6 research outputs found
Cell Surface Profiling Using High-Throughput Flow Cytometry: A Platform for Biomarker Discovery and Analysis of Cellular Heterogeneity
<div><p>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.</p></div
HT-FC allows phenotypic segregation and identification of cell samples from diverse lineages.
<p>Unsupervised hierarchical clustering of percent-positive marker expression values generated on 119 samples was performed. Colours indicate emergence of biologically related samples into clusters based on surface marker profiles. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105602#pone.0105602.s002" target="_blank">Figure S2</a> for a magnified image of the dendrogram.</p
HT-FC allows intratumoral analysis of stromal and cancer cell subsets within primary ccRCC samples.
<p>(<b>A</b>) Heatmap showing expression of each of the 363 antibodies in four subpopulations of ccRCC samples: CD45<sup>+</sup> immune cells, CD45<sup>−</sup>CD31<sup>+</sup>CD34<sup>+</sup> vascular endothelial cells, CD45<sup>−</sup>TE7<sup>+</sup> fibroblasts, and CD45<sup>−</sup>TE7<sup>−</sup>CD31<sup>−</sup>CD34<sup>−</sup> cancer cells. Antibodies are simply arranged in alphabetical order on the vertical axis and the four populations for each sample are ordered across the top. A low resolution overview demonstrates a surprisingly reproducible “fingerprint” of tumor cell subpopulations from one sample to the next. (<b>B</b>) Supervised hierarchical clustering reveals clusters of antigens corresponding to specific cell subsets within tumors (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105602#pone.0105602.s011" target="_blank">Table S6</a> for details). (<b>C</b>) Principal components analysis of the entire data set further illustrates how effectively the cell surface profile delineates the 4 distinct cell populations within primary ccRCC samples. Red: immune cells; Green: endothelial cells; Blue: fibroblasts; Orange: cancer cells.</p
The HT-FC platform is highly reproducible.
<p>MDA-MB-231 breast cancer cells, 22RV1 prostate cancer cells, or PBMCs were each run 3 times on the HT-FC platform. When the same cell line or peripheral blood sample was analyzed 3 independent times, consistently reproducible results were obtained, with Spearman correlation coefficients between runs ranging from 0.847 to 0.973, and p<0.001 for all comparisons.</p
Antibodies with no staining across all cell types analyzed.
<p>Antibodies with no staining across all cell types analyzed.</p