Enrichment of rare circulating tumor cells (CTCs) in blood is typically achieved using antibodies to epithelial cell adhesion molecule (EpCAM), with detection using cytokeratin (CK) antibodies. However, EpCAM and CK are not expressed in some tumors and can be downregulated during epithelial-to-mesenchymal transition. A micro-fluidic system, not limited to EpCAM or CK, was developed to use multiple antibodies for capture followed by detection using CEE-Enhanced (CE), a novel in situ staining method that fluorescently labels the capture antibodies bound to CTCs. Higher recovery of CTCs was demonstrated using antibody mixtures compared to anti-EpCAM. In addition, CK-positive breast cancer cells were found in 15 of 24 samples (63%; range 1–60 CTCs), while all samples contained additional CE-positive cells (range 1–41; median = 11; P = .02). Thus, antibody mixtures against a range of cell surface antigens enables capture of more CTCs than anti-EpCAM alone and CE staining enables the detection of CK-negative CTCs.
Ability to perform cytogenetic interrogations on circulating tumor cells (CTCs) from the blood of cancer patients is vital for progressing toward targeted, individualized treatments. CTCs are rare compared to normal (bystander) blood cells, found in ratios as low as 1:10 9 . The most successful isolation techniques have been immunocytochemical technologies that label CTCs for separation based on unique surface antigens that distinguish them from normal bystander cells. The method discussed here utilizes biotin-tagged antibodies that bind selectively to CTCs. The antibodies are introduced into a suspension of blood cells intending that only CTCs will display surface biotin molecules. Next, the cell suspension is passed through a microfluidic channel that contains about 9000 transverse, streptavidin coated posts. A CTC making contact with a post has the opportunity to engage in a biotin-streptavidin reaction that immobilizes the cell. Bystander blood cells remain in suspension and pass through the channel. The goal of the present study is to establish the technical performance of these channels as a function of antigen density and operating conditions, especially flow rate. At 18 lL/min, over 70% of cells are captured at antigen densities greater than 30 000 sites/cell while 50% of cells are captured at antigen densities greater than 10 000. It is found that lower flow rates lead to decreasing cell capture probabilities, indicating that some streamlines develop which are never close enough to a post to allow cell-post contact. Future modeling and streamline studies using computational fluid dynamics software could aid in optimization of channel performance for capture of rare cells.
T hree dimensional (3D) microarrays utilizing hydrogel matrixes are becoming increasingly attractive as a desired format for bio-analysis. These materials offer significant advantages as a scaffolding for capture agents over more conventional two dimensional (2D) printed formats in both captures per site and the ability to provide an environment more closely resembling that of a free solution. Biocept has developed a flexible three dimensional polyethylene glycol (PEG) polymer based platform suitable for a variety of biological assays. This novel approach is simple, biocompatible and provides a high degree of reproducibility and very low variability in the final array.
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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<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
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