Immunomagnetic Isolation of HER2-Positive Breast Cancer Cells Using a Microfluidic Device

Abstract

Analysis of circulating tumor cells (CTCs) as a tool for monitoring metastatic cancers, early diagnosis, and evaluation of disease prognosis paves the way toward personalized cancer treatment. Developing an effective, feasible, and low-cost method to facilitate CTC isolation is, therefore, vital. In the present study, we integrated magnetic nanoparticles (MNPs) with microfluidics and used them for the isolation of HER2-positive breast cancer cells. Iron oxide MNPs were synthesized and functionalized with the anti-HER2 antibody. The chemical conjugation was verified by Fourier transform infrared spectroscopy, energy-dispersive X-ray spectroscopy, and dynamic light scattering/zeta potential analysis. The specificity of the functionalized NPs for the separation of HER2-positive from HER2-negative cells was demonstrated in an off-chip test setting. The off-chip isolation efficiency was 59.38%. The efficiency of SK-BR-3 cell isolation using a microfluidic chip with a S-shaped microchannel was considerably enhanced to 96% (a flow rate of 0.5 mL/h) without chip clogging. Besides, the analysis time for the on-chip cell separation was 50% faster. The clear advantages of the present microfluidic system offer a competitive solution in clinical applications.

Figure 1

MNPs’ surface modification by HER2 investigated through (A) FTIR, (B) DLS/zeta potential analysis, and (C) EDS analysis.

Figure 2

Representative fluorescent microscopy images of off-chip cell isolation, (A) SKBR3 cells stained with DAPI, (B,C) stained SKBR3 cells incubated with HER2-MNPs before and after magnetic washing, (D) MDA-MB-231 cells stained with fluorescein dye, (E,F) stained MDA-MB-231 cells incubated with HER2-MNPs before and after magnetic washing, (G–I) blue, green, and merged fluorescent images of stained SKBR3 and MDA-MB-231 cell cocktail incubated with HER2-MNPs before magnetic washing, (J–L) blue, green, and merged fluorescent images of stained SKBR3 and MDA-MB-231 cell cocktail incubated with HER2-MNPs after magnetic washing, aand (M,N) stained SKBR3 cells incubated with bare MNPs before and after magnetic washing.

Figure 3

Capture efficiency of the off-chip isolation by flow cytometry. Forward and side scatter density plots of (A) 5 × 10⁵ unstained SK-BR-3, (B) 5 × 10⁵ unstained MDA-MB-231, (C) 2.5 × 10⁵ unstained SK-BR-3 mixed with 2.5 × 10⁵ unstained MDA-MB-231, (D) the overlay of (A–C), the fluorescein signal of (E) 5 × 10⁵ unstained SK-BR-3, (F) 5 × 10⁵ stained SK-BR-3, 2.5 × 10⁵ stained SK-BR-3 + 2.5 × 10⁵ unstained MDA-MB-231 incubated with (G) HER2-MNPs, (H) bare MNPs, and (I) HER2.

Figure 4

Fluorescence microscopy images of on-chip cell isolation, (A,B) movement of stained SK-BR-3 cells and HER2-MNPs inside the chip, (C) magnetic enrichment of the cells attached to the HER2-MNPs in the trap region, (D) high magnification of fluorescent SK-BR-3 cells in the trap region, (E) fluorescence and bright-field images of SK-BR-3 cells separated after magnetic washing, and (F) SK-BR-3 cell isolation efficiency versus fluid’s flow rate inside the chip (the green bar shows the optimum flow rate value).