This is a revision to a proposal submitted in response to PA-04-035 concerning the development of novel technologies for collecting, enriching, and detecting exfoliated tumor cells in bodily fluids. Our proposal will address two important issues in molecular diagnosis of cancer cells: generation of molecular probes for the identification of cancer cells and the sensitive detection of exfoliated tumor cells. Molecular level differences are present between any two given types of cells. Making use of these differences to generate novel cell-specific molecular probes for the efficient capturing and detection of cancer cells is the major theme of this proposal. Recently, we have developed a novel cell-based aptamer selection strategy (cell-SELEX) to produce a group of aptamers for the specific recognition of individual cells without prior knowledge of the biomarkers on the cells. The cell-SELEX uses whole cells as targets to select aptamers that can distinguish target from control cells. Using leukemia as a model, we have generated more than 20 aptamers. The selection process is fast and reproducible. The newly generated aptamers recognize target cells in a variety of samples including bone marrow and leukemia patient samples. ? ? In this proposal, we will develop effective molecular probes for small cell lung cancer (SCLC) using cell-SELEX. SCLC is a very aggressive form of lung cancer with only 15% long-term disease-free survival rate, partially due to the lack of methods for early diagnosis. SCLC and non-SCLC cells will be used as target and control cells for cell-SELEX, respectively. Once the aptamers highly specific for SCLC are generated, they will be conjugated to the magnetic nanoparticles (MNPs) and fluorescent nanoparticles (NPs) developed in our laboratory. The two NPs will then be incubated with SCLC samples for the enrichment and sensitive detection of exfoliated SCLC cells. The fluorescent NPs can report the SCLC cells with excellent sensitivity as each NP has tens of thousands of fluorophores embedded. The two- particle approach will allow fast enrichment and sensitive detection of trace amount of exfoliated SCLC cells in bodily fluids, thus enabling the potential for early diagnosis of the lung cancer. Once completed, the proposed work will not only show that cell-based aptamer selection can be widely applicable to various types of cells, but also convincingly demonstrate the advantages of using cell-SELEX to generate aptamers for effective cancer studies and early cancer diagnosis. ? ? This proposal details a highly innovative method for the collection, enrichment, and detection of exfoliated small cell lung cancer cells from complex samples. This methodology has great medical relevance including allowing earlier detection of small cell lung cancer and could even be used as a screening method for the disease. If successful the proposed research could have a great impact in cancer detection in that the protocols developed will be applicable to nearly all exfoliated cancer cell types. ? ? ?
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