Strategies for Spatial Multi-omics: Co-detection of protein and RNA biomarkers on a single FFPE tissue section powered by InSituPlex® Technology
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1. Background and Introduction:
Understanding the complexities of a particular tumor microenvironment can vastly improve the accuracy of immuno-oncology clinical prognosis and accelerate the discovery of immunotherapy targets towards patient-focused personalized medicine, as well as help researchers gain a more complete understanding of mechanisms of action and cellular phenotypes that may be promoting or hindering response. To this end, biomarker detection approaches capable of rapid identification, quantification, and spatial mapping of the many cell types of the tumor environment within FFPE tissue sections are becoming increasingly valuable for investigating the highly complex biology of tumor microenvironments.
Multiplexed immunofluorescence (mIF) approaches enable the detection of multiple protein targets with their spatial context in a single tissue sample, which in turn enables the identification of diverse cellular sub-types, shedding light on the phenotypic microenvironment of the tumor. Although many mIF staining methods exist, most require a long assay development time, provide limited information on small regions of heterogenous tissue samples or require specialized instrumentation and long imaging times. In contrast, the Ultivue® InSituPlex® (ISP) technology enables rapid, pre-optimized staining of multiple targets simultaneously in a single FFPE tissue section. Furthermore, Ultivue’s flexible portfolio of ISP-based kits, including FixVUE™ kits (formerly called UltiMapper) include ready-to-use panels with conventional manual and automated workflows for use with a variety of commercially available automated imaging systems. Additionally, ISP technology facilitates high-multiplex kits staining on single slides through sequential imaging of multiple targets, where up to four targets are imaged in one round, followed by gentle signal removal and application of a different set of probes for the next round of imaging.
In the field of spatial biology, phenotypic and transcriptomic analyses are evolving in parallel. However, to investigate complex cellular interactions, in-depth multi-omics analyses often require detecting several protein and RNA targets on a single tissue section. Co-detection of protein and RNA can also be valuable for contextualizing the tissue microenvironment by visualizing specific secretory proteins like cytokines. Additionally, co-detection of protein and RNA enables correlative analyses of different markers based on protein and RNA expression levels. Further, detecting RNAs for specific biomarkers can serve as a proxy for detecting proteins using antibodies when antibody use is unfeasible (e.g., when antibodies are not available, have suboptimal specificity, or have longer production timelines). Because FixVUE™ reagents efficiently preserve the tissue microenvironment throughout the entire assay workflow, ISP assays can be integrated with other spatial detection assays for combined protein and RNA detection.
In this white paper, we demonstrate the compatibility of ISP assays for protein detection with a commercially available RNAscope® assay (Advanced Cell Diagnostics, Inc.) for RNA detection. This study highlights the value of an integrated ISP-in situ hybridization (ISH) workflow for co-detection of protein and RNA targets on a single FFPE tissue section.
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