Abstract 2376: Development of a novel microfluidic device for the capture and molecular characterization of circulating tumor cells from metastatic breast cancer patients

Abstract

Abstract Breast cancer (BC) is the most commonly diagnosed malignancy and the second leading cause of cancer death among women in the United States. Microtubule targeting drugs (MTDs) represent the most active chemotherapy against BC and are standard therapy for locally advanced and metastatic disease. However, metastatic BC (MBC) remains an incurable disease due to acquired drug resistance. The molecular basis of clinical resistance to MTD therapy is not well understood, primarily due to the lack of readily accessible BC tissue during therapy and at the time of relapse precluding the development of rational approaches to overcome it. Circulating Tumor Cells (CTCs) have emerged as a reliable and accessible source of tumor tissue enabling molecular analysis of CTCs during disease progression. However, the extremely low purity associated with the use of the commercially available EpCAM-based CTC capture technology, precludes accurate CTC molecular analyses. To overcome this limitation, we are developing a novel BC-specific microfluidic device that operates on the principle of geometrically enhanced differential immunocapture (GEDI), using antibody-coated microposts. This innovative design achieves capture of viable CTCs using only 1 ml of blood and minimizes leucocyte contamination. We selected HER2 as an alternative to EpCAM-based capture, as HER2 is expressed in 90% of breast tumors and overexpressed in 30%, whereas it is absent from leucocytes. In addition, our preliminary data suggest that EpCAM can be downregulated during epithelial-to-mesenchymal transition (EMT) that often precedes dissemination of cancer cells to the circulation. HER2, on the other hand, is expressed in EMT cells further indicating its suitability for CTC capture from most of the clinically relevant BC subtypes. We tested the performance of various HER-2 antibodies using immunoblot and confocal microscopy analyses of 10 different BC cell lines with varying HER2 expression levels. Surprisingly, the antibody that provided the best dynamic range of HER2 detection in these assays, exhibited poor capture efficiency and lack of discrimination between HER2-positive and negative cell lines. Next, we tested antibody performance using flow cytometry and live-cell imaging in order to preserve the native conformation of HER2 extracellular epitope. These assays identified a different HER2 antibody that showed high capture efficiency and specificity for HER2 positive cells when applied to the GEDI microfluidic device. Currently, we are using the HER2-based GEDI to isolate CTCs from MBC patients receiving MTD-based chemotherapy. In this prospective study, we are characterizing the GEDI-captured CTCs isolated from MBC patients at baseline (before chemotherapy), during the course of therapy and at disease progression in order to gain insights into mechanisms of MTD-resistance. 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 2376. doi:1538-7445.AM2012-2376