Surface antigen profiling of colorectal cancer using antibody microarrays with fluorescence multiplexing
A procedure is described for the disaggregation of colorectal cancers (CRC) and normal intestinal mucosal tissues to produce suspensions of viable single cells, which are then captured on customized antibody microarrays recognising 122 different surface antigens (DotScan™ CRC microarray). Cell bindi...
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Published in | Journal of immunological methods Vol. 355; no. 1; pp. 40 - 51 |
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Main Authors | , , , , , , , , , , |
Format | Journal Article |
Language | English |
Published |
Amsterdam
Elsevier B.V
15.04.2010
Elsevier |
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Abstract | A procedure is described for the disaggregation of colorectal cancers (CRC) and normal intestinal mucosal tissues to produce suspensions of viable single cells, which are then captured on customized antibody microarrays recognising 122 different surface antigens (DotScan™ CRC microarray). Cell binding patterns recorded by optical scanning of microarrays provide a surface profile of antigens on the cells. Sub-populations of cells bound on the microarray can be profiled by fluorescence multiplexing using monoclonal antibodies tagged with Quantum Dots or other fluorescent dyes. Surface profiles are presented for 6 CRC cell lines (T84, LIM1215, SW480, HT29, CaCo and SW620) and surgical samples from 40 CRC patients. Statistical analysis revealed significant differences between profiles for CRC samples and mucosal controls. Hierarchical clustering of CRC data identified several disease clusters that showed some correlation with clinico-pathological stage as determined by conventional histopathological analysis. Fluorescence multiplexing using Phycoerythrin- or Alexa Fluor 647-conjugated antibodies was more effective than multiplexing with antibodies labelled with Quantum Dots. This relatively simple method yields a large amount of information for each patient sample and, with further application, should provide disease signatures and enable the identification of patients with good or poor prognosis. |
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AbstractList | A procedure is described for the disaggregation of colorectal cancers (CRC) and normal intestinal mucosal tissues to produce suspensions of viable single cells, which are then captured on customized antibody microarrays recognising 122 different surface antigens (DotScan+ CRC microarray). Cell binding patterns recorded by optical scanning of microarrays provide a surface profile of antigens on the cells. Sub-populations of cells bound on the microarray can be profiled by fluorescence multiplexing using monoclonal antibodies tagged with Quantum Dots or other fluorescent dyes. Surface profiles are presented for 6 CRC cell lines (T84, LIM1215, SW480, HT29, CaCo and SW620) and surgical samples from 40 CRC patients. Statistical analysis revealed significant differences between profiles for CRC samples and mucosal controls. Hierarchical clustering of CRC data identified several disease clusters that showed some correlation with clinico-pathological stage as determined by conventional histopathological analysis. Fluorescence multiplexing using Phycoerythrin- or Alexa Fluor 647-conjugated antibodies was more effective than multiplexing with antibodies labelled with Quantum Dots. This relatively simple method yields a large amount of information for each patient sample and, with further application, should provide disease signatures and enable the identification of patients with good or poor prognosis. A procedure is described for the disaggregation of colorectal cancers (CRC) and normal intestinal mucosal tissues to produce suspensions of viable single cells, which are then captured on customized antibody microarrays recognising 122 different surface antigens (DotScan™ CRC microarray). Cell binding patterns recorded by optical scanning of microarrays provide a surface profile of antigens on the cells. Sub-populations of cells bound on the microarray can be profiled by fluorescence multiplexing using monoclonal antibodies tagged with Quantum Dots or other fluorescent dyes. Surface profiles are presented for 6 CRC cell lines (T84, LIM1215, SW480, HT29, CaCo and SW620) and surgical samples from 40 CRC patients. Statistical analysis revealed significant differences between profiles for CRC samples and mucosal controls. Hierarchical clustering of CRC data identified several disease clusters that showed some correlation with clinico-pathological stage as determined by conventional histopathological analysis. Fluorescence multiplexing using Phycoerythrin- or Alexa Fluor 647-conjugated antibodies was more effective than multiplexing with antibodies labelled with Quantum Dots. This relatively simple method yields a large amount of information for each patient sample and, with further application, should provide disease signatures and enable the identification of patients with good or poor prognosis. |
Author | Huang, Pauline Y. Bokey, Leslie Clarke, Candice Zhou, Jerry Belov, Larissa Chan, Charles Clarke, Stephen J. Chapuis, Pierre H. Christopherson, Richard I. Solomon, Michael J. Shin, Joo-Shik |
Author_xml | – sequence: 1 givenname: Jerry surname: Zhou fullname: Zhou, Jerry organization: School of Molecular & Microbial Biosciences, University of Sydney, NSW 2006, Australia – sequence: 2 givenname: Larissa surname: Belov fullname: Belov, Larissa email: L.Belov@usyd.edu.au organization: School of Molecular & Microbial Biosciences, University of Sydney, NSW 2006, Australia – sequence: 3 givenname: Pauline Y. surname: Huang fullname: Huang, Pauline Y. organization: School of Molecular & Microbial Biosciences, University of Sydney, NSW 2006, Australia – sequence: 4 givenname: Joo-Shik surname: Shin fullname: Shin, Joo-Shik organization: Royal Prince Alfred Hospital, Camperdown, NSW 2050, Australia – sequence: 5 givenname: Michael J. surname: Solomon fullname: Solomon, Michael J. organization: Royal Prince Alfred Hospital, Camperdown, NSW 2050, Australia – sequence: 6 givenname: Pierre H. surname: Chapuis fullname: Chapuis, Pierre H. organization: Department of Colorectal Surgery, Concord Hospital, NSW 2319, Australia – sequence: 7 givenname: Leslie surname: Bokey fullname: Bokey, Leslie organization: Department of Colorectal Surgery, Concord Hospital, NSW 2319, Australia – sequence: 8 givenname: Charles surname: Chan fullname: Chan, Charles organization: Department of Anatomical Pathology, Concord Hospital, NSW 2139, Australia – sequence: 9 givenname: Candice surname: Clarke fullname: Clarke, Candice organization: Department of Anatomical Pathology, Concord Hospital, NSW 2139, Australia – sequence: 10 givenname: Stephen J. surname: Clarke fullname: Clarke, Stephen J. organization: Department of Medicine, Concord Hospital, NSW 2319, and Department of Medicine, University of Sydney, NSW 2006, Australia – sequence: 11 givenname: Richard I. surname: Christopherson fullname: Christopherson, Richard I. organization: School of Molecular & Microbial Biosciences, University of Sydney, NSW 2006, Australia |
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Keywords | Multiplexing CD QDs EpCAM IHC Colorectal cancer Antibody microarrays Fluorescence Leukocytes ACP EGFR CD antigens BSA TAMs CRC FCS TILs HBSS CEA MeV Rectal disease Antibody Leukocyte Malignant tumor Immunological method Colonic disease Antigen Digestive diseases Intestinal disease Cancer |
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SubjectTerms | Adult Aged Aged, 80 and over Antibodies, Monoclonal - chemistry Antibodies, Neoplasm - chemistry Antibody microarrays Antigens, Neoplasm - metabolism Biological and medical sciences CD antigens Cell Line, Tumor Colorectal cancer Colorectal Neoplasms - metabolism Female Fluorescence Fluorescent Dyes - chemistry Fundamental and applied biological sciences. Psychology Fundamental immunology Humans Leukocytes Male Middle Aged Molecular immunology Multiplexing Protein Array Analysis - methods Quantum Dots Techniques |
Title | Surface antigen profiling of colorectal cancer using antibody microarrays with fluorescence multiplexing |
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