서강대학교

초록/요약 도움말

Circulating tumor cells (CTCs), which exist in extremely rare numbers in the bloodstream of cancer patients, may indicate the likelihood and severity of metastatic progression. Identification, enumeration, and characterization of CTCs may provide a minimally invasive method for assessing cancer status of patients and prescribing personalized anti-cancer therapy. However, examination of CTCs requires isolation of these cells from whole blood of patients, which is difficult due to their low quantity (estimated to be around one CTC per 109 non-cancerous hematopoietic cells in patient blood). Some CTCs are reported to be nearly identical in size—or even smaller—than leukocytes, making them difficult to discriminate by size alone. As for protein expression, epithelial markers, such as EpCAM (epithelial cell adhesion molecule), are downregulated during the progression of epithelial-to-mesenchymal transition (EMT). Due to the heterogeneous nature of CTCs, it may be practically impossible to isolate them with high isolation efficiency using conventional isolation methods. To resolve these issues and maximize isolation efficiency, I devised a dual mode isolation platform that combines affinity-based enrichment and size-based exclusion. This strategy involves size augmentation of CTCs using microbeads conjugated with CTC-specific antibodies to enable better size discrimination between CTCs and leukocytes. Subsequent size filtration isolates bead-bound CTCs, allowing the recovery of even smaller sized CTCs. The performance of the dual mode isolation platform was validated with cancer cells of various sizes and surface protein expression level, and very high isolation efficiency was achieved. However, all bead–based capture methods, including this one, have the inherent limitation of prohibiting accurate image analysis, due to optical distortion created by the presence of beads attached to cells. The attached beads not only impede observation of cellular morphology but can actually alter fluorescence signal intensities. This means incompatibility of in situ quantitative analysis with bead-based capture methods. Therefore, I devised a novel approach for isolation and subsequent in situ protein expression analysis of CTCs using these detachable beads termed RIA (Reversible Size-Augmented Isolation and Analysis). The RIA platform make possible to increase isolation efficiency of tumor cells from blood and allow accurate quantification of protein expression for each individual cell to provide more information for suitable therapy. RIA was then used to isolate CTCs from whole blood of metastatic breast cancer patients, and at least one CTC was successfully obtained from all clinical samples. HER2 expression in isolated CTCs was analyzed by immunofluorescence and subsequent image analysis, and 67% of samples showed concordant HER2 status between primary breast cancer tissue and CTC. These results demonstrate that RIA provides not only high isolation efficiency of CTCs but also proficient assessment of HER2 status from isolated CTCs. The type and expression levels of surface proteins may vary greatly depending on the cancer histological subtype, therefore, to achieve efficient capture of all CTCs, multiple antibodies need to be used in conjunction. Considering the limited number of known surface proteins specific to each cancer subtype and the extremely rare nature of CTCs, it would be beneficial to use the same marker in both positive selection and in situ expression analysis. To solve this challenge, I developed a hybrid nanoparticle (HNP), which consists of three parts: affinity part, signal part, and capture/release part. This platform demonstrated simultaneous capture, in situ protein expression analysis, and cellular phenotype identification of CTC. The performance of the HNP platform was validated with cancer cells of various subtypes, and high capture efficiency and identification accuracy was achieved. Furthermore, by cleaving the DNA portion with restriction enzymes, captured cells were released from the isolation platform and re-collected with high efficiencies. Thus, HNPs can make it possible to count, analyze in situ protein expression, and culture CTCs, all from the same set of cells, enabling a wider range of molecular- and cellular-based studies using CTCs. To expand the modalities of CTC characterization, analysis of genomic DNA found in CTCs was considered. For this, it is necessary that the DNA extraction process take place on the isolation chip itself to minimize any losses due to manual handling of the samples and ineffectiveness caused by sample over-dilution. Thus, I present an electrochemical cell lysis platform to prepare DNA samples for molecular analysis of cells. It utilizes electrolysis of saline to generate hydroxide ions (OH-) at the cathode as alkaline lytic agents. Using the cell lysis platform, cell lysis of Chinese Hamster Ovary (CHO) cells was performed in a very short amount of time. I directly electrolyzed four bacterial cell types and human cancer cell lines suspended in saline. This cell lysis platform has significant advantages over conventional methods, which include microliter-level sample volume, higher lysis efficiencies, absence of cell lysis chemicals and heating, no adverse effects on PCR amplification, low DNA loss, low voltage and power consumption, and rapid processing. The device could potentially be applied as an on-chip component for DNA extraction. With further studies and development of the methods presented in this dissertation, I expect that these platforms can play a critical role in the early diagnosis of cancer, personalized anti-cancer therapy, and monitoring of cancer recurrence.