In the past years, we have established 1) the SimpleWesternTM capillary nano-immunoassay (CNIA) system; and (2) the bead-based Luminex xMAP multiplexed-immunoassay system for high-performance quantitative proteomic profiling. 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 comprehensive and quantitative signaling molecule profiling at the protein level and high-performance assays for preclinical/clinical applications. In response to the demand of CCR investigators, a validated panel of about three hundred CNIA assays (https://cptr.cancer.gov/technologies/simple_western/assays) were developed, covering many key biological processes such as signaling transduction, transcriptional regulation, cell cycle control and apoptosis. This provides a powerful profiling platform for applications such as protein activity characterization, biomarker and therapeutic target identification and drug selectivity determination. The development of these assays helped the advancement of multiple projects that had been challenging when conventional methods were used. The CNIA technology offers a platform for clinical investigators to: 1) incorporate companion diagnostics at the protein level; 2) evaluate effects of targeted therapy; 3) determine pharmacodynamics and biologically relevant dose; and 4) determine prognosis and refinements for treatment strategies. Collaborating with CCR investigators, we have developed approaches applying CNIA assays for clinical/pre-clinical research, and successfully supported multiple clinical trial and drug development projects. A panel of about 120 CNIA assays were also established with peripheral blood mononuclear cells (PBMCs) covering major pathways for targeted therapy and drug resistance. This noninvasive approach enables the monitor of protein levels and state of activation at more frequent intervals. The Luminex xMAP bead-based multiplex-immunoassay system provides 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 or other treatment responses in cancer therapies. Since the beginning of 2018, we are joining the effort of CCR Office of Science and Technology Resource (OSTR) evaluate, develop, and implement single-cell analysis technologies to accommodate the increasing demands for both discovery and clinical applications. Single-cell analysis technologies are emerging as powerful tool-kits 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 and implemented 1) Single-cell Western technology; and 2) the CO-Detection by Indexing (CODEX) technology. 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. Assay reproducibility, specificity and dynamic range were evaluated and optimized. A panel of assays were developed for the MAP kinase and NF-kappa-B pathways, transcription factors, PKC isoforms, cyclin targets and loading controls. Conditions were also established for 12-plex protein probing from a single experiment run. This effort established us as pioneer in single-cell western assay development, and the work was presented to technology users in a national wide Webinar. We are currently working with NIH investigators to explore the feasibility of using the technology to study single-cell signaling and population heterogeneity. The CODEX Technology is an immunofluorescence imaging platform for highly multiplexed (more than 20-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 were approached by researchers from both the CCR/NCI/NIH community and other academic institutes. In the past year, collaborating with CCR investigators, CODEX assay performance and feasibility were assessed on a multitude of human and mouse tissues and tumors (such as breast, lung, prostate, heart and lymphoid tissues). Ongoing research projects include profiling of immune-landscape in tumors and its response to treatment, investigation of the dynamics of immune cell populations and the stem cell niche in metastasis etc. We are also working with multi-discipline teams of experts involving other CCR technology and bioinformatics specialists and industry collaborators to establish the infrastructure for data storage and collaborative data sharing and analysis, as well as developing image viewing and quantitative analysis tools. In the 2019 fiscal year, we have actively worked with 31 PIs from different CCR branches/laboratories and supported 35 research projects. Our work was presented at several national and international meetings, such as the American Association for Cancer Research (AACR) annual meeting, Keystone Symposia, Focus on Microscopy meeting and Microscience Microscopy Congress. We also facilitated multiple publications on peer reviewed journals, such as Molecular Cancer Research, PLoS ONE, Cancer Research, Molecular Biology of the Cell, Science Translational Medicine, Chembiochem and Oncoimmunology. Several other manuscripts are currently under review or in-preparation.
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