Abstract 5172: Fluorescence labeling of cytokeratin-negative circulating tumor cells in peripheral blood: A new paradigm for the study of cancer progression
Authors: Mikolajczyk Stephen D. , Millar Lisa S. , Zomorrodi Maryam , Bischoff Farideh Z. , Scoggin Jayne , Pham Tam , Wong Karina , Pircher Tony J. 
Published: June 2012

Abstract
Introduction: The measurement of circulating tumor cells (CTCs) in blood generally requires either anti-EpCAM for capture, anti-cytokeratin (CK) for detection, or both. However, EpCAM and CK are absent in some tumor cells, and both may be down-regulated during neoplastic progression or during epithelial-to-mesenchymal transition (EMT). Here we show capture on a micro-fluidic system using single or multiple antibodies, and CTC detection with CEE-Enhanced (CE), a novel in situ staining method that fluorescently labels the capture antibodies bound to the CTCs.
Methods: Buffy coat cells isolated from 8 mL of blood were pre-incubated with either anti-EpCAM alone or with a mixture of antibodies to epithelial and mesenchymal cell surface antigens. CTCs were captured on a micro-fluidic channel. Identification of CTCs was determined with anti-CK and with CE to detect cells with the capture antibodies bound to the cell surface antigens. All cells scored as positive for CK or CE were, by definition, CD45-negative and DAPI-positive.
Results: Using anti-EpCAM alone for capture, significantly more CTCs were detected by CE staining than with anti-CK in breast and prostate cancers. This indicated that CK-negative CTCs were captured but not detected and that some EpCAM-positive CTCs were CK-negative. Results using capture antibody mixtures varied from sample to sample but gave up to 2-fold higher CK-positive cells than anti-EpCAM only, and as much as 4-fold higher CE-positive cells. Control blood from healthy donors was CK and CE-negative. All CK-positive cells co-stained with CE, as determined with different fluorescent labels. In a clinical study of stage IV breast cancer, CTCs were isolated with an antibody mixture and sequentially stained with CK and CE. Fifteen of 24 samples (63%) contained CK-positive cells (range 1-60 CTCs) while 24 of 24 samples (100%) contained additional CE-positive cells (range 1-41; median=11; Wilcoxon test, p=0.02). The modest correlation coefficient (r = 0.57, p=0.004) for the number of CE-positive and CK-positive cells in each sample suggests that one or more different phenotypes of CTCs were being detected. Amplified Her2 was detected by FISH in isolated CK-positive CTCs, and also in CK-negative, CE-positive CTCs from Her2-positive patients, indicating these were tumor cells.
Conclusions: The CEE-Enhanced staining technology enables the detection of CK-negative CTCs. This novel detection method, based on the in situ labeling of antibodies used for capture, greatly expands single and multi-antibody approaches to the study of rare circulating cells. Specifically, this allows the exploration and study of circulating cells not expressing CK such as highly de-differentiated tumor cells, stem cells or those tumor cells undergoing EMT. The clinical significance of these CK-negative CTCs is under investigation.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5172. doi:10.1158/1538-7445.AM2011-5172