Circulating Tumor Cells Enriched by the Depletion of Leukocytes with Bi-Antibodies in Non-Small Cell Lung Cancer: Potential Clinical Application

Abstract

Background: It has been considered that the detection methods for circulating tumor cells (CTCs) based on epithelial cell adhesion molecule (EpCAM) underestimate the number of CTCs and may miss a metastatic subpopulation with cancer stem cell (CSC) properties. Therefore, we investigated EpCAM-positive and -negative CTCs in non-small cell lung cancer (NSCLC) patients at different stages, assessed the clinical value of these CTCs and explored their capacity in the following CSC model.

Methods: CTCs were enriched by the depletion of leukocytes with bi-antibodies using a magnetic bead separation technique and then identified by the expression of EpCAM and cytokeratin 7 and 8 using multi-parameter flow cytometry. We determined the distribution of CTCs classified by the expression of EpCAM in 46 NSCLC patients with stages I to IV, assessed the diagnostic value of these CTCs by longitudinal monitoring in 4 index patients during adjuvant therapy and characterized the stemness of these CTCs by the expression of CXCR4 and CD133 in 10 patients.

Results: EpCAM-negative (E-) CTCs were detected to be significantly higher than EpCAM-positive (E+) CTCs in stage IV (p = 0.003). The patients with the percentage of E-CTCs more than 95% (r > 95%) were detected to be significantly increased from 13.3% in stage I-II to 61.1% in stage IV (p = 0.006). Kaplan-Meier analysis indicated that the patients with r > 95% had significantly shorter survival time than those with r ≤ 0.95 (p = 0.041). Longitudinal monitoring of CTCs indicated that the patients with a high percentage of E-CTCs in the blood were not responsive to either chemotherapy or targeted therapy. Further characterization of CTCs revealed that a stem-like subpopulation of CXCR4+CD133+ CTCs were detected to be significantly more prevalent in E-CTCs than that in E+CTCs (p = 0.005).

Conclusions: The enrichment of CTCs by the depletion of leukocytes with bi-antibodies is a valuable method for estimating the number of CTCs, which can be potentially applied in predicting the prognosis, monitoring the therapeutic effect of NSCLC patients and further analyzing the biology of CTCs.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1

Calibration curves in spiking experiments using (A) PC9 cell line and (B) PC9-EMT cells; (C) FACS analysis for the expression of EpCAM in PC9 and PC9-EMT cells stimulated by 5ng/mL TGF-β for 3 days.

Fig 2. Distribution of CTCs classified by the expression of EpCAM in NSCLC patients.

(A) Distribution of the detection rates of E+ and E-CTCs in patients at different stages; (B) Percentage of detected patients with a percentage of E-CTCs higher than 95% (r > 95%); (C) Distribution of the number of E+ and E-CTCs in patients at different stages; (D) Distribution of the Percentage of E-CTCs in different stages. (+/+, Cytokeratin+EpCAM+; +/-, Cytokeratin+EpCAM-).

Fig 3. Longitudinal monitoring of CTCs in adjuvant therapy.

(A) Gefitinib and then ALIMTA + cisplatin treatment; (B) Paclitaxel liposome + cisplatin and then radiotherapy + erlotinib treatment; (C) ALIMTA + carboplatin and then erlotinib treatment; (D) Erlotinib treatment. Each period of treatment was one month.

Fig 4. Kaplan-Meier survival analyses of E+ and E-CTCs in NSCLC patients.

Fig 5. Characterization of CTCs in stemness.

Distribution of (A) the number of CXCR4+ and CXCR4+CD133+ cells and (B) the positivity detection rates of CXCR4+CD133+ cells in CTCs classified by the expression of EpCAM in 10 NSCLC patients.