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 \u3e82% and \u3e78%, 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
