Aptamer-cocktail Functionalized Nano-microfluidic Chip for Enhancing Isolation and Characterization of Circulating Cancer Cells

Abstract

Background/Aim: Circulating tumor cells (CTCs) have been shown to have a correlation to metastasis and prognosis of patients with cancer. The enumeration and downstream gene analysis of CTCs have attracted efforts for personalized medicine. However, enumeration and phenotypic profiling in most capture devices are challenging due to the rarity and heterogeneity of CTCs. Here, we report an aptamer-cocktail strategy coupled in nano-microfluidic chip for enhancing isolation performance and characterizing the phenotypes of CTCs. Materials and Methods: Aptamer-cocktail recognizing EpCAM/Vimentin/EGFR/CD44 were bound to a nanopillar array on a nano-microfluidic chip. The recognition was validated with cancer cells by flow cytometry and the critical parameters were optimized with the nano-microfluidic chip. Finally, the system was applied to clinical samples. Results: The proposed aptamer-cocktail showed the predominant affinity with MDA-MB-231 and SK-BR-3. When utilized to capture artificial clinical samples, it showed 71% to 83% capture efficiency. CTC detection rate was 100% in five pre-treatment and five post-treatment breast cancer patients. The enumeration data ranged from 6-33 per 2 ml. The number of CTCs in breast cancer patients before therapy was 1.27-2 times higher than that after therapy. CTCs with epithelial and mesenchymal phenotype were both detected and identified; interestingly, the mean diameter of CTC_pck^pos acquired in these cases was much larger than that of CTC_vimentin^pos. Conclusion: The nano-microfluidic chip not only made it easier to phenotyping epithelial-like or mesenchymal-like CTCs, but can also be used to detect downstream genetic variation. The established platform can be applied in clinical research and facilitate auxiliary diagnosis with tumor recurrence and metastasis in advance.