RESEARCH ARTICLE Multiplex detection of surface molecules on colorectal cancers Peter Ellmark 1 , Larissa Belov 1 , Pauline Huang 1 , C. Soon Lee 2 , Michael J. Solomon 3 , Daniel K. Morgan 1 and Richard I. Christopherson 1 1 School of Molecular and Microbial Biosciences G08, University of Sydney, Sydney, NSW, Australia 2 Department of Anatomical Pathology, Royal Prince Alfred Hospital, Camperdown, NSW, Australia 3 Department of Colorectal Surgery, Royal Prince Alfred Hospital, Camperdown, NSW, Australia A technique of fluorescence multiplexing is described for analysis of the plasma membrane proteome of colorectal cancer cells from surgically resected specimens, enabling detection and immunophenotyping when the cancer cells are in the minority. A single-cell suspension was prepared from a colorectal tumour, and the mixed population of cells was captured on a CD antibody microarray. The cancer cells were detected using a fluorescently tagged antibody for carcinoembryonic antigen (CEA-Alexa647) or epithelial cell adhesion marker (EpCAM- Alexa488). Using this multiplexing procedure, dot patterns from colorectal cancers were distinct from those of adjacent normal tissue. Subtraction of the expression levels for each antigen from normal tissue from those for the cancer shows differential expression in the cancer of CD66c, CD15s, CD55, CD45, CD71, CD45RO, CD11b and CEA, in descending order. Cells captured on the same microarray were also labelled with fluorescent CD3-phycoerythrin antibody revealing the presence of tumour-infiltrating lymphocytes. The immunophenotypes of T lymphocytes from the tumour samples showed differential expression of HLA-DR, TCR a/b, CD49d, CD52, CD49e, CD5, CD95, CD28, CD38 and CD71, in descending order. Fluorescence multiplexing of mixed cell populations captured on a single antibody microarray enables expression profiling of multiple sub-populations of cells within a tumour sample. Received: June 24, 2005 Revised: August 23, 2005 Accepted: September 13, 2005 Keywords: Antibody microarrays / Cluster of differentiation antigens / Colorectal carcinoma / Immunophenotyping / Multiplexing Proteomics 2006, 6, 1791–1802 1791 1 Introduction Colorectal carcinoma is a common cause of cancer deaths in Western countries [1]. The majority of colorectal cancers are adenocarcinomas that have arisen from benign adenomas due to accumulation of multiple mutations [2, 3]. An efficient and reliable staging system is critical for prognosis and clin- ical decisions for post-operative adjuvant treatment. The current staging methods rely on modifications of the Dukes classification system [4, 5] based on clinical, radiological and histopathological assessments. An alternative and more accurate staging system for the cancer could be based upon an extensive immunophenotype (partial membrane pro- teome) determined using an antibody microarray, where patterns of protein expression can be correlated with sub- types of the cancer. Such an immunophenotype would reflect the genetic program of the malignant cells. A cluster of differentiation (CD) antibody microarray (DotScan) has been developed that enables identification of 82 surface antigens on leukaemia cells, providing an extensive immunophenotype from a single analysis [6, 7]. Immunophe- notypes have been obtained for leukaemias from more than Correspondence: Professor Richard I. Christopherson, School of Molecular and Microbial Biosciences, University of Sydney, NSW 2006, Australia E-mail: ric@mmb.usyd.edu.au Fax: 161-2-9351-4726 Abbreviations: ALL, acute lymphocytic leukaemia; AML, acute myeloid leukaemia; CD, cluster of differentiation; CEA, carci- noembryonic antigen; CLL, chronic lymphocytic leukaemia; DotScan, a microarray of antibodies for cell capture; EpCAM, epi- thelial cell adhesion marker; TIL, tumour-infiltrating lymphocytes DOI 10.1002/pmic.200500468  2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com