High-throughput label-free viable isolation of circulating tumor cells for applications to cancer heterogeneity and exosome research

Abstract

The thesis presents a microfilter device for the high-throughput label-free viable isolation of circulating tumor cells (CTCs) and its applications to cancer heterogeneity and exosome research. In spite of the clinical importance of CTCs, the lack of simple and non-biased CTC isolation method is a big hurdle for implementing CTC studies into clinical use. The present microfilter having tapered-slit is designed not only to increase sample flow rate with the minimal captured cell stress but also to isolate CTC selectively, thus achieving high-throughput viable CTC isolation. We verified our device performance for 11 types of cancer cells at the 5 different spiked cancer cell concentrations (5-100 cells/1ml). The present device shows the capture efficiency of 77.7% with the viability of 80.6% at the extremely rare cell (5 cells/ml) and high-throughput (30ml/h) condition. The grafting of poly (ethylene glycol) (PEG) onto the microfilter has been demonstrated for the efficient capture and release of the CTCs. The PEG, a hydrophilic polymeric compound mainly used to form non-fouling thin films on silicon surfaces, generates repulsive force so that the nonspecific adsorption of the surface is incomparably reduced than unmodified filter surfaces. The effectiveness of the PEG-modified filters was verified using lung (H358) and colorectal (SW620) cancer cells spiked in the phosphate-buffered saline (PBS) and the whole bloods. The PEG-modified filters showed approximately 37.7% and 22.8% improvement of release efficiency compared to unmodified filters, without significant changes in cell viability and capture efficiency. Thanks to the device’s heterogeneous viable circulating tumor cell isolation performance, we extended our study to cancer heterogeneity and circulating exosome researches. Using clinical samples from patients with lung, colorectal, breast, pancreatic and renal cell carcinoma, we evaluated the heterogeneity of cancer in terms of CTC’s surface protein expression, cytopathological characteristics, and genetic information. From this study, we showed that CTCs expressed different level of surface protein, cancer associated mRNAs and heterogeneous cytopathological characteristics depending on patient’s pathological characteristic, disease stage, or types of tumor. At the last, we induced the circulating tumor exosomes from the captured viable circulating tumor cells then analyzed both circulating markers to see their similarity and epithelial/mesenchymal properties. The circulating tumor exosomes derived from the CTCs showed the great similarity (80-100%) to CTCs with even higher quantity than CTCs, thus suggesting new way to examine the status of patients using more than one type of circulating markers. This simple CTC isolation device and experimental setups for cancer heterogeneity and exosome researches can be applicable to comprehensive study of cancer, reflecting its innate heterogeneity and complexity.