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.
Highlights
Since the paradigm shift to disentangle the underlying mechanisms of oncogenesis from solely tumor-centric principles to a more conceivably dynamic interplay and crosstalk involving normal and cancerous epithelial cells, fibroblasts, immune cells, and extracellular matrix (ECM) back in the mid-1980s, cumulative works were in progress to underpin the significant roles of the tumor microenvironment (TME) in localized neoplasm as well as a metastatic invasion [1]
Two formalin-fixed paraffin-embedded (FFPE) HEK293 cell pellet slides were used at Mills Institute for Personalized Cancer Care (MIPCC) to validate the Digital spatial profiling (DSP) protein profiling workflow, with the core protein module consisting of 20 antibodies, together with IO Drug Target module and Immune Cell Typing module (38 antibodies in total)
We set up the first DSP platform in China at MIPCC, Fynn Biotechnologies (FynnBio) in 2019, right after nanoString Technologies launched the technology
Summary
Since the paradigm shift to disentangle the underlying mechanisms of oncogenesis from solely tumor-centric principles to a more conceivably dynamic interplay and crosstalk involving normal and cancerous epithelial cells, fibroblasts, immune cells, and extracellular matrix (ECM) back in the mid-1980s, cumulative works were in progress to underpin the significant roles of the tumor microenvironment (TME) in localized neoplasm as well as a metastatic invasion [1]. Chemotherapy, radiotherapy, genotoxic agents, and anti-angiogenic drugs pose negative feedback effects on TME, many of which include reprogramming of immune cells, fibroblasts, and interstitial humoral regulation, involving inflammatory cytokines, tumor-initiating growth factors, and antibodies [1, 6]. It is not surprising from oncology perspectives that targeting tumor-TME interactions has become a research hotspot in the past 15 years and therapeutic interventions targeting tumorigenic TME have been extensively investigated. Expression of these RTKs on designated cells in regional and distal TME promotes tumor proliferation and vasculogenesis, which further strengthens the concept of mutual influence between evading cancer cells and surrounding TME [7,8,9]
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