Collaborative Protein Technology Resource - Nano-Scale Protein Analysis Section

Division Of Basic Sciences - Nci

Investigators

Linked publications, trials & patents

Abstract

In the past years, we have 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 up to 40 protein markers in tissues at single cell level; and 4) GeoMx Digital Spatial Profiler (DSP) enabling detection of up to 100s of protein and thousands of RNA targets from a single tissue. 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 2021-2022 fiscal year, we have been working with 6 PIs to support their projects. The data generated facilitates preparation of multiple manuscripts (three have been submitted). 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 inflammations 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 two years we offer the CO-Detection by Indexing (CODEX) spatial ultra-highplex immunofluorescence technology as service for CCR/NCI/NIH branches/laboratories. The ability to quantitatively measure up to 40 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. We have assessed CODEX assay performance and feasibility in multiple human/mouse organ and tumor tissues (such as lung and breast cancers, 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 successfully implemented 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 to characterize the tumor microenvironment of human liver tumors. There is increased interest in using the technology in basic as well as clinical studies. This fiscal year, we continued to work with 8 PIs to support their research projects. Our work has been presented at several national and international meetings, such as the American Association for Cancer Research (AACR) annual meeting, Society for Immunotherapy of cancer (SITC), etc. Several manuscripts are in-preparation, close to be submitted. 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 and NCI HALO 2D digital pathology image analysis platform. With the help and support from CCR Office of Science and Technology Resource (OSTR) and in collaboration with CCR Genomics Core, recently we 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 whole transcriptome atlas (WTA) RNA panel. This fiscal year, we have supported 10 intra and extramural investigators for cancer discovery research projects and clinical specimen profiling. The data were included in two published/accepted manuscripts with multiple other in preparation.