Detection of AXL expression in circulating tumor cells of lung cancer patients using an automated microcavity array system

Abstract

Noninvasive diagnostics using circulating tumor cells (CTCs) are expected to be useful for decision making in precision cancer therapy. AXL, a receptor tyrosine kinase is associated with tumor progression, epithelial‐to‐mesenchymal transition (EMT), and drug resistance, and is a potential therapeutic target. However, the epithelial markers generally used for CTC detection may be not enough to detect AXL‐expressing CTCs due to EMT. Here, we evaluated the detection of AXL‐expressing CTCs using the mesenchymal marker vimentin with a microcavity array system. To evaluate the recovery of cancer cells, spike‐in experiments were performed using cell lines with varying cytokeratin (CK) or vimentin (VM) expression levels. With high CK and low VM‐expressing cell lines, PC‐9 and HCC827, the recovery rate of AXL‐expressing cancer cells was 1%‐17% using either CK or VM as markers. Whereas, with low CK and high VM‐expressing cell lines, MDA‐MB231 and H1299, it was 52%‐75% using CK and 72%‐88% using VM as a marker. For clinical evaluation, peripheral blood was collected from 20 non–small cell lung cancer patients and CTCs were detected using CK or VM as markers in parallel. Significantly more AXL‐expressing single CTCs were detected in VM‐positive than CK‐positive CTCs (P < .001). Furthermore, CTC clusters were identified only among VM‐positive CTCs in 20% of patients. Patients with one or more prior treatments harbored significantly more VM‐positive AXL‐expressing CTCs, suggesting the involvement of these CTCs in drug resistance. These results indicate the necessity of integrating mesenchymal markers with CTC detection and this should be further evaluated clinically.

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

“…A pivotal aspect of CTC analysis resides on the possibility to perform successive liquid biopsies to monitor the progression of the disease and to assess treatment efficacy. Importantly, the emergence of EMT-shifted CTC phenotypes after one or several lines of treatment was found to correlate with drug resistance in several studies [52,104,112,121,124,179,360]. This finding is in agreement with a large amount of experimental data demonstrating an enhanced ability of EMT-shifted cells to resist most existing therapeutic options (chemo-radio-resistance, and resistance to existing targeted therapies) [81,[162][163][164][165][166].…”

“…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].…”

“…EMT transcription factors (Twist, ZEB or Snail) have also been frequently detected in CTCs and associated with a poor prognosis in different cancer types [101,174,353,357,358]. Axl was found in CTCs isolated from lung cancers, particularly in those expressing vimentin [52], and it was similarly associated with a poor prognosis [132]. Interestingly, high expression of PD-L1 and EMT markers in CTCs was reported to be a sign of a grim prognosis in patients with complete surgically resected lung cancer [130].…”

“…In addition, a deeper molecular characterization of these drug-resistant phenotypes may also point to the emergence of other targetable pathways, or identify new potential therapeutic targets. For instance, NSCLC patients treated with one or several lines of therapies, progressively presented significantly more vimentin-positive Axl-expressing CTCs [52]. Overexpression of Axl in CTCs isolated from lung cancer patients who developed resistance to EGFR inhibitor therapies has also been reported [361].…”

“…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 ].…”