Abstract 2369: Isolation of circulating tumor cells by size exclusion using lithography fabricated precision microfilters

Abstract

Abstract Background: We present a novel precision microfilter that achieves rapid and highly efficient isolation of circulating tumor cells (CTCs) from peripheral blood. Isolation of CTCs by size exclusion is a widely researched technique with the advantage of capturing cells without reliance on cell surface expression markers. For many years CTC filtration technology has relied on track-etch microfilters with randomly located pores and low-porosity. More recently, precision microfilters with high porosity have been tested for CTC isolation, but they are not widely available. We have developed a new method of fabricating precision, high porosity microfilters that are capable of high volume manufacturing. These microfilters are strong, clear, non-fluorescent, and have high CTC capture efficiencies. Methods: We describe an assay to capture and enumerate prefixed and live CTCs. MCF-7 human breast adenocarcinoma cells (ATCC) were cultured in DMEM until confluent. Cells were stained in a fixative/staining solution containing paraformaldehyde, acridine orange, and DAPI dilacetate. After incubation, a cell sample was placed on a microscope slide and cells counted to obtain the exact input. Cells were then washed into 7.5 mL whole human blood with 7.5 mL PBS and placed into a syringe. An 8 µm pore precision microfilter was placed into Swinnex filter holder, and the sample drawn by negative pressure through the filter at ∼10 mL/min. The microfilter was mounted onto a microscope slide and counted using a fluorescence microscope under FITC and DAPI settings. Cells were defined by their shape and coloration under FITC fluorescence and nuclear signature under DAPI fluorescence. The procedure was then repeated using a staining solution without fixative to capture live MCF-7 cells. Both live and fixed MCF-7 cell isolation by microfiltration were performed in triplicate. Results: Precision microfilters were able to capture MCF-7 cells spiked into 7.5 mL human blood with a recovery rate of 98±3% for fixed cells, and 90±2% for live cells. The filtration was performed in 2 minutes, and cell counts were obtained in 10 minutes. By contrast, track-etched microfilters captured only 73% of fixed cells and cell counts required 20 minutes. Conclusions: Creatv's precision microfilters are shown to recover MCF-7 cells more efficiently than track-etch filters. The pore uniformity and low fluorescent background allowed for faster and more accurate counts of spiked CTC in human peripheral blood compared to track-etch microfilters. 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 2369. doi:10.1158/1538-7445.AM2011-2369