High-Throughput Label-Free Isolation of Heterogeneous Circulating Tumor Cells and CTC Clusters from Non-Small-Cell Lung Cancer Patients

Abstract

(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−) 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 ± 1023 CTCs/mL). EpCAM− CTCs were significantly greater than EpCAM+ CTCs. Cell clusters of ≥2 CTCs were observed in 96% of patients—of which, 75% were EpCAM−. 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−, suggesting that these would have been missed using traditional antibody-based capture methods.

Gene expression in circulating tumor cells reveals a dynamic role of EMT and PD-L1 during osimertinib treatment in NSCLC patients

“…Vimentin positive CTCs in advanced NSCLC patients are of high abundance indicating the involvement of EMT in drug resistance 30 , 46 . According to a very recent study, based on a similar size-based CTC enrichment methodology, the majority of recovered CTCs/clusters were EpCAM-negative, suggesting that these cells would have been missed using traditional antibody-based capture methods 47 .…”

EMT-Associated Heterogeneity in Circulating Tumor Cells: Sticky Friends on the Road to Metastasis

“…A recent study of CTCs isolated from breast cancer patients and mouse models, reported that the binding sites for CSC transcription factors such as OCT4, SOX2, or NANOG, are hypomethylated in CTC clusters compared with isolated CTCs [49]. Other data collected from human samples also emphasize the presence of hybrid E/M phenotypes in heterogeneous CTC clusters, particularly in lung cancers [51,52,109,190]. Very elegantly, a study by Yu and coworkers identified cells expressing both epithelial (such as EpCAM or cytokeratins) and mesenchymal markers (including fibronectin, N-cadherin or PAI-1) in isolated CTCs but also in CTC clusters from breast cancer patients [179].…”

“…Several filter-based assays exploit the knowledge that most hematopoietic cells are smaller (<10 µm) than CTCs (>10 µm) [ 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 ]. A profusion of recently conceived microfluidic devices also uses the size and/or the deformability parameters [ 46 , 47 , 48 , 49 , 50 , 51 , 52 , 53 , 54 , 55 , 56 , 57 , 58 ]. Finally, CTC isolation devices, many also being microfluidic-based [ 59 , 60 , 61 , 62 , 63 ], use dielectrophoresis (DEP) to differentiate tumor cells from normal cells by their electric charges [ 64 ].…”

Epithelial/Mesenchymal Characteristics and PD-L1 Co-Expression in CTCs of Metastatic Breast Cancer Patients Treated with Eribulin: Correlation with Clinical Outcome

“…CTCs are found either as single cells or in clusters, while their phenotypic analysis has shown significant intra- and inter-patient heterogeneity [ 4 ]. The detection of CTCs in clusters and their contribution in metastasis and poor outcome of patients has been highlighted in various cancers [ 5 , 6 , 7 , 8 ]. The molecular characteristics and properties of clusters are different from those of single CTCs [ 6 , 7 ] and their size considerably varies, from 2 to 100 CTCs [ 4 , 6 , 9 ].…”