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Single-Cell “Unbiased Discovery” Proteomic Technologies


Fast-Track proposals will NOT be accepted. Direct-to-Phase II proposals will NOT be accepted. Number of anticipated awards: 3-5 Budget (total costs, per award): Phase I: up to $400,000 for up to 9 months Phase II: up to $2,000,000 for up to 2 years PROPOSALS THAT EXCEED THE BUDGET OR PROJECT DURATION LISTED ABOVE MAY NOT BE FUNDED. Summary This proposal is for the development of high-throughput, single-cell, unbiased (i.e. untargeted) discovery proteomic technologies to advance our knowledge in cancer development and progression, to enable robust cancer biomarker discovery and clinical application. So far, the identification of genomic changes in cancer has led to successful therapy-biomarker matches. It has also become integral to the design of clinical trials of molecularly targeted agents. However, it has only succeeded in identifying “actionable” abnormalities in a minority of cancer patients, and robust predictive biomarkers are still lacking for key targeted therapeutics. Since proteins are the targets of most anti-cancer therapies, and potentially meaningful changes at the proteomic level are not always present at the genomic level, it is becoming increasingly clear that the cancer proteome is an underexplored domain with significant potential for novel biomarker discovery. To date, the contribution of cancer tissue proteomics to biomarker discovery has been hampered by the requirement for substantial amounts of tissue and the need for more sensitive and high-throughput techniques. The current bulk proteomic analysis does not account for the heterogeneity within a tumor, or the proteomic profiling of rare or low-abundance cells. Single-cell proteomic technologies will have this capacity and will facilitate the development of better diagnosis and more efficient, individualized treatment. Although it is too early to predict the ultimate form and potential of such technology, some initial steps towards high- throughput single-cell proteomic approaches have already been taken. For example, researchers in the mass spectrometry field are rethinking sample preparation (cell lysis, protein purification, digestion and clean-up) and separation approaches to reduce sample losses during processing, to be able to quantify over a thousand proteins in single cells. New innovative proteomic approaches that use the principles of parallel-in-space fluorescence imaging (developed for next-generation DNA sequencing) are also being developed. Project Goals The short-term goal of this concept is to stimulate the development of unbiased (i.e. untargeted) discovery proteomic technologies with the capacity to identify proteins in a single cell with a typical size (~10 μm in diameter). The mid-term goal is to provide efficient research tools with the ability to generate more complete and accurate human cancer proteome information without relying on antibodies or inferring proteomes from mRNA sequencing. Protein sequencing at the single-cell level will allow a better understanding of tumor heterogeneity and microenvironment. Single-cell proteomics will also enable capturing proteomic information from rare and low-abundant cells such as circulating tumor cells and migratory dendritic cells. This will open the door to new biomarker and therapeutic target discoveries in cancers. The long-term goals also include providing efficient clinical tools for precision medicine by matching patients to therapies based on their proteomic results from clinically relevant samples; earlier cancer detection with the ability to better differentiate healthy normal cells from cancerous cells by adding proteomic information to the genomic and transcriptomic data; and better assessment of treatment response and monitoring with the capacity to get more precise clonal information. Activities not responsive to announcement: • Technologies that are solely based on computational approaches • Bulk proteomic technologies using bioinformatic approaches to deconvolve different cell and clone types in the bulk tumor sample • Targeted methods for identifying and quantifying proteins including, but not limited to, antibody-based methods • Technologies incapable to identify and quantify at least 500 proteins in a single cell Phase I Activities and Deliverables: Phase I activities should generate proof-of-concept data that demonstrates the capability of the technology to identify and quantify, at least, 500 proteins in a single cell with a typical size (i.e. ~10 μm in diameter): • Benchmark the new technology against existing approaches. • Provide an analytical validation report that describes the studies performed for analytical validation of your technology and its performance characteristics including: o Accuracy o Reproducibility o Repeatability o Sensitivity for low and high abundance protein o Specificity for low and high abundance protein o Single-cell proteome depth of coverage. • Address signal-to-noise issues by evaluating and interpreting “noise” of the measurements. • Deliver detailed SOPs related to the sample preparation and sequencing protocols used for your single-cell proteomic technology to NCI for evaluation. Note: SOPs for isolation of single cells are not required. • Describe the potential pitfalls of the experimental measurements. • Develop a proof-of-principle prototype. • Present assay performance and validation results and demonstrate the workflow of the technology during NCI SBIR site visit. Phase II Activities and Deliverables: Phase II activities should support establishing commercial prototype of the technology, including but not limited by the following activities: • Demonstrate that the technology is identifying and quantifying at least 500 proteins in a single cell with a typical size (i.e.~10 μm in diameter) • Demonstrate system performance and functionality by adopting commercially relevant quantitative milestones: o Offerors should specify quantitative technical and commercially-relevant milestones that can be used to evaluate the success of the tool or technology being developed. o Offerors should also provide appropriate justification relevant to both the development and commercialization of these technologies. o Quantitative milestones may be relative metrics (e.g. compared to benchmarks, alternative assays or minimization of the pitfalls of the experimental measurements described in phase I) or absolute metrics (e.g. minimum level of detection in a clinically meaningful indication). • Demonstrate that the technology can analyze the proteomes from the routinely collected cancer samples (fresh, frozen, fixed tissue and/or blood samples). • Report the throughput of the technology and the cost of the proteomic analysis of a single cell • Show feasibility to scale up the technology at a throughput compatible with widespread adoption by the clinical research community. • Establish QA/QC parameters at every step of the process to ensure reliability of results generated by your technology. • Develop a working prototype kit/tool/device for the single-cell proteomic technology (e.g. a sample preparation product and/or a protein sequencing product) and/or establish a marketing partnership/alliance with an established strategic business partner (e.g. diagnostic or device company)
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