Combination of negative and positive microfluidic immunofiltration for the selective enrichment of circulating tumor cells and exosomes

Abstract

The selective enrichment of circulating tumor markers is required for estimating the metastatic relapse and tumor burden of cancer patients. However, the isolation of circulating tumor cells (CTCs) and circulating tumor exosomes (CTExs) are still challenging due to their own issues. This dissertation presents dual-patterned immunofiltration (DIF) devices applicable for marker-specific enrichment with negative and positive strategy. The DIF device is designed not only to maximize the binding rate but also minimize the loss rate, by enhancing flow whirling, resulting in effective isolation in terms of high-throughput and high enrichment ratio. In the first chapter, the DIF device is modified with anti-CD45 antibodies for negative enrichment of CTCs, thus resolving rarity and heterogeneity (NS-CTC-DIF). The device eliminated 97.07 ± 2.79 % of leukocytes, with the loss of less than 10% loss of cancerous cells

consequently, the untouched CTCs were thoroughly enriched 23,188 times by 1-hour process. The device was further verified using the blood samples from 11 cancer patients, and average 283.3 CTC-like cells were identified with heterogeneous characteristics. Those cells which are isolated by the devices included relatively small cells (<15 um), EpCAM-negative cancer cells and triangular cells having multi-vacuolation. Meanwhile, less than 1 CTC-like cells were found in the samples from healthy donors. These results implies that most CTC-like cells can be lost under the approach focused on specific characteristics. In the second chapter, the DIF device is modified with anti-CD63 antibodies through cleavable linker for positive enrichment of circulating exosomes, thus resolving low purity and high variety (PS-CTX-DIF). The device isolated approximately 16.0 % higher amount of MCF-7 secreted exosomes, compared to the conventional exosome isolation kit

accordingly, the captured exosomes were subsequently released via reduction of cleavable linkers. The device was further verified using plasma samples from 6 cancer patients and 4 healthy donors. The cancer patients showed 2.7 times higher concentration of exosome-sized vesicles, compared to healthy donors. In spite of deviation among cases, the gap in enrichment yield between cancer patients (4.00) and healthy donors (0.80) are clearly distinguishable. These results implies that increment of exosome level might be related to the disease. In conclusion, the present DIF devices shows rapid and effective immunofiltration performance for the circulating markers. On the basis of the enhanced binding rate and reduced loss rate, heterogeneous CTCs and CTExs were efficiently enriched within a short time. Therefore, the flexible strategy combined with NS-CTC-DIF and PS-CTX-DIF devices is capable to the effective collection of highly purified circulating markers for the downstream analysis, making possible to understand the unknown properties or undiscovered roles of circulating tumor markers.