Abstract A21: High-throughput isolation of circulating tumor cells (CTCs) from non-small cell lung cancer (NSCLC) patients for personalized treatments
Authors: Zeinali Mina , Nadhan Arthi , Mathur Anvya , Lee Maggie , Huang Wei , Hafner Mathias , Ramnath Nithya , Nagrath Sunitha 
Published: February 2019

Abstract
It has been recognized that circulating tumor cells (CTCs) shed by a primary tumor can be a valuable surrogate to tissue biopsies. Our project goal is to develop a CTC-based liquid biopsy platform as a noninvasive method for identifying primary and secondary mutations in non-small lung cancer to direct personalized therapies.
The high-throughput, label-free and size-based inertial microfluidic separation device “Labyrinth” developed in the Nagrath lab has been used for simultaneous isolation of epithelial (EpCs) and mesenchymal (EMTCs) CTCs from lung cancer patients. The total channel length of Labyrinth is 637 mm, with 500 mm in width and 100 mm in height. The device consists of complex of small and high ratio of curvature structures (11 long loops and sharp right-angle 56 corners) to enhance differential focusing of both CTCs and white blood cells into separate fluid streams in the outlet area. The high recoveries and advantage of size-based biomarker independent separation are crucial for the downstream applications including molecular characterization of CTCs (mutation and mRNA profiling) and expansion of CTCs in vitro.
Labyrinth was optimized and tested for inertial separation of cancer cells using the human lung cancer cell line PC-9. The percentage of recovery and purity was &gt;93% with 2400 µL/min flow rate. We have enrolled 15 metastatic patients for this study and isolated CTCs from baseline (n=15) and post-therapy follow-up visits (range 1-4). To be able to detect different subpopulations of CTCs including EpCs and EMTCs from patients, we have employed immunofluorescence to label EpCAM (for EpCs) and Vim (for EMTCs), in addition to PanCK (tumor marker) and CD45 (leukocyte marker). CTCs were defined as EpCAM+/-/ Vim+/-/ PanCK+/ CD45-. All 15 patients, with different mutations/fusion, including EGFR, RET, ROS1, and ALK, had detectable CTCs at all visits, including 156±175 (at baseline, n=15), 141±133 (at visit 1, n=14), 255±218 (at visit 2, n=8), 84±81 (at visit 3, n=4), and 157±177 per mL (at visit 4, n=2). Patients with EGFR mutants had an average of 112±97 total CTCs per mL at their baseline and 66±57 after they were treated with either erlotinib or osimertanib. Patients with ROS1 fusion had slightly higher total CTCs per mL at their baseline (185±250) and during treatment with crizotinib (120±113) compared to EGFR mutant patients. To expand patient-derived CTCs for ex vivo drug testing, three different CTC culture conditions were tested: a plain, a “Fibronectin coated” (2D), and a “Matrigel-collagen-irradiated mice fibroblast coated” (3D) 48-well plate. After 6 weeks in culture, the growth rate was significantly higher in the 3D model. For CTC expansion, the enrichment methods require further optimization prior to adopting the 3D culture model for future samples.
The cell surface marker independent microfluidic device, Labyrinth, isolated heterogeneous CTC subpopulations that were characterized and expanded in vitro. This could allow ex vivo drug testing to direct patient-specific therapies.
Citation Format: Mina Zeinali, Arthi Nadhan, Anvya Mathur, Maggie Lee, Wei Huang, Mathias Hafner, Nithya Ramnath, Sunitha Nagrath. High-throughput isolation of circulating tumor cells (CTCs) from non-small cell lung cancer (NSCLC) patients for personalized treatments [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr A21.