Wedge-shaped microfluidic chip for circulating tumor cells isolation and its clinical significance in gastric cancer

Abstract

Background: Circulating tumor cells (CTCs) have great potential in both basic research and clinical application for the managements of cancer. However, the complicated fabrication processes and expensive materials of the existing CTCs isolation devices, to a large extent, limit their clinical translation and CTCs' clinical value. Therefore, it remains to be urgently needed to develop a new platform for achieving CTCs detection with low-cost, mass-producible but high performance.

Methods: In the present study, we introduced a novel wedge-shaped microfluidic chip (named CTC-ΔChip) fabricated by two pieces of glass through wet etching and thermal bonding technique for CTCs isolation, which achieved CTCs enrichment by different size without cell surface expression markers and CTCs identification with three-color immunocytochemistry method (CK+/CD45-/Nucleus+). We validated the feasibility of CTC-ΔChip for detecting CTCs from different types of solid tumor. Furthermore, we applied the newly-developed platform to investigate the clinical significance of CTCs in gastric cancer (GC).

Results: Based on "label-free" characteristic, the capture efficiency of CTC-ΔChip can be as high as 93.7 ± 3.2% in DMEM and 91.0 ± 3.0% in whole blood sample under optimized conditions. Clinically, CTC-ΔChip exhibited the feasibility of detecting CTCs from different types of solid tumor, and it identified 7.30 ± 7.29 CTCs from 2 mL peripheral blood with a positive rate of 75% (30/40) in GC patients. Interestingly, we found that GC CTCs count was significantly correlated with multiple systemic inflammation indexes, including the lymphocyte count, platelet count, the level of neutrophil to lymphocyte ratio and platelet to lymphocyte ratio. In addition, we also found that both the positivity rate and CTCs count were significantly associated with multiple clinicopathology parameters.

Conclusions: Our novel CTC-ΔChip shows high performance for detecting CTCs from less volume of blood samples of cancer patients and important clinical significance in GC. Owing to the advantages of low-cost and mass-producible, CTC-ΔChip holds great potential of clinical application for cancer therapeutic guidance and prognostic monitoring in the future.

Keywords: Cell capture; Circulating tumor cells; Gastric cancer; Microfluidic; Wedge-shaped chip.

Fig. 1

Schematic diagram of CTCs isolation using the wedge-shaped microfluidic chip. a Overview of the wedge-shaped microfluidic chip; b detailed structural parameters of the microfluidic chip; c schematic diagram of CTCs isolation using the microfluidic chip with a wedge-shaped microchamber

Fig. 2

Optimization assays of the microfluidic chip. A Flow chart of the optimization assays: (a) isolating BGC823 cancer cells from blood sample using the microfluidic chip: ➀ blood sample, ➁ sampling tube, ➂ waste tube, ➃ microfluidic chip, ➄ sampling needle, ➅ clamping device; (b) Identification cancer cells captured by the microfluidic chip with three-color immunofluorescence staining (Pan-CK, red; FITC-CD45, green; DAPI nuclear staining, blue) using fluoresce microscope (scale bar, 10×); and (c) Three-color immunofluorescence image of BGC823 cancer cell (CK+, CD45− and DAPI+) and WBC (CK−, CD45+ and DAPI+) in two different height areas (red area: about 1 μm; green area: about 4 μm) of the chip. Scale bar, 100 μm; B the capture efficiency of the microfluidic chip at different outlet heights (4, 5, 6, 7, 8 μm, respectively); C the capture efficiency of the microfluidic chip at different flow rates (50, 100, 200, 300, 400, 600, 800 μL/min). The error bar represents standard deviation from three repeats

Fig. 3

Capturing performance of the CTCs enrichment platform equipped with a wedge-shaped microchamber. a The fluorescent micrographs of BGC823 captured from DMEM and healthy blood sample. Three-color immunocytochemistry method based on CK, CD45, and nuclear staining was applied to identify and enumerate BGC823 (red arrow) from WBCs (green arrow). Scale bars are 10 μm; b the recovered number of BGC823 was validated from DMEM and healthy blood sample with different spiking level (25, 50, 100, 150, 200 cells/2 mL, respectively); c the capture efficiency of four tumor cells lines (BGC823, HCT116, PC3, SKBR3) spiked in DMEM and healthy blood samples. All experiments were performed under optimal condition. The error bar represents standard deviation from three repeats

Fig. 4

Result of CTCs isolation from 40 GC patients. a CTCs detected from a GC patient. Three-color immunocytochemistry method based on CK, CD45, and nuclear staining was applied to identify and enumerate CTCs (red arrow) from non-specifically trapped WBCs (green arrow). Scale bars are 10 μm; b CTCs enumeration results obtained from 40 GC patients. Scatter plot for CTCs number of GC patients; c different tumor differential status; d with and without lymphovascular invasion; e with and without perineural invasion; f different TNM stage, each red dot stands for one GC patient. The error bars represent standard error of the mean

Fig. 5

Association of CTCs and blood microenvironment indexes. The difference of a LYM count, b PLT count, c NLR and d PLR between CTCs-positive and negative group. ***Represents P < 0.001. The relationship of CTC count and e LYM count, f PLT count, g NLR and h PLR