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Development of CTC Isolation Technologies Enabling Downstream Single Cell Molecular Analysis


Summary Circulating tumor cells (CTCs) are cancer cells shed from either the primary tumor or its metastases and are circulating in the peripheral blood. While metastases are directly responsible for the majority of cancer deaths, CTCs may constitute seeds for metastases and may be instrumental for the spread of the disease. Many studies have shown that the presence of CTCs in peripheral blood or bone marrow is of prognostic significance in different types of solid tumors, and that the number and molecular changes of CTCs may help predict or monitor response to treatment. An increasing number of studies have shown large molecular and cellular heterogeneity of CTCs from the same types of cancer and even from CTCs from the same patient. This phenomenon has made the interpretation of cancer status very difficult. Current FDA-approved CTC analysis is based on immunological capture of CTCs by magnetic beads. This method does not capture all types of CTCs, and the recovery of the captured cells for subsequent molecular or cellular analysis is limited; hence, it is important to develop improved methodologies for CTC isolation that enable subsequent genomic, proteomic, or metabolomic analysis at the single cell level in order to understand the origin and role of these subpopulations of CTCs in cancer progression and treatment response. Enabling CTC analysis at the single-cell level will significantly contribute to cancer research and the selection of treatment options for patients based on changes in CTC numbers and molecular characteristics before and during treatment. Project Goals The long-term goal of the project is to integrate new or established technologies to enable molecular characterization and analysis of individual CTCs isolated from blood or bone marrow. An ideal system will be a modular platform combining a CTC capture and separation module with several other modules for downstream molecular analysis such as genomic, metabolomic, proteomic and mutation analysis at the individual cell level. Non-modular systems will also be acceptable. The short-term goal is to demonstrate the technical viability of the proposed technology to isolate and analyze CTCs at the single-cell level in an experimental setting. If molecular analysis is not performed with the proposed device, a detailed description about compatible downstream analysis technology(/ies), including manufacturers and model numbers, is required. Acceptable studies include but are not limited to: • CTC isolation and enrichment technologies such as magnetic separation, microfluidics, size separation, and negative or positive selection • Integrated CTC devices which combine CTC capture and molecular analysis • Viable CTC cell isolation and/or culturing for treatment assessment • Low-cost multichannel scanning, imaging, flow cytometry, spectral analysis or equivalent technologies for CTC molecular analysis with the potential to combine with innovative single-cell isolation (e.g. micro dissection) • Non-separation-based technologies for CTCs that enable molecular analysis Phase I Activities and Expected Deliverables • Develop a method for CTC isolation or identification amenable to downstream single-cell analysis • The technology/device should be able to isolate or identify CTCs from samples with CTC counts as low as one cell/ml of blood (for Phase I, seeding experiments are acceptable) • The technology/device should be able to perform single-cell molecular analysis (or whole genome amplification) for more than 100 CTCs, or isolate more than 100 CTCs individually in a format and volume that is compatible with existing downstream single-cell molecular analysis o In the latter case, please specify the format, volume, and intended downstream analysis • Characterize the variation, reproducibility, and accuracy of the method; the method must demonstrate at least 80% recovery and 70% purity • When applicable (e.g., when downstream analysis is gene expression), determine the viability of CTCs • Demonstrate feasibility that the device (including imaging, spectral analysis or equivalent technologies) can provide CTCs for molecular analysis at the single-cell level, and at least 10 biomarkers (including markers to confirm that the isolated cells are CTCs) can be measured (preferably simultaneously) for the same cell • Implement a QA/QC plan • The establishment of a collaboration or partnership with established diagnostic or pharmaceutical companies is strongly encouraged • Provide the NCI with SOPs, including sample collection, shipping, storage conditions, consumables used, and molecular analysis, for evaluation • Provide the NCI detailed design specifications (including components) and estimations of the cost of producing the proposed devices and/or reagents, including an analysis/breakdown of vendors and/or sources of raw materials Phase II Activities and Expected Deliverables • Develop a prototype of the device incorporating the technology demonstrated in Phase I with at least two of the applications below or other applications with significant clinical utility: o Single CTC whole genome sequencing o Single CTC molecular phenotyping o Single CTC proteomic analysis o Single CTC metabolomic analysis o Single CTC targeted multiplex gene expression analysis o Single CTC targeted multiplex mutation analysis o Single CTC targeted multiplex epigenetic analysis o Culture of individual CTCs with sufficient percent of viable cells for ex vivo analysis (e.g. drug treatment) • Test the device with a sufficient number of patient samples to demonstrate clinical utility and advantages, with appropriate consideration of statistical significance • Establish a marketing partnership or alliance with an established diagnostic or device company
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