery and response, including immune cell infiltrate, dysfunctional vasculature and dense extracellular matrix[9] In response to this analytical need, a number of highly-multiplexed immunostaining techniques have been developed as a means for quantitative, spatial proteomics to fully elucidate and characterize proximity interactions between cells of all functions that drive tumor evolution. By the laser spot size, limiting detection of single cells and low abundant antigens (e.g., phosphoproteins) In response to these challenges, hybrid techniques that use unique antibody tags, such as oligonucleotide “barcodes,” analogous to the rare earth metal-tagged antibodies have been d eveloped[24]. Available Cu-free click chemistry labeling kits enable facile site-specific addition of azide groups to the antibodies minimizing disruption of the antigen binding site
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