Multiplex detection of surface molecules on colorectal cancers.
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 alpha/beta, 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.
School of Molecular and Microbial Biosciences G08, University of Sydney, Sydney, NSW, Australia.
Peter
P
Ellmark
Larissa
L
Belov
Pauline
P
Huang
C Soon
CS
Lee
Michael J
MJ
Solomon
Daniel K
DK
Morgan
Richard I
RI
Christopherson
D015704:CD4 Antigens / Q000276:immunology / Q000378:metabolism; D018938:Caco-2 Cells; D002272:Carcinoembryonic Antigen / Q000276:immunology / Q000378:metabolism; D018929:Cell Culture Techniques; D045744:Cell Line, Tumor; D016000:Cluster Analysis; D015179:Colorectal Neoplasms / Q000737:chemistry* / Q000473:pathology; D003470:Culture Media; D016650:Fluorescein-5-isothiocyanate; D005455:Fluorescent Antibody Technique; D005456:Fluorescent Dyes; D020869:Gene Expression Profiling; D006801:Humans; D016130:Immunophenotyping; D019012:Integrin beta1 / Q000276:immunology / Q000378:metabolism; D040081:Protein Array Analysis / Q000379:methods*; D020543:Proteome / Q000032:analysis*; D017464:Receptors, Complement 3d / Q000276:immunology / Q000378:metabolism; D012680:Sensitivity and Specificity
D015704:CD4 Antigens; D002272:Carcinoembryonic Antigen; D003470:Culture Media; D005456:Fluorescent Dyes; D019012:Integrin beta1; D020543:Proteome; D017464:Receptors, Complement 3d; D016650:Fluorescein-5-isothiocyanate