High multiplex, digital spatial profiling of proteins and RNA in fixed tissue using genomic detection methods

Abstract

We have developed Digital Spatial Profiling (DSP), a non-destructive method for high-plex spatial profiling of proteins and RNA, using oligonucleotide detection technologies with unlimited multiplexing capability. The key breakthroughs underlying DSP are threefold: (1) multiplexed readout of proteins/RNA using oligo-tags; (2) oligo-tags attached to affinity reagents (antibodies/RNA probes) through a photocleavable (PC) linker; (3) photocleaving light projected onto the tissue sample to release PC-oligos in any spatial pattern. Here we show precise analyte reproducibility, validation, and cellular resolution using DSP. We also demonstrate biological proofof-concept using lymphoid, colorectal tumor, and autoimmune tissue as models to profile immune cell populations, stroma, and cancer cells to identify factors specific for the diseased microenvironment. DSP utilizes the unlimited multiplexing capability of modern genomic approaches, while simultaneously providing spatial context of protein and RNA to examine biological questions based on analyte location and distribution.

High multiplex analysis of the immune microenvironment in bone marrow trephine samples using GeoMX™ digital spatial profiling

“…However, correlation between DSP and alternative analysis methods has been described recently, indicating strong correlation with standard IHC techniques. 23,27 Indeed, when the mean CD3 and CD45 counts of the ROIs in the healthy BM sample were compared with DSP counts, good correlation was seen for both antigens ( Figure 1C).…”

“…Technologies laboratories under an early technology access programme, as described recently. 22,26,27 ROI selection and subsequent statistical and correlative analyses were undertaken in the Australian Cancer Research Foundation translational research laboratory. Briefly, a multiplexed cocktail of primary antibodies, each with a unique, UV-photocleavable indexing oligo (Table 1), mouse anti-human CD3E antibody clone UMAB54 (Origene Technologies, Inc., Rockville, MD) conjugated to AF647 using AF647 antibody labelling kit (Thermo Fisher Scientific, Waltham, MA), rabbit anti-human CD45 antibody clone D9M81 (Cell Signalling Technology, Inc., Danvers, MA) conjugated to AF594 using AF594 antibody labelling kit (Thermo Fisher Scientific) and Syto83 nucleic acid stain (Thermo Fisher Scientific), was applied to a slide-mounted BM trephine tissue section.…”

Single-Cell Transcriptomic Analysis of mIHC Images via Antigen Mapping

“…STvEA is complementary to emerging technologies for simultaneous highly-multiplexed spatial profiling of proteins and transcripts, such as Digital Spatial Profiling 40…”

Overview of multiplex immunohistochemistry/immunofluorescence techniques in the era of cancer immunotherapy

“…Profiling every cell at single‐cell resolution using DSP may be impractical and cost‐prohibitive. Thus, a general advantage of imaging‐based methods compared to DSP is the ability to get multiplexed information on each cell in the tissue slice rather than high‐plex information of regions of interest as in DSP [91]. In other words, the DSP can only be visualized based on its ‘morphology kit’ of up to 3 markers, including cytokeratin and CD45.…”

“…HALO (Indica Labs, Albuquerque, New Mexico, USA, ; https://www.indicalab.com/halo/) is an image analysis platform for quantitative tissue analysis in digital pathology, mainly used for analysis in oncology, toxicology, metabolism, neuroscience, and more [90‐94]. It reports the morphology and multiplexed expression data of each and every cell across the entire tissue sections.…”