Isolation of Rare Tumor Cells from Blood Cells with Buoyant Immuno-Microbubbles

et al. (2013) Isolation of Rare Tumor Cells from Blood Cells with Buoyant Immuno-Microbubbles. PLoS ONE 8(3): e58017. doi:10.1371/journal.pone.0058017 Isolation of Rare Tumor Cells from Blood Cells with Buoyant Immuno-Microbubbles Guixin Shi 0 Wenjin Cui 0 Michael Benchimol 0 Yu-Tsueng Liu 0 Robert F. Mattrey 0 Rajesh Mukthavaram 0 Santosh Kesari 0 Sadik C. Esener 0 Dmitri Simberg 0 Gayle E. Woloschak, Northwestern University Feinberg School of Medicine, United States of America 0 1 Moores Cancer Center, University of California San Diego, La Jolla, California, United States of America, 2 Department of Radiology, University of California San Diego, San Diego, California, United States of America, 3 Department of Electrical and Computer Engineering, University of California San Diego, La Jolla, California, United States of America, 4 Neuro-Oncology Program, Moores Cancer Center, University of California San Diego, La Jolla, California, United States of America, 5 Department of Neurosciences, University of California San Diego, La Jolla, California, United States of America, 6 Department of NanoEngineering, University of California San Diego, La Jolla, California, United States of America, 7 Solid Tumor Therapeutics Program, Moores Cancer Center, University of California San Diego , La Jolla, California , United States of America Circulating tumor cells (CTCs) are exfoliated at various stages of cancer, and could provide invaluable information for the diagnosis and prognosis of cancers. There is an urgent need for the development of cost-efficient and scalable technologies for rare CTC enrichment from blood. Here we report a novel method for isolation of rare tumor cells from excess of blood cells using gas-filled buoyant immuno-microbubbles (MBs). MBs were prepared by emulsification of perfluorocarbon gas in phospholipids and decorated with anti-epithelial cell adhesion molecule (EpCAM) antibody. EpCAM-targeted MBs efficiently (85%) and rapidly (within 15 minutes) bound to various epithelial tumor cells suspended in cell medium. EpCAM-targeted MBs efficiently (88%) isolated frequent tumor cells that were spiked at 100,000 cells/ml into plasma-depleted blood. AntiEpCAM MBs efficiently (.77%) isolated rare mouse breast 4T1, human prostate PC-3 and pancreatic cancer BxPC-3 cells spiked into 1, 3 and 7 ml (respectively) of plasma-depleted blood. Using EpCAM targeted MBs CTCs from metastatic cancer patients were isolated, suggesting that this technique could be developed into a valuable clinical tool for isolation, enumeration and analysis of rare cells. - Funding: This project was funded by the United States National Institutes of Health (NIH) 1R21CA137721-01 IMAT grant awarded to D.S. and by the UCSD Cancer Center Specialized Support Grant P30 CA23100. W.C. was partially supported by the NIH P50CA128346 (ICMIC) grant. M.B. was partially supported by the NCI 5U54CA119335 grant. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. As cancer progresses, malignant cells are shed into the blood [1,2,3]. Circulating tumor cells (CTCs) could provide invaluable information for the monitoring of tumor progression and recurrence in cancer patients [1,2,3]. The successful identification and isolation of CTCs is a major challenge akin to finding a needle in a haystack: there are only a few CTCs per million of blood cells circulating throughout the body. At present, several assays for CTC isolation and analysis are on the market or in clinical development. The most common strategy for isolating CTCs from blood is based on the use of immunomagnetic beads coated with anti-epithelial EpCAM [4,5,6,7,8], the most commonly used marker for detecting circulating tumor cells [7,9]. An immunomagnetic bead-based CellSearch Assay (Veridex) has received U.S. Food and Drug Administration approval for the detection of epithelial CTCs in metastatic cancer patients. At present, this assay is the gold standard for CTC isolation. The capturing efficiency of rare tumor cells with magnetic beads ranges between 6090% [10,11]. The most significant limitations of the assay are its relatively long processing time, non-specific carryover and contamination with leukocytes [8,12,13,14]. Recently, the field of CTC isolation witnessed a surge of technologies, including microfluidics and filtration. These state-of-the-art technologies allow to isolate, count and even to manipulate single CTCs [15,16,17,18]. At the same time, there is a continuing interest in development and testing of cost-efficient, scalable and simple technologies for CTC isolation. Perfluorocarbon gas-filled microbubbles (MBs) are clinically approved for injection as ultrasound contrast agents [19,20]. A typical microbubble consists of a gas interior coated by a soft shell, which could consist of either a lipid monolayer or protein (albumin). Perfluorocarbon gas maintains the stability of MBs in the aqueous phase and confers buoyancy [19]. Recently, we demonstrated that anti-fluorescein antibody-coated buoyant MBs efficiently bound and separated fluorescein-labeled erythrocytes in mouse blood [21]. Here we set out to test whether EpCAM-targeted MBs are capable of sensitive and specific isolation of rare tumor cells from mouse and human blood. Our data suggest that MBs efficiently and specifically isolate tumor cells from plasma-depleted blood. We demonstrate that buoyancy-based separation of tumor cells from complex cell mixtures is feasible and could become a promising strategy to immune marker-based fractionation and isolation of rare cells. Materials and Methods 1. Ethics statement Collection of healthy blood from anonymous volunteers was approved by the UC San Diego Institutional Review Board (protocol 081077XT). Collection and usage of human specimens from consenting patients was approved by the UC San Diego Institutional Review Board (protocol 100936). All the participants had to sign approved IRB approved consent form prior to blood collection. All animal studies were conducted under UCSD IACUC protocol (protocol S07388). 2. Reagents 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) was purchased from Avanti Polar Lipids (Alabaster, AL, USA), 2-distearoyl-sn-glycero-3-phospho-ethanolamine-N-[maleimide (polyethylene glycol)-3400] (DSPE-PEG3400-Malemide) and maleimide-polyethylene glycol 3400-succinimidyl valerate (Mal-PEG-SVA) were purchased from Laysan Bio, Inc. (Arab, AL, USA), polyoxyethylene (40) stearate was purchased from Sigma. All lipids were stored as chloroform solution under argon at 220uC. Trauts reagent (22Iminothiolane) was purchased from Thermo Fisher Scientific (Rockford, IL, USA). The reagent was dissolved in double-distilled water at 5 mg/ml and stored in aliquots at 220uC. Ellmans reagent (5,5-dithiobis-(2-nitrobenzoic acid), or DTNB) was purchased from T (...truncated)