Circulating tumor cells enrichment and single cell analysis in non-small cell lung cancer

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Circulating tumor cells enrichment and single cell analysis in non-small cell lung cancer
Lim, Minji
Cho, Yoon-Kyoung
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Graduate School of UNIST
Compared to general-used tissue biopsy, less invasive “liquid biopsy” can provide insight which related to the real-time dynamics of cancer by more frequent analysis of circulating biomarkers including circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA). Furthermore, liquid biopsy is expected to offer a more comprehensive information of the disease, because circulating biomarkers may include cancer-associated materials from multiple disease sites in the body. However, the extreme rarity of cancer-associated circulating biomarkers in blood has been a great challenge to the achievement of the goals for liquid biopsy. CTC refers to tumor cell which disseminate from the primary tumor site to blood stream. Although CTC enumeration provides potential utility as a promising prognostic liquid biopsy marker, it has not been established yet due to its extreme rarity in comparison to other types of blood cells (1–10 CTCs/10^6 blood cells in 1 mL of blood). Representatively, CellSearch has been approved by the FDA about CTC enumeration system for breast, colorectal, and prostate cancer testing. And various microfluidic chips have been developed for CTC isolation based on physical and biochemical characteristics of CTCs. For example, some chips used epithelial cell adhesion molecule (EpCAM) antibody based capture and others used a size-selective manner. Many clinical papers were reported about the role of CTCs according to the remarkable improvement of the device. Notably, a high CTC count is associated with poor prognosis for cancer patients. Despite the clinical importance and progress of CTC-based cancer diagnostics, most of the current methods of enriching CTCs are difficult to implement in general hospital settings due to complex and time-consuming protocols. Among existing technologies, size-based isolation methods provide antibody-independent, relatively simple, and high throughput protocols. However, the clogging issues and lower than desired recovery rates and purity are the key challenges. In this thesis, fluid-assisted separation technology (FAST), which applied to centrifugal microfluidics with size-based membrane filtration, was suggested to solve clogging issues. FAST was inspired by antifouling membranes with liquid-filled pores in nature and it achieved high sensitivity (95.9 ± 3.1% recovery rate) and high selectivity (>2.5 log depletion of white blood cells) without clogging. Moreover, rapid (>3 mL/min) isolation of viable CTCs from whole blood without prior sample treatment enabled simple and ultrafast process which could be applied in clinical field. Numerical simulation and experiments demonstrated that FAST disc achieved uniform, clog-free, ultrafast cell enrichment with pressure drops much less than in conventional size-based filtration, at 1 kPa. We validated the clinical utility of the point-of-care detection of CTCs with 142 cancer patients’ samples. For the depth study, we selected lung cancer to apply molecular analysis based on CTCs. In particularly, lung cancer is the most common cause of worldwide cancer-related mortality with 1.8 million new cases reported every year in both men and women. NSCLC accounts for 85% of all lung cancers and epidermal growth factor receptor (EGFR) mutations are the representative mutation in 10-30% of patients. According to the mutant type of NSCLC patients, targeted therapies including EGFR tyrosine kinase inhibitor (TKI) drugs can induce improved clinical outcomes. For obtaining molecular information about their tumors, detection of EGFR mutation from patient-driven CTCs should be validated through the confirmation of concordance with tissue biopsy results. Successive downstream analysis of single CTCs from each patient during treatment is needed for observing a change of characteristics related with therapeutic response. To make it possible, robust CTC isolation system with high efficiency is required. With the above needs in mind, we report the clinical validation and demonstration of a FAST disc capable of rapid CTC isolation and easy single cell picking for individual CTC characterization. We report here a proof-of-concept demonstration showing clinical meaning of CTCs included multigene expression as a monitoring marker in NSCLC.
Department of Biomedical Engineering
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