Abstract 3307: Label-free high throughput microfluidic device for the isolation and single cell multiplex gene expression analysis of circulating tumor cells from breast cancer patients
Authors: Lin Eric , Rivera Lianette , Fouladdel Shamileh , Yoon Hyeun Joong , Guthrie Stephanie , Weiner Jacob , Deol Yadwinder S. , Keller Evan , Sahai Vaibhav , Simeone Diane M. , Burness Monika L. , Azizi Ebrahim , Wicha Max S. , Nagrath Sunitha 
Published: July 2016

Abstract
Introduction and Objective: The metastasis of cancer is preceded by the dissemination of cancer cells from the primary tumor site to remote sites via the blood circulation. The presence of circulating tumor cells (CTCs) in the peripheral blood represents a strong and independent prognostic factor for decreased disease-free and overall survival. Immune-affinity based capture, although being the most commonly used method for the isolation of CTCs, offers low throughput (∼1mL/hr) and have considerably cell loss caused by the heterogeneous expression of biomarkers on CTCs. Various label-free approaches utilizing the physical properties of CTCs have been developed to overcome the limitations, such as micro-filters, microscale laminar vortices, inertial migration of particles, and integrated systems. Here we present an inertial microfluidic-based separation technique for high throughput and label-free isolation of CTCs yielding the highest throughput with high CTC recovery and high blood cell removal among all the label-free technologies. The isolated CTC populations were further analyzed for single cell multiplex gene expression analysis.
Methods: The PDMS-made inertial microfluidic device has 637 mm in length with 56 corners, 500 μm in width, and 100 μm in height. The separation of CTCs is driven by two main forces: (i) inertial force that focuses the cells into streamlines, and (ii) drag force from Dean flow that migrates the focused cells to various positions based on size. Device is optimized with MCF-7 and Panc-1 cell line within PBS buffer solution and diluted blood, and is tested in patients with breast cancer on an average of 5 mL of whole blood processed through double devices in series. CTCs isolated were analyzed for tumor specific protein markers and genomic characterization is done using singe cell analysis techniques.
Results: Samples are processed through the inertial microfluidic device and CTCs are enriched in second outlet based on size difference between CTCs and blood cells. Device is optimized to operate at an extremely high throughput of 2500 μL/min with high recovery (greater than 90%) and high white blood cells (WBCs) removal (5 log orders). In patient samples, we identified CTCs in 38 of 40 (95%) breast patients with metastatic disease (5.4±4.6 CTC/mL) with low WBC contamination (663±647 WBC/mL). Based on the gene expression, both inter and intra patient heterogeneity of CTCs at the single cell level were discovered among the tested patient samples.
Conclusion: The study of CTCs could have a direct impact upon society by presenting novel ways to address one of the major hurdles in cancer research - early detection - and will foster the advancement of science and engineering via the exploration of new druggable targets approaches and the further understanding of the pharmacodynamics.
Citation Format: Eric Lin, Lianette Rivera, Shamileh Fouladdel, Hyeun Joong Yoon, Stephanie Guthrie, Jacob Weiner, Yadwinder S. Deol, Evan Keller, Vaibhav Sahai, Diane M. Simeone, Monika L. Burness, Ebrahim Azizi, Max S. Wicha, Sunitha Nagrath. Label-free high throughput microfluidic device for the isolation and single cell multiplex gene expression analysis of circulating tumor cells from breast cancer patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3307.