Abstract 2373: An integrated approach for isolating CTCs to enable the interrogation of cellular heterogeneity using IFAST
Authors: Casavant Benjamin P. , Lang Joshua , Berry Scott M. , Maccoux Lindsey , McNeel Douglas , Beebe David J. 
Published: August 2012

Abstract
Background: The isolation of circulating tumor cells (CTCs) from whole blood is enabling for the study of malignant cells from patients with metastatic disease through a minimally invasive procedure. The analytical methods following the isolation of CTCs can be used to gain information about the disease, but require a highly purified population of cells that are minimally disturbed from the isolation procedures. While enumeration of CTCs during therapy has been shown to predict overall survival in men with metastatic prostate cancer, current methodology is limited in its ability to obtain information about these cells beyond count. Recent findings also suggest that CTC populations are heterogeneous (e.g. EMT, stem cell markers), and enumeration of CTCs based solely on EpCAM surface expression does not permit evaluation of CTC subpopulations. Therefore, methods that can isolate these cells based on their heterogeneity and further evaluate surface markers in an integrated platform will be beneficial in progressing the clinical relevance of CTC collection. Method: We present a method to sequentially isolate cells from a single input sample by cellular surface markers. Using immiscible filtration assisted by surface tension (IFAST) technology, we have demonstrated the ability to purify cells from a mixed cell population while maintaining the viability of these cells. The principle of the technology is that a high interfacial energy between the cell sample and oil creates a barrier that is normally impassable due to surface tension. Magnetic beads are added that have antibodies specific to a cellular surface marker and bind to the cells. A magnetic force is then applied to move the cells to and across the interface between the fluid and the oil, leaving anything unbound to the beads behind, thus isolating the cells of interest. CTC isolation is challenging due to potential cell loss in pipette transfer and wash steps. Using the IFAST device, we are able to add whole buffycoat into the input of the device and sequentially isolate, stain, and image CTCs without any pipette transfer or wash steps. Results: We have demonstrated the ability to have a single source solution in which two unique cell populations are present representing a basal and luminal cell type (DU145s, LNCaPs) and demonstrate the ability to individually isolate each of the two populations using a cellular surface antibody unique to one of the cell types (DU145 - CD44, LNCaPs - EpCAM) from a buffycoat containing high background PBMCs with greater than 80% of the resulting cells being the cell of interest in each situation, and demonstrate immunostaining of the isolated cell population (vimentin, HLA-A2). Conclusions: IFAST technology can be used to isolate multiple fractions from a single buffycoat solution using sequential bead additions and pull-throughs, demonstrating an advantage of this technology in the assessment of CTC heterogeneity.
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 2373. doi:1538-7445.AM2012-2373