title: Microfluidic immunocapture of circulating pancreatic cells using parallel EpCAM and MUC1 capture: characterization, optimization and downstream analysis.
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.
authors.affiliation: Department of Biomedical Engineering, College of Engineering, Cornell University, Ithaca, NY 14853, USA. fit5@cornell.edu.
authors.forename: Fredrik I
authors.initials: FI
authors.lastname: Thege
authors.forename: Timothy B
authors.initials: TB
authors.lastname: Lannin
authors.forename: Trisha N
authors.initials: TN
authors.lastname: Saha
authors.forename: Shannon
authors.initials: S
authors.lastname: Tsai
authors.forename: Michael L
authors.initials: ML
authors.lastname: Kochman
authors.forename: Michael A
authors.initials: MA
authors.lastname: Hollingsworth
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Rhim
authors.forename: Brian J
authors.initials: BJ
authors.lastname: Kirby
mesh_terms: D055910:Antibodies, Immobilized / Q000276:immunology; D000951:Antigens, Neoplasm / Q000032:analysis* / Q000276:immunology; D014408:Biomarkers, Tumor / Q000032:analysis / Q000276:immunology; D015815:Cell Adhesion Molecules / Q000032:analysis* / Q000276:immunology; D002460:Cell Line; D000071858:Epithelial Cell Adhesion Molecule; D006801:Humans; D007211:Indoles / Q000032:analysis / Q000276:immunology; D007515:Islets of Langerhans / Q000166:cytology / Q000378:metabolism*; D007633:Keratins / Q000032:analysis / Q000276:immunology; D017493:Leukocyte Common Antigens / Q000032:analysis / Q000276:immunology; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation / Q000379:methods*; D018396:Mucin-1 / Q000032:analysis* / Q000276:immunology; D055264:Mucin-4 / Q000032:analysis / Q000276:immunology; D009360:Neoplastic Cells, Circulating / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000097:blood / Q000235:genetics / Q000276:immunology; D016283:Proto-Oncogene Proteins p21(ras); D012313:RNA / Q000097:blood / Q000302:isolation & purification; D012825:Silicon / Q000737:chemistry; D018631:ras Proteins / Q000097:blood / Q000235:genetics / Q000276:immunology
chemical_list: D055910:Antibodies, Immobilized; D000951:Antigens, Neoplasm; D014408:Biomarkers, Tumor; D015815:Cell Adhesion Molecules; C499379:EPCAM protein, human; D000071858:Epithelial Cell Adhesion Molecule; D007211:Indoles; C117307:KRAS protein, human; D018396:Mucin-1; D055264:Mucin-4; D011518:Proto-Oncogene Proteins; C007293:DAPI; D012313:RNA; D007633:Keratins; D017493:Leukocyte Common Antigens; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins; D012825:Silicon