Two main technology platforms have been established and are now in routine use in the core. These are 1) the SimpleWesternTM capillary nano-immunoassay (CNIA) system (PeggySue, ProteinSimple) and (2) the bead-based Luminex xMAP multiplexed immunoassay system (MAGPIX, Bio-Rad). 1) The SimpleWestern 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. In response to CCR investigator needs, we have developed a validated panel of close to three hundred CNIA assays (https://cptr.cancer.gov/technologies/simple_western/assays), covering many key biological processes such as signaling transduction, transcriptional regulation, cell cycle control and apoptosis. This allows us to provide CCR investigators with a powerful profiling platform for applications such as protein activity characterization, biomarker and therapeutic target identification and drug selectivity determination. The CNIA technology has been successfully applied in clinical/pre-clinical research. An increasing number of collaborators are working with us to develop assays to facilitate and inform drug development programs and clinical diagnosis. A panel of 120 CNIA assays were validated for use with peripheral blood mononuclear cells (PBMCs) allowing analysis of samples collected with non-invasive methods. 2) The Luminex xMAP bead-based multiplex immunoassay system allows us to provide a complementary high-performance analysis platform for the measurement of targets such as cytokines, metabolites and other serum/plasma biomarkers. Using this technology, we have been supporting increasing number of projects on clinical and pre-clinical studies to monitor immune responses in cancer therapies. In an ongoing new initiative, we are joining the CCR effort to evaluate single-cell proteomic technologies to accommodate the increasing demand in this field. Single-cell analysis technologies are emerging as powerful toolkits to advance our understanding of cell heterogeneity and subtype-specific protein expressions, as well as facilitate the identification of cellular lineage, cell-cell interaction and tissue organization. Working with technology inventors and collaborating with CCR investigators, two innovative technologies are recently acquired, 1) Single-cell Western technology (Milo, ProteinSimple); and 2) the CO-Detection by Indexing (CODEX) technology (Akoya). 1) The single-cell western system performs western analysis on 1000-2000 single cells in parallel. The system measures proteins hard-to-detect by conventional single cell analysis platforms (e.g. FACS), such as isoforms, phosphorylated proteins, transcription factors, intracellular proteins etc. It offers a cost-effective tool for single cell signaling study and population heterogeneity dissection at the protein level. Besides establishing analysis conditions and validating assay performance, we have started collaborating with CCR investigators on a variety of projects, such as investigating discrete signaling events associated with different cell populations and studying stem cell specific signaling and markers. 2) The CODEX Technology is an immunofluorescence imaging platform for highly multiplexed (30-50 plex) IHC-analysis with single-cell resolution and retention of spatial context. It allows comprehensive protein profiling in their native spatial contexts and enables multi-parametric readouts from a single tissue section. The approach provides information not available from alternative techniques like analysis of single markers in serial sections or conventional single-cell analysis technologies using suspended cell samples. It offers a strategy to deep phenotyping cellular microenvironment and study its impact on cell fate and function. Even the technology is still at early development stage, we have received multiple collaboration interest to study immune cell infiltration and tumor response to cancer therapy in both preclinical mouse models and clinical patient specimens, as well as migrate current 6-8 multiplex IHC assays to allow a higher plex analysis. Multidisciplinary collaborations are also established with CCR investigators, bioinformatics team, and technology experts to develop new methods, algorithms and infrastructures to facilitate large data sharing/storage and image processing/quantitation, as well as in-depth data analysis and presentation. We actively communicate with investigators and offer our expertise throughout all project stages, including project feasibility, experiment design, method/analysis strategy development, sample preparation/analysis, data evaluation/summary, further project advancement, and assistance with manuscript preparation. The concepts of Good Laboratory Practice (GLP) are integral to the core operation, to ensure data accuracy & reliability and assay readiness for transferring from bench to bedside. A web-based interface (https://cptr.cancer.gov) is employed for ease of accessibility to our technologies and protocols, as well as more efficient project review, communication and management.
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