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.

Rare cancer stem cells (CSC) are proposed to be responsible for tumour propagation and re-initiation and are functionally defined by identifying tumour-initiating cells (TICs) using the xenotransplantation limiting dilution assay (LDA). While TICs in clear cell renal cell carcinoma (ccRCC) appeared rare in NOD/SCID/IL2Rγ−/− (NSG) mice, xenografts formed more efficiently from small tumour fragments, indicating the LDA underestimated ccRCC TIC frequency. Mechanistic interrogation of the LDA identified multiple steps that influence ccRCC TIC quantitation. For example, tissue disaggregation destroys most ccRCC cells, common assays significantly overestimate tumour cell viability, and microenvironmental supplementation with human extracellular factors or pharmacological inhibition of anoikis increase clonogenicity and tumourigenicity of ccRCC cell lines and primary tumour cells. Identification of these previously uncharacterized concerns that cumulatively lead to substantial underestimation of TICs in ccRCC provides a framework for development of more accurate TIC assays in the future, both for this disease and for other cancers.

4568 Background: To study “cancer stem cells” it is imperative to account for all stromal cell populations within the tumour. The existence of “cancer stem cells” in clear cell renal cell carcinoma (ccRCC) has not been examined in ex vivo patient samples. Methods: We established a multiplex flow cytometry (FC) antibody panel in ccRCC, which reliably identified stromal lineages including CD45+ immune, CD31+/CD144+ endothelial and fibroblast-marker-positive subpopulations, thus allowing isolation of "lineage-negative" tumor cells. To verify the identity of tumour-derived populations as either cancer cells or normal stromal cells, we took advantage of the fact that mutations in VHL occur early during ccRCC tumorigenesis and are found in two-thirds of patients. Results: We sequenced 18 patient tumor samples, 12 of which had VHL exome mutations. Targeted re-sequencing of FC sorted subpopulations from these patients’ samples revealed that while CD45+ immune cells and CD31+/CD144+ endothelial cells were genetically normal, a population of VHL-mutant fibroblast-marker positive cells was consistently identified in every patient’s tumour. Immunohistochemistry showed that fibroblast marker-positive VHL-mutant cells do not have the large “clear cell” morphology typical of the majority of the cancer cells in these tumours. When purified and cultured, these fibroblast marker-positive VHL-mutant cells proliferate extensively under mesenchymal culture conditions, but displayed different morphologies to lineage-negative VHL-mutant tumor cells. Functional characterization of these FC sorted cell subpopulations is ongoing, including proliferation, migration, invasion, differentiation and treatment resistance. Conclusions: The phenotype and preliminary functional characterization of these VHL-mutant fibroblast-marker positive cells suggests a mesenchymal differentiation program in ccRCC, with implications for the ontogeny, biology and clinical management of VHL-mutant renal cancer.