Abstract
We have developed and optimized a microfluidic device platform for the capture and analysis of circulating pancreatic cells (CPCs) and pancreatic circulating tumor cells (CTCs). Our platform uses parallel anti-EpCAM and cancer-specific mucin 1 (MUC1) immunocapture in a silicon microdevice. Using a combination of anti-EpCAM and anti-MUC1 capture in a single device, we are able to achieve efficient capture while extending immunocapture beyond single marker recognition. We also have detected a known oncogenic KRAS mutation in cells spiked in whole blood using immunocapture, RNA extraction, RT-PCR and Sanger sequencing. To allow for downstream single-cell genetic analysis, intact nuclei were released from captured cells by using targeted membrane lysis. We have developed a staining protocol for clinical samples, including standard CTC markers; DAPI, cytokeratin (CK) and CD45, and a novel marker of carcinogenesis in CPCs, mucin 4 (MUC4). We have also demonstrated a semi-automated approach to image analysis and CPC identification, suitable for clinical hypothesis generation. Initial results from immunocapture of a clinical pancreatic cancer patient sample show that parallel capture may capture more of the heterogeneity of the CPC population. With this platform, we aim to develop a diagnostic biomarker for early pancreatic carcinogenesis and patient risk stratification.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, Non-P.H.S.
MeSH terms
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Antibodies, Immobilized / immunology
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Antigens, Neoplasm / analysis*
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Antigens, Neoplasm / immunology
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Biomarkers, Tumor / analysis
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Biomarkers, Tumor / immunology
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Cell Adhesion Molecules / analysis*
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Cell Adhesion Molecules / immunology
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Cell Line
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Epithelial Cell Adhesion Molecule
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Humans
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Indoles / analysis
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Indoles / immunology
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Islets of Langerhans / cytology
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Islets of Langerhans / metabolism*
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Keratins / analysis
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Keratins / immunology
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Leukocyte Common Antigens / analysis
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Leukocyte Common Antigens / immunology
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Microfluidic Analytical Techniques / instrumentation
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Microfluidic Analytical Techniques / methods*
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Mucin-1 / analysis*
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Mucin-1 / immunology
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Mucin-4 / analysis
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Mucin-4 / immunology
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Neoplastic Cells, Circulating / metabolism
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Proto-Oncogene Proteins / blood
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Proto-Oncogene Proteins / genetics
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Proto-Oncogene Proteins / immunology
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Proto-Oncogene Proteins p21(ras)
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RNA / blood
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RNA / isolation & purification
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Silicon / chemistry
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ras Proteins / blood
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ras Proteins / genetics
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ras Proteins / immunology
Substances
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Antibodies, Immobilized
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Antigens, Neoplasm
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Biomarkers, Tumor
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Cell Adhesion Molecules
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EPCAM protein, human
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Epithelial Cell Adhesion Molecule
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Indoles
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KRAS protein, human
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Mucin-1
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Mucin-4
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Proto-Oncogene Proteins
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DAPI
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RNA
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Keratins
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Leukocyte Common Antigens
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Proto-Oncogene Proteins p21(ras)
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ras Proteins
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Silicon