Spatially-resolved proteomics and transcriptomics: An emerging digital spatial profiling approach for tumor microenvironment

Abstract

Digital spatial profiling (DSP) is an emerging powerful technology for proteomics and transcriptomics analyses in a spatially resolved manner for formalin-fixed paraffin-embedded (FFPE) samples developed by nanoString Technologies. DSP applies several advanced technologies, including high-throughput readout technologies (digital optical barcodes by nCounter instruments or next generation sequencing (NGS)), programmable digital micromirror device (DMD) technology, and microfluidic sampling technologies into traditional immunohistochemistry (IHC) and RNA in situ hybridization (ISH) approaches, creating an innovative tool for discovery, translational research, and clinical uses. Since its launch in 2019, DSP has been rapidly adopted, especially in immuno-oncology and tumor microenvironment research areas, and has revealed valuable information that was inaccessible before. In this article, we report the successful setup and validation of the first DSP technology platform in China. Both DSP spatial protein and RNA profiling approaches were validated using FFPE colorectal cancer tissues. Regions of interest (ROIs) were selected in the areas enriched with tumor cells, stroma/immune cells, or normal epithelial cells, and multiplex spatial profiling of both proteins and RNAs were performed. DSP spatial profiling data were processed and normalized accordingly, validating the high quality and consistency of the data. Unsupervised hierarchical clustering as well as principal component analysis (PCA) grouped tumor, stroma/immune cells, and normal epithelial cells into distinct clusters, indicating that the DSP approach effectively captured the spatially resolved proteomics and transcriptomics profiles of different compartments within the tumor microenvironment. In summary, the results confirmed the expected sensitivity and robustness of the DSP approach in profiling both proteins and RNAs in a spatially resolved manner. As a novel technology in highly complex spatial analyses, DSP endows refined analytical power from the tumor microenvironment perspective with the potential of scaling up to more analyzable targets at relatively low cell input levels. We expect that the DSP technology will greatly advance a wide range of biomedical research, especially in immuno-oncology and tumor microenvironment research areas.

Best Practices for Spatial Profiling for Breast Cancer Research with the GeoMx® Digital Spatial Profiler

“…However, some non-coding RNA species can be profiled using custom probes in parallel with the CTA/WTA assays. The protein menu could also benefit from additional reagents to detect more proteins, especially post-translationally modified proteins [92]. In addition, the development of detection capabilities for DNA sequences, such as somatic and germline mutations or copy number variations, would offer an intriguing dataset that could be integrated with the RNA and protein data.…”

“…In parallel with improving the number of analytes that can be measured, improvements to the resolution of the platform could yield richer datasets. Near single-cell sensitivity for highly expressed proteins is achievable, but this is not universally true for all analytes [42,92]. Current recommendations of the number of cells to profile enable reasonably fine-grained characterization, although not to the level of single-cell profiling.…”

Tumor Microenvironment Profiles Reveal Distinct Therapy-Oriented Proteogenomic Characteristics in Colorectal Cancer

“…Paired pretreatment biopsy samples may reveal more information to identify how tumors evolve and adapt in response to chemotherapy with or without coadministration of checkpoint blockades and may help design future clinical strategies in CRC. Nevertheless, by employing DSP, with only limited samples, high-plex proteogenomic profiling of over 40plex proteins and 84-plex mRNA on CRC patients was depicted at a very high resolution, which has never been achieved with previous methods (Wang et al, 2021b). In summary, we provided valuable research resources and preliminary data by focusing on specific TME regions histologically unobtainable with other technologies and ensured future application in various contexts to disentangle underlying biology within the complex and heterogenous TMEs.…”

“…Conjugated target-specific oligos were released upon UV light illumination and collected in 96-well plates. Heat-dried oligos were then hybridized to unique NanoString barcodes, purified on the nCounter Prep station, and counted on the nCounter analysis system using standard procedures (Merritt et al, 2020;Wang et al, 2021b).…”

Spatial transcriptomics and proteomics technologies for deconvoluting the tumor microenvironment

“…We also share our extensive experience on Digital Spatial Profiling (DSP) technology and elaborate on its unique features to analyze both protein and RNA, anticipating its broad application from TME perspectives. [ 16 ]…”