Feasibility of a novel one-stop ISET device to capture CTCs and its clinical application

Abstract

Introduction: Circulating tumor cells (CTCs) play a crucial role in cancer metastasis. In this study, we introduced a novel isolation method by size of epithelial tumor cells (ISET) device with automatic isolation and staining procedure, named one-stop ISET (osISET) and validated its feasibility to capture CTCs from cancer patients. Moreover, we aim to investigate the correlation between clinicopathologic features and CTCs in colorectal cancer (CRC) in order to explore its clinical application.

Results: The capture efficiency ranged from 80.3% to 88% with tumor cells spiked into medium while 67% to 78.3% with tumor cells spiked into healthy donors' blood. In detection blood samples of 72 CRC patients, CTCs and clusters of circulating tumor cells (CTC-clusters) were detected with a positive rate of 52.8% (38/72) and 18.1% (13/72) respectively. Moreover, CTC positive rate was associated with factors of lymphatic or venous invasion, tumor depth, lymph node metastasis and TNM stage in CRC patients (p < 0.01). Lymphocyte count and neutrophil to lymphocyte ratio (NLR) were significantly different between CTC positive and negative groups (p < 0.01).

Materials and methods: The capture efficiency of the device was tested by spiking cancer cells (MCF-7, A549, SW480, Hela) into medium or blood samples of healthy donors. Blood samples of 72 CRC patients were detected by osISET device. The clinicopathologic characteristics of 72 CRC patients were collected and the association with CTC positive rate or CTC count were analyzed.

Conclusions: Our osISET device was feasible to capture and identify CTCs and CTC-clusters from cancer patients. In addition, our device holds a potential for application in cancer management.

Keywords: circulating tumor cells (CTCs); clusters of circulating tumor cells (CTC-clusters); colorectal cancer (CRC); epithelial-mesenchymal transition (EMT); isolation method by size of epithelial tumor cells (ISET).

Figure 1. The results of capture efficiency tests

(A) The capture efficiencies of different tumor cells sipked into DMEM or blood samples. (B) Captured SW480 cell number against the number of spiked in DMEM or blood samples at different concentrations. The error bars represent a mean ± standard deviation from three repeats. (C) Wright's staining of captured MCF-7, A549, SW480 and Hela cells. The arrows indicated tumor cells and the triangle (▲) indicated white blood cells (WBCs).

Figure 2. The images of IF staining and Wright's staining for the same samples

MCF-7 tumor cells spiked into healthy blood samples were set as positive control. CK+/CD45-/Hoechst+ cell was scored as CTCs and CK-/CD45+/Hoechst+ cell as WBCs.

Figure 3. The detection results of blood samples from 15 cancer patients

(A) Images of Wright's staining for isolated CTCs and CTC clusters from cancer patients. (B) The number of captured CTCs and CTC clusters in blood samples from 15 cancer patients. The arrows indicated CTCs and the triangle (▲) indicated WBCs.

Figure 4. Detection results of CTCs and CTC clusters in different stages of 72 CRC patients

Figure 5. Correlationship of CTC counts with number of lymphocytes and NLR

Figure 6. Introduction of the osISET device

(A) The blood samples were waiting for isolation. (B) The filter membrane picked by the forceps was at the bottom of a cylindrical filtration. (C) The calibrated 8-μm-diameter pores were shown. (D) The transparent filter membrane was placed on slide after automated isolation.