Flow cytometric determination of stem/progenitor content in epithelial tissues: an example from nonsmall lung cancer and normal lung.
Single cell analysis and cell sorting has enabled the study of development, growth, differentiation, repair and maintenance of "liquid" tissues and their cancers. The application of these methods to solid tissues is equally promising, but several unique technical challenges must be addressed. This report illustrates the application of multidimensional flow cytometry to the identification of candidate stem/progenitor populations in non-small cell lung cancer and paired normal lung tissue. Seventeen paired tumor/normal lung samples were collected at the time of surgical excision and processed immediately. Tissues were mechanically and enzymatically dissociated into single cell suspension and stained with a panel of antibodies used for negative gating (CD45, CD14, CD33, glycophorin A), identification of epithelial cells (intracellular cytokeratin), and detection of stem/progenitor markers (CD44, CD90, CD117, CD133). DAPI was added to measure DNA content. Formalin fixed paraffin embedded tissue samples were stained with key markers (cytokeratin, CD117, DAPI) for immunofluorescent tissue localization of populations detected by flow cytometry. Disaggregated tumor and lung preparations contained a high proportion of events that would interfere with analysis, were they not eliminated by logical gating. We demonstrate how inclusion of doublets, events with hypodiploid DNA, and cytokeratin+ events also staining for hematopoietic markers reduces the ability to quantify epithelial cells and their precursors. Using the lung cancer/normal lung data set, we present an approach to multidimensional data analysis that consists of artifact removal, identification of classes of cells to be studied further (classifiers) and the measurement of outcome variables on these cell classes. The results of bivariate analysis show a striking similarity between the expression of stem/progenitor markers on lung tumor and adjacent tumor-free lung.
Department of Cardiothoracic Surgery, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA. donnenbergvs@upmc.edu
Vera S
VS
Donnenberg
Rodney J
RJ
Landreneau
Melanie E
ME
Pfeifer
Albert D
AD
Donnenberg
D000230:Adenocarcinoma / Q000378:metabolism / Q000473:pathology; D015415:Biomarkers / Q000378:metabolism; D014408:Biomarkers, Tumor / Q000378:metabolism; D002289:Carcinoma, Non-Small-Cell Lung / Q000378:metabolism / Q000473:pathology*; D004247:DNA / Q000378:metabolism; D004273:DNA, Neoplasm / Q000378:metabolism; D004847:Epithelial Cells / Q000378:metabolism / Q000473:pathology*; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D007633:Keratins / Q000378:metabolism; D008168:Lung / Q000378:metabolism / Q000473:pathology*; D008175:Lung Neoplasms / Q000378:metabolism / Q000473:pathology*; D019009:Proto-Oncogene Proteins c-kit / Q000378:metabolism; D013234:Stem Cells / Q000378:metabolism / Q000473:pathology*
D015415:Biomarkers; D014408:Biomarkers, Tumor; D004273:DNA, Neoplasm; D007633:Keratins; D004247:DNA; D019009:Proto-Oncogene Proteins c-kit