Massimo Cristofanilli 1 Savitri Krishnamurthy 2 Chandra M. Das 3 James M. Reuben 4 William Spohn 5 Jamileh Noshari 6 Frederick Becker 6 Peter R. Gascoyne 6 1 Department of Breast Medical Oncology, University of Texas, Houston, TX, USA 2 Department of Pathology, University of Texas, Houston, TX, USA 3 Department of Pediatrics Research, University of Texas, Houston, TX, USA 4 Department of Hematopathology, University of Texas, Houston, TX, USA 5 Department of Tumor Biology, University of Texas, Houston, TX, USA 6 Department of Molecular Pathology, University of Texas, Houston, TX, USA Original Paper Dielectric cell separation of fine needle aspirates from tumor xenografts As an approach to isolating tumor cells from fine needle biopsy specimens, we inves- tigated a dielectric cell preparation method using an in vivo xenographic tumor model. Cultured human MDA-MB-435 tumor cells were grown as solid tumors in nude mice and fine needle aspiration biopsies were conducted. Biopsied cells were suspended in sucrose medium and collected on slides patterned with microelec- trode arrays (electrosmears) energized by electrical signals in the range 10 to 960 kHz. The unlabeled cells adhered to characteristic regions of the slides in accordance with their morphology as a result of dielectric forces. Tumor cells were trapped between 40 and 60 kHz and were separated according to whether they were mitotic, large and complex, or small. Damaged tumor cells were captured at between 60 and 120 kHz; granulocytes between 70 and 90 kHz; lymphocytes between 85 and 105 kHz; healthy erythrocytes between 140 and 180 kHz, and dam- aged erythrocytes above 180 kHz. Using intrinsic cell characteristics, the electro- smear presented cell subpopulations from fine needle aspiration biopsy specimens in a manner that is compatible with automated slide-based analysis systems. The approach has the potential to facilitate the analysis of the role of cell subpopula- tions in disease. Keywords: Cancer / Cell sorting / Dielectrophoresis / Fine needle aspiration biopsy / Received: June 26, 2008; revised: August 14, 2008; accepted: August 15, 2008 DOI 10.1002/jssc.200800366 1 Introduction Isolating and characterizing tumor cells against a back- ground of normal cells is extremely challenging although methods including PCR [1, 2], fluorescent-acti- vated cell sorting [3], laser-scanning cytometry [4], mag- netic-activated cell sorting (MACS) [5, 6], and cell filtering [5, 7] represent progress in isolating and quantifying tumor cell sub-populations. Additional methods that would facilitate more sensitive, reliable, rapid, and con- venient isolation of tumor cells are desirable especially if they could introduce to the clinic novel and complemen- tary methods of cell discrimination. Dielectrophoretic (DEP) sorting exploits cell size and membrane morphology as cooperative markers and can be applied without the need for cell labeling with dyes or antibodies [8]. DEP methods attract or repel cells from a surface patterned with microelectrodes energized by alternating electric fields [8, 9]. Several dielectric meth- ods have been used to sort small concentrations of cells [9–15], and we showed recently that dielectrophoretic field-flow fractionation (depFFF) could be used to isolate cultured tumor cells from clinically-relevant numbers of peripheral blood mononuclear cells (PBMNs) [14]. We report fine needle aspiration biopsy (FNAB) experi- ments on a xenographic cancer model in which human MDA-MB-435 cells grew as solid tumors in mice. The DEP- based electrosmear method [16], which we previously proved capable of isolating cultured tumor cells from mixtures with blood, was able to isolate the tumor cells from other cell types in the fine needle biopsies. This sug- gests the method can also assist in the isolation and char- acterization of cell subpopulations in tumor biopsy specimens. 2 Materials and methods 2.1 Cell culture The human cancer line MDA-MB-435 (American Type Cul- ture Collection, Rockville, MD) was the source of tumor cells. Cultures were grown in DMEM-F12 medium with Correspondence: Dr. Peter R. C. Gascoyne, Department of Mo- lecular Pathology, Unit 0951, The University of Texas M. D. An- derson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA E-mail: pgascoyn@mdanderson.org Fax: +1-713-834-6103 Abbreviations: DEP, dielectrophoresis; depFFF, dielectrophoret- ic field-flow fractionation; FNAB, fine needle aspiration biopsy; MACS, magnetic-activated cell sorting; PBMNs, peripheral blood mononuclear cells i 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com 3732 M. Cristofanilli et al. J. Sep. Sci. 2008, 31, 3732 – 3739