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(1) Background: Circulating tumor cell (CTC) clusters are emerging as clinically significant harbingers of metastases in solid organ cancers. Prior to engaging these CTC clusters in animal models of metastases, it is imperative for technology to identify them with high sensitivity. These clusters often present heterogeneous surface markers and current methods for isolation of clusters may fall short. (2) Methods: We applied an inertial microfluidic Labyrinth device for high-throughput, biomarker-independent, size-based isolation of CTCs/CTC clusters from patients with metastatic non-small-cell lung cancer (NSCLC). (3) Results: Using Labyrinth, CTCs (PanCK+/DAPI+/CD45&minus, ) were isolated from patients (n = 25). Heterogeneous CTC populations, including CTCs expressing epithelial (EpCAM), mesenchymal (Vimentin) or both markers were detected. CTCs were isolated from 100% of patients (417 &plusmn, 1023 CTCs/mL). EpCAM&minus, CTCs were significantly greater than EpCAM+ CTCs. Cell clusters of &ge, 2 CTCs were observed in 96% of patients&mdash, of which, 75% were EpCAM&minus, CTCs revealed identical genetic aberrations as the primary tumor for RET, ROS1 and ALK genes using fluorescence in situ hybridization (FISH) analysis. (4) Conclusions: The Labyrinth device recovered heterogeneous CTCs in 100% and CTC clusters in 96% of patients with metastatic NSCLC. The majority of recovered CTCs/clusters were EpCAM&minus, suggesting that these would have been missed using traditional antibody-based capture methods.

Background: Circulating tumor cell (CTC) clusters are emerging as clinically significant harbingers of metastases in solid organ cancers. Prior to engaging these CTC clusters in animal models of metastases, it is imperative for technology to identify them with high sensitivity. These clusters often present heterogeneous surface markers, and current methods for isolation of clusters may fall short. Methods: We have applied the inertial microfluidic Labyrinth device for high-throughput, biomarker-independent, size-based isolation of CTCs and CTC clusters from patients with metastatic non-small cell lung cancer (NSCLC). Results: Using the Labyrinth device, CTCs (PanCK+/DAPI+/CD45-) were isolated from metastatic NSCLC patients (n=25). Heterogeneous CTC populations were detected, including CTCs expressing epithelial (EpCAM), mesenchymal (vimentin), or both markers. CTCs were isolated from 100% of patients (417±1023 CTCs/mL), and CTCs that were EpCAM negative were significantly higher in numbers than EpCAM+ CTCs. Cell clusters of ≥2 CTCs were observed in 96% of patients, of which 75% were negative for EpCAM. Patients with higher number of CTC clusters than single CTCs had worse progression-free survival (PFS) (p=0.05). Recovered CTCs from patients with RET, ROS1, and ALK-rearranged tumors revealed identical genetic aberrations as the primary tumor for each gene using FISH analysis. We have successfully expanded the recovered CTCs from 2 patients and screened for therapeutic targeting. We have found that TPX-0005 might be effective in these patients and would direct them to a clinical trial using this compound. Conclusions: The label-free Labyrinth device demonstrated the capability of collecting recovered CTCs from the device using a continuous processing technique while in a suspension state. This advantage opens the opportunities not only for CTC expansion off-chip, but also for ex vivo drug testing to direct patient-specific therapies. Citation Format: Mina Zeinali, Wei Huang, Maggie Lee, Arthi Nadhan, Anvya Mathur, Casey Hedman, Eric Lin, Ramdane Harouaka, Max S. Wicha, Lili Zhao, Nallasivam Palanisamy, Mathias Hafner, Rishindra Reddy, Gregory P. Kalemkerian, Bryan J. Schneider, Khaled A. Hassan, Nithya Ramnath, Sunitha Nagrath. High-throughput label-free isolation and expansion of circulating tumor cells (CTCs) from non-small cell lung cancer (NSCLC) patients for personalized treatments [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr B45.

Background: Circulating tumor cells (CTCs) have emerged as important blood-based surrogate markers of primary tumors. Current methods for isolation of lung CTCs mostly rely on biomarker dependent antibody-based capture, missing populations that may be stem-like in nature. Results: We have applied the microfluidic Labyrinth device for high throughput, label-free, size-based isolation of CTCs from non-small cell lung cancer patients (NSCLC). The Labyrinth device was optimized and tested for inertial separation of cancer cells using the human lung cancer cell line H1650. The recovery and purity were >82% and >78%, respectively, operating at a flow rate of 2.5 mL/min. Using the biomarker-independent Labyrinth separation device, heterogeneous CTC populations were isolated from metastatic NSCLC patients (n=21). Heterogeneous CTC populations were detected, including CTCs (PanCK+ and CD45-), CTCs expressing EpCAM or Vimentin, and CTCs expressing both markers representing an EMT-like population of CTCs. Using Labyrinth, we were able to isolate CTCs from 100% of patients with an average yield of 180±168 CTCs/mL. Among the captured CTCs, EpCAM- CTCs were significantly more common than EpCAM+ CTCs (115.7 vs. 39.1 CTCs/mL respectively). Cell clusters of 2 or more CTCs were also observed in 95% of patients; 79% of these clusters were negative for EpCAM expression, whereas 35% expressed Vimentin, suggestive of an EMT phenotype. Recovered CTCs from patients with RET, ROS1 and ALK rearranged tumors showed aberrations matching with the primary tumor for each gene using FISH analysis. We have successfully expanded the recovered CTCs from 2 patients and screened for therapeutic targeting. We have found that TPX-0005 might be effective in these patients and would direct them to a clinical trial using this compound. Conclusion: The label-free Labyrinth device demonstrated the capability of collecting recovered CTCs from the device using a continuous processing technique while in a suspension state. This advantage opens the opportunities not only for CTC expansion off-chip, but also for ex-vivo drug testing to direct patient-specific therapies. Citation Format: Mina Zeinali, Maggie Lee, Arthi Nadhan, Anvya Mathur, Wei Huang, Eric Lin, Ramdane Harouaka, Max S. Wicha, Nallasivam Palanisamy, Mathias Hafner, Rishindra Reddy, Gregory P. Kalemkerian, Bryan J. Schneider, Khaled A. Hassan, Nithya Ramnath, Sunitha Nagrath. High Throughput isolation and expansion of circulating tumor cells (CTCs) from Non-small cell lung cancer (NSCLC) patients for personalized treatments [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1332.

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 >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.