Abstract 2390: Increased detection of circulating prostate epithelial tumor cells on microfluidic channels using enhanced staining and automated scanning
Authors: Pircher Tony J. , Millar Lisa S. , Tsinberg Pavel , Wong Brian , Mikolajczyk Stephen D. 
Published: August 2012

Abstract
INTRODUCTION Anti-cytokeratin antibodies are typically used to detect circulating tumor cells (CTC) of epithelial origin in the blood of cancer patients. Cytokeratin (CK) is a cytoplasmic structural protein. We have previously described the CEE Enhanced TM (CE) technology for in situ fluorescence labeling of the capture antibodies used to isolate CTCs in our microfluidic channel. The signal intensity of cells stained by CE therefore reflects the number of antibody-targeted surface antigens on each cell. In this study we show enhanced and increased detection of prostate CTCs by co-staining CTCs with anti-CK, CE and anti-PSA in combination with automated digital analysis. METHODS Matched tubes of prostate cancer blood were processed and the CTCs captured on micro-channels using a cocktail of antibodies. CTCs were fluorescently stained with anti-CK and anti-CD45 labeled with AlexaFluor 488 and 594, respectively. Micro-channels were rapidly scanned using a Cyntellect Celigo cell scanner in combination with an automated Applied Spectral Imaging microscope system. CTCs were identified as intact cells that were DAPI+, CK+ and CD45-negative. After CTCs were confirmed by image analysis, the same channels were further stained with CE-AlexaFluor 488 and anti-PSA labeled with AlexaFluor 647. The cell XY coordinates and cell fluorescence intensity for scan 1 and scan 2 of the same micro-channel were matched using proprietary software. Changes in fluorescence intensity and co-localization of PSA staining were determined. All reported CTCs were DAPI + and CD45 negative. RESULTS Seventy-four of 100 prostate CTCs matched after CK stain and subsequent staining with CE showed a CE increase in fluorescence intensity greater than 10% above CK. The mean increase in fluorescence intensity for these CTCs was 5.8-fold (95% CI 4.0-7.6), with a median increase of 2.6-fold (95% CI 2.0-3.5). The mean and median increase in CTC fluorescence intensity after CE was significant (p&lt;0.001). In addition to increased fluorescence intensity of CK-stained CTCs after CE, a 40% increase in the number of CTCs was observed as CK-negative cells that had become positively stained with CE. Of the CK+ CTCs, 70% were PSA positive, while CK-negative cells detected as CTCs with CE-only were 54% PSA positive. Staining first with CE on matched blood samples gave similar numbers of CTCs as staining with anti-CK. CONCLUSIONS These results demonstrate that most CD45-, CK+ prostate CTCs co-stained with CE and PSA. In addition, new CK-, PSA+ CTCs were detectable as CE positive and CD45 negative. CE can increase the fluorescence signal of weakly CK stained cells, and allows detection of CTCs previously below the CK detection threshold. Studies are underway to characterize other CE-positive, CK-negative cells with epithelial-to-mesenchymal cellular markers.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2390. doi:1538-7445.AM2012-2390