Collaborative Protein Technology Resource - Nano-Scale Protein Analysis Section

Division Of Basic Sciences - Nci

Investigators

Linked publications & trials

Abstract

IIn the past years, the core has established 1) the SimpleWesternTM capillary nano-immunoassay (CNIA) system; 2) the bead-based Luminex xMAP multiplexed-immunoassay system for high-performance, quantitative protein measurement with small sample consumption; 3) the CO-Detection by Indexing (CODEX) technology for quantitative detection of 40-60 protein markers in tissues at single cell level; 4) GeoMx Digital Spatial Profiler (DSP) enabling detection of 100s of proteins and thousands of RNA targets including cancer transcriptome and whole transcriptome from a single tissue; and 5) CosMx Spatial Molecular Imager (SMI) to image expression of 1000s of RNA transcripts or 64 proteins at single cell level in tissues. 1) The SimpleWesternTM CNIA system is an automated capillary immunoassay system for highly quantitative, reproducible detection of target proteins and their post-translational modifications in a format that is applicable to very small sample sizes. The CNIA analysis platform fills a need for high-performance assays for comprehensive and quantitative signaling molecule profiling at the protein level and facilitates transferring those assays from discovery research into preclinical/clinical practice. CPTR is one of the earliest adopters of the CNIA technology. We established assay conditions for more than three hundred signaling molecules covering key pathways, including targets in apoptosis/cell death, cell cycle and checkpoint control, gene and RNA regulation, cell proliferation and development. Collaborating with CCR investigators, we have successfully developed protocols/strategies supporting multiple clinical trial and drug-development programs, including the validation of a panel of 120 CNIA assays in peripheral blood mononuclear cells (PBMCs) allowing analysis of samples collected with non-invasive methods and monitoring of protein levels and state of activation at more frequent intervals. In the 2022-2023 fiscal year, we have been working with 5 PIs to support their projects. The data generated facilitated preparation of multiple manuscripts: 1 published, 1 accepted for publication. 2) The Luminex xMAP bead-based multiplex-immunoassay system provides a highly-quantitative multiplex-analysis of cytokines, metabolites and other serum/plasma biomarkers. The technology has been broadly applied in life-science research including cancer, immunology, cardiovascular disease, metabolic disease, and inflammation with clinically applicable assay performance. Using this technology, we have been supporting projects on clinical and pre-clinical studies to monitor immune or other treatment responses in cancer therapies. In addition to full support for the technology, we also offer walk-up access to the instrument, so users do not need to sustain multiple individual instruments at high cost in their laboratories. This fiscal year, we have been working with 3 PIs on their research projects. 3) For the last 4 years we offer the CO-Detection by Indexing (CODEX) spatial ultra-highplex immunofluorescence technology to detect expression of tens of protein markers in a tissue as service for CCR/NCI/NIH branches/laboratories. The ability to quantitatively measure 40-60 protein targets from the same tissue at single cell level preserving spatial context allows for deep phenotyping of immune cells in the tumor microenvironment, characterize their interaction with tumor cells and identify cellular neighborhoods determining cancer fate and progression, as well as response to cancer treatment. CPTR is an early adopter of the CODEX technology and became one of most advanced sites, spearheading the development and implementation of improved imaging, processing and data analysis workflows. This year we updated one of the two CODEX systems to Phenocycler Fusion that allows for imaging of tissues and tissues microarrays (TMAs) on slides and is reported to be the fastest commercially available imaging modality for multiplex staining. We have assessed CODEX assay performance and feasibility in multiple human/mouse organ and tumor tissues (such as lung and breast cancer, ovarian and liver tumors, melanoma, neurofibroma, lung metastasis, lymphoid tissues, etc.). Previously most of our experience has been with imaging fresh frozen tissues, this year we continued to implement assay protocols for the clinically more relevant FFPE tissues and tissue microarrays. Collaborating with CCR/NCI/NIH investigators, we customized multiple antibodies to extend the commercially available antibody panel to better support cancer research and therapy. We established a 39-plex antibody panel for fresh frozen human liver tumors and a 41-plex antibody panel for FFPE liver samples to characterize the tumor microenvironment. There is increased interest in using the technology in basic as well as clinical studies. In the 2022-2023 fiscal year, we continued to work with 10 PIs to support their research projects. Several manuscripts containing CODEX data generated are in-preparation (n=3) close to be submitted, submitted and accepted (n=2) and published (1). We also have been actively working with multi-disciplinary teams of experts involving other CCR technology and bioinformatics specialists and industry collaborators to establish the infrastructure for data storage, collaborative data sharing and analysis, and implementing image viewing and quantitative analysis tools, such as NCI HALO 2D digital pathology image analysis platform. 4) With the help and support from CCR Office of Science and Technology Resource (OSTR) and in collaboration with CCR Genomics Core, recently we have established the GeoMx Digital Spatial Profiler (DSP) platform at CPTR. GeoMx DSP combines standard immunofluorescence techniques with the nCounter digital optical barcoding or NGS readout to perform highly-multiplexed, spatially-resolved protein and transcript profiling of tissues. Both RNA and protein panels have been developed by the vendor for immune-oncology, cancer pathways and neuroscience, as well as the 1800-target 'cancer transcriptome atlas' (CTA) and the 18,000-target whole transcriptome atlas (WTA) RNA panel. CPTR's unique contribution to this technology is customization of morphology markers based on the project needs to allow for improved selection of regions of interest (ROIs) as well as enabling analysis on the whole slides, not only selected ROIs. This fiscal year, we have supported 10 intramural investigators for cancer discovery research projects and clinical specimen profiling. The generated results are included in multiple manuscripts in preparation (5), under submission (2), or published (1). 5) Starting September 2022, our laboratory spearheaded the evaluation and implementation of CosMx Spatial Molecular Imager (SMI) to enable high resolution (single cell level) detection of cancer related RNA transcripts (1000) or different protein targets (64). This technology complements the ROI-based GeoMx DSP to detect RNA transcripts and the CODEX multiplex protein detection technology. Imaging of two pilot projects and two early access projects have been finished, with the current effort now focusing on establishing data storage and data analysis pipeline.