title: Cell surface profiling using high-throughput flow cytometry: a platform for biomarker discovery and analysis of cellular heterogeneity.
abstract: Cell surface proteins have a wide range of biological functions, and are often used as lineage-specific markers. Antibodies that recognize cell surface antigens are widely used as research tools, diagnostic markers, and even therapeutic agents. The ability to obtain broad cell surface protein profiles would thus be of great value in a wide range of fields. There are however currently few available methods for high-throughput analysis of large numbers of cell surface proteins. We describe here a high-throughput flow cytometry (HT-FC) platform for rapid analysis of 363 cell surface antigens. Here we demonstrate that HT-FC provides reproducible results, and use the platform to identify cell surface antigens that are influenced by common cell preparation methods. We show that multiple populations within complex samples such as primary tumors can be simultaneously analyzed by co-staining of cells with lineage-specific antibodies, allowing unprecedented depth of analysis of heterogeneous cell populations. Furthermore, standard informatics methods can be used to visualize, cluster and downsample HT-FC data to reveal novel signatures and biomarkers. We show that the cell surface profile provides sufficient molecular information to classify samples from different cancers and tissue types into biologically relevant clusters using unsupervised hierarchical clustering. Finally, we describe the identification of a candidate lineage marker and its subsequent validation. In summary, HT-FC combines the advantages of a high-throughput screen with a detection method that is sensitive, quantitative, highly reproducible, and allows in-depth analysis of heterogeneous samples. The use of commercially available antibodies means that high quality reagents are immediately available for follow-up studies. HT-FC has a wide range of applications, including biomarker discovery, molecular classification of cancers, or identification of novel lineage specific or stem cell markers.
authors.affiliation: Princess Margaret Cancer Centre, University Health Network, Toronto, ON, Canada.
authors.forename: Craig A
authors.initials: CA
authors.lastname: Gedye
authors.affiliation: Dept. of Medical Biophysics, University of Toronto, Toronto, ON, Canada.
authors.forename: Ali
authors.initials: A
authors.lastname: Hussain
authors.affiliation: Princess Margaret Cancer Centre, University Health Network, Toronto, ON, Canada.
authors.forename: Joshua
authors.initials: J
authors.lastname: Paterson
authors.affiliation: Princess Margaret Cancer Centre, University Health Network, Toronto, ON, Canada.
authors.forename: Alannah
authors.initials: A
authors.lastname: Smrke
authors.affiliation: Princess Margaret Cancer Centre, University Health Network, Toronto, ON, Canada.
authors.forename: Harleen
authors.initials: H
authors.lastname: Saini
authors.affiliation: Princess Margaret Cancer Centre, University Health Network, Toronto, ON, Canada.
authors.forename: Danylo
authors.initials: D
authors.lastname: Sirskyj
authors.affiliation: Dept. of Medical Biophysics, University of Toronto, Toronto, ON, Canada.
authors.forename: Keira
authors.initials: K
authors.lastname: Pereira
authors.affiliation: Dept. of Medical Biophysics, University of Toronto, Toronto, ON, Canada.
authors.forename: Nazleen
authors.initials: N
authors.lastname: Lobo
authors.affiliation: Dept. of Medical Biophysics, University of Toronto, Toronto, ON, Canada.
authors.forename: Jocelyn
authors.initials: J
authors.lastname: Stewart
authors.affiliation: Princess Margaret Cancer Centre, University Health Network, Toronto, ON, Canada.
authors.forename: Christopher
authors.initials: C
authors.lastname: Go
authors.affiliation: Princess Margaret Cancer Centre, University Health Network, Toronto, ON, Canada.
authors.forename: Jenny
authors.initials: J
authors.lastname: Ho
authors.affiliation: Dept. of Medical Biophysics, University of Toronto, Toronto, ON, Canada.
authors.forename: Mauricio
authors.initials: M
authors.lastname: Medrano
authors.affiliation: Hospital for Sick Children Research Institute, Toronto, ON, Canada.
authors.forename: Elzbieta
authors.initials: E
authors.lastname: Hyatt
authors.affiliation: Hospital for Sick Children Research Institute, Toronto, ON, Canada.
authors.forename: Julie
authors.initials: J
authors.lastname: Yuan
authors.affiliation: Hospital for Sick Children Research Institute, Toronto, ON, Canada.
authors.forename: Stevan
authors.initials: S
authors.lastname: Lauriault
authors.forename: Mona
authors.initials: M
authors.lastname: Meyer
authors.affiliation: Princess Margaret Cancer Centre, University Health Network, Toronto, ON, Canada.
authors.forename: Maria
authors.initials: M
authors.lastname: Kondratyev
authors.affiliation: Princess Margaret Cancer Centre, University Health Network, Toronto, ON, Canada.
authors.forename: Twan
authors.initials: T
authors.lastname: van den Beucken
authors.affiliation: Princess Margaret Cancer Centre, University Health Network, Toronto, ON, Canada.
authors.forename: Michael
authors.initials: M
authors.lastname: Jewett
authors.affiliation: Hospital for Sick Children Research Institute, Toronto, ON, Canada.
authors.forename: Peter
authors.initials: P
authors.lastname: Dirks
authors.affiliation: Hospital for Sick Children Research Institute, Toronto, ON, Canada.
authors.forename: Cynthia J
authors.initials: CJ
authors.lastname: Guidos
authors.affiliation: Hospital for Sick Children Research Institute, Toronto, ON, Canada.
authors.forename: Jayne
authors.initials: J
authors.lastname: Danska
authors.affiliation: Princess Margaret Cancer Centre, University Health Network, Toronto, ON, Canada.
authors.forename: Jean
authors.initials: J
authors.lastname: Wang
authors.affiliation: Princess Margaret Cancer Centre, University Health Network, Toronto, ON, Canada; Dept. of Medical Biophysics, University of Toronto, Toronto, ON, Canada.
authors.forename: Bradly
authors.initials: B
authors.lastname: Wouters
authors.affiliation: Princess Margaret Cancer Centre, University Health Network, Toronto, ON, Canada; Dept. of Medical Biophysics, University of Toronto, Toronto, ON, Canada.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Neel
authors.affiliation: Princess Margaret Cancer Centre, University Health Network, Toronto, ON, Canada.
authors.forename: Robert
authors.initials: R
authors.lastname: Rottapel
authors.affiliation: Princess Margaret Cancer Centre, University Health Network, Toronto, ON, Canada; Dept. of Medical Biophysics, University of Toronto, Toronto, ON, Canada.
authors.forename: Laurie E
authors.initials: LE
authors.lastname: Ailles
mesh_terms: D000954:Antigens, Surface / Q000032:analysis*; D015415:Biomarkers / Q000032:analysis; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D016000:Cluster Analysis; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D019169:Jurkat Cells; D061986:MCF-7 Cells; D008856:Microscopy, Fluorescence; D020543:Proteome / Q000032:analysis* / Q000145:classification / Q000276:immunology; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results
chemical_list: D000954:Antigens, Surface; D015415:Biomarkers; D020543:Proteome