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High-Plex Spatially Resolved RNA and Protein Detection Using Digital Spatial Profiling: A Technology Designed for Immuno-oncology Biomarker Discovery and Translational Research

  • Joseph M. BeechemEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2055)

Abstract

Digital spatial profiling (DSP) is a nondestructive method for high-plex spatial profiling of proteins and RNA from a wide variety of sample types, including formalin-fixed, paraffin-embedded (FFPE) tissue sections. This method uses small photocleavable oligonucleotide “barcodes” (PC-oligos) covalently attached to in-situ affinity reagents (antibodies and RNA-probes) to provide unlimited multiplexing capability. The photocleavage light is projected onto the tissue slice using two-digital micromirror devices (DMD), containing one-million semiconductor-based micromirrors allowing complete flexibility in the pattern of light utilized for high-plex digital profiling of the tissue. These spatial light-patterns can be automatically configured to profile (1) “tumor-only” cells plus “tumor-microenvironment-only” cells; (2) unique cell types and rare cell features (e.g., macrophages, CD8, CD3, CD45, PD-L1 on macrophages, PD-L1 on tumors, etc.); (3) spatial gradients around cell-features or tumor features (e.g., excluded boundaries); (4) hypothesis-free spatial grids; (5) simple hand-selected geometric areas (e.g., free-hand software-based “drawing” on tissue regions); and (6) or any combination of the above modalities. These DMDs can automatically configure themselves to “align” to the biology presented by each individual tissue section. Advanced validated high-plex panels of proteins (~100-plex) and RNA (up to 20,000-plex) specifically designed for immuno-oncology (IO) have been developed. Immuno-oncology clinical trial samples examined using DSP have already provided key insights into the mechanism of action of combination therapy in melanoma, appearing recently in back-to-back articles published in Nature Medicine. DSP has been developed with knowledge of key immuno-oncology terms (tumor, tumor microenvironment, stroma, etc.) and prevalidated high-plex panels of affinity markers (antibodies and in situ RNA probes) and has the potential to bring the full power of high-plex molecular profiling to spatially resolved studies.

Key words

Digital spatial profiling Imaging Biomarkers Immuno-oncology High-plex RNA Proteins 

Notes

Acknowledgments

To the entire GeoMx team at NanoString (Seattle WA). Also, the original concept for DSP was developed together with Dr. Gordon Mills (OHSU, Portland OR). Extensive interactions with Dr. David Rimm (Yale University) during the early development of GeoMx greatly enhanced the overall capability of the system.

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  1. 1.NanoString Technologies Inc.SeattleUSA

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