Abstract 1182: Single cell analysis of primary and disseminated tumor cells isolated from tissue and blood
Authors: Deng Glenn , Powell Ashley A. , Zhang Haiyu , Krishnakumar Sujatha , Talasaz AmirAli H. , Mondrinos Michael , Davis Ronald W. , Jeffrey Stefanie S. 
Published: February 2011

Abstract
Background: Cancer evolves through the accumulation of multiple genetic alterations. How such alterations are related to tumor growth and metastatic dissemination is being widely investigated. Tissue level analyses, however, may be confounded by tumor cell heterogeneity and contamination by wildtype stromal and immune cells. Single cell analysis of tumor cells may contribute to a better understanding of tumorigenesis and metastasis. Single cell analyses using flow cytometry or microdissected cells from tissues require large numbers of cells, expensive equipment, laborious technical training, and the analyzed single cell information may be impacted by technical processing artifacts. We developed an efficient, reliable and simple new method to isolate fresh single tumor cells from tissues and blood for mutational analysis and to evaluate additional biomarkers.
Materials and Methods: Tumor tissues and blood samples were obtained from breast cancer patients after informed consent. Primary or metastatic tissue was used for preparation of single cell suspensions for subsequent isolation. Single cells were isolated using EpCAM-conjugated microbeads and the MagSweeper, a device invented by our group. Individual microbead-labeled single cells were easy to observe and extract. Single cells were placed into individual PCR tubes for further DNA and RNA analyses. Single cell DNA mutational analysis was performed by sequencing using multiplex pre-amplification followed by PCR amplification. Multiplex RNA expression analyses were performed by qRT-PCR from single tumor cells. Enriched and unenriched single cell suspensions were used for biomarker evaluation and results were compared with normal tissues. Tumor cells with mutations were compared on a single cell level among multiple compartments: primary tumor, blood, and metastatic tumor.
Results and Discussion: Single cells were isolated from suspensions prepared from primary and metastatic tumor tissues, EpCAM-enriched and unenriched single cells microscopically observed without requiring additional treatment and used for immunostaining for tumor cells identification. Some single cells not bound to EpCAM microbeads still met tumor cell criteria (cytokeratin positive, CD45 negative, and DAPI positive). The percentage of EpCAM -positive and cytokeratin-positive was determined in the single cell suspensions. Single cell DNA showing a single nucleotide mutation in the PIK3CA gene was identified in individual tumor cells from different tissues but not white blood cells. Results from our model indicate that isolating single tumor cells from tumor tissues or blood provides more detailed information about different tumor compartments in individual cancer patients. Comparison of individual tumor cells from primary and/or metastatic sites to tumor cells in blood should provide further insight into the metastatic process.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1182.