Circulating tumor cells (CTCs) are known to escape from the primary tumor site and may settle down at the distant organ to grow a second tumor. CTCs are one of causes initiating carcinoma metastasis. Detection of CTCs has been considered to be valuable for cancer management, including diagnosis, prognosis, and clinical treatment management. However, efficient isolation, enumeration, characterization, and genetic analysis of CTCs in whole-blood samples from cancer patients are very challenging due to their extremely low concentration and rare nature (per CTC in blood cells is 1:106–109). With the increasing worldwide death rate associated with cancer, there is a desperate demand for a high-sensitivity, high-throughput, and low-cost detection and separation system. My doctoral research focused on the design and fabrications of the screening system for the detection of CTCs with further analysis of captured CTCs, such as immunofluoresce staining and fluorescence in-situ hybridization (FISH). The distinct significance of this research is that the development of the computer-controlled rotational holder with a series of six inverted microfluidic chips reduced the cost by significantly reducing the consumption of magnetic carriers (25% of the consumed amount used in the commercial CellSearch® system), increasing the capture efficiency by manipulating the blood sedimentation in the microchannel, enhancing the system stability by integrating the micromagnets on the plain glass slide substrate, and achieving high throughput because of the high flow rate (2.5 mL/hr) and large screening volume (screening up to six chips in parallel with each containing 2.5 mL of blood). Immunofluorescence staining and the FISH method have been performed to prove the capability of the system. In addition, the system has been successfully applied for patient samples screening. The incorporation of micromagnets has demonstrated that micromagnets provide localized magnetic forces to scatter the target cancer cells and free nanoparticles throughout the whole channel substrate to increase the channel space usage by 13%. Four cancer cell lines, including COLO 205 (colorectal cancer), SK-BR-3 (breast cancer), MCF-7 (breast cancer), and PC3 (prostate cancer), were spiked in blood samples from healthy donors to verify high capture efficiency of the developed system. On average, over a 97% capture rate was demonstrated for all cell lines. Moreover, the developed screening system has been successfully screened over 40 patient samples, including metastatic lung cancer, breast cancer, prostate cancer, and colorectal cancer. After capture of CTCs, immunofluorescence staining was used to identified the captured cancer cells and the FISH method was performed to characterize the isolated cancer cells by studying the gene expression of CTCs from breast cancer. The proposed automated immunomagnetic microchip-based screening system shows high capture efficiency (average 97% for three spiked cell lines), high throughput (15 mL of blood sample per screening), high sensitivity, high specificity, and low nanoparticle consumption (75% less than CellSearch® system). The screening system provides great promise as a clinical tool for early cancer diagnosis, diagnosis, personalized therapy, and treatment monitoring. / text

Epigenetic modifications, such as DNA methylation and histone modifications, play important roles in gene expression and regulation, and are highly involved in cellular processes such as stem cell pluripotency/differentiation and tumorigenesis. Chromatin immunoprecipitation (ChIP) is the technique of choice for examining in vivo DNA-protein interactions and has been a great tool for studying epigenetic mechanisms. However, conventional ChIP assays require millions of cells for tests and are not practical for examination of samples from lab animals and patients. Automated microfluidic chips offer the advantage to handle small sample sizes and facilitate rapid reaction. They also eliminate cumbersome manual handling. In this report, I will talk about three different projects that utilized microfluidic immunoprecipitation followed by next genereation sequencing technologies to enable low input and high through epigenomics profiling. First, I examined RNA polymerase II transcriptional regulation with microfluidic chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) assays. Second, I probed the temporal dynamics in the DNA methylome during cancer development using a transgenic mouse model with microfluidic methylated DNA immunoprecipitation followed by next generation sequencing (MeDIP-seq) assays. Third, I explored negative enrichment of circulating tumor cells (CTCs) followed by microfluidic ChIP-seq technology for studying temporal dynamic histone modification (H3K4me3) of patient-derived tumor xenograft on an immunodeficient mouse model during the course of cancer metastasis. In the first study, I adapted microfluidic ChIP-seq devices to achieve ultrahigh sensitivity to study Pol2 transcriptional regulation from scarce cell samples. I dramatically increased the assay sensitivity to an unprecedented level (~50 K cells for pol2 ChIP-seq). Importantly, this is three orders of magnitude more sensitive than the prevailing pol2 ChIP-seq assays. I showed that MNase digestion provided better ChIP-seq signal than sonication, and two-steps fixation with MNase digestion provided the best ChIP-seq quality followed by one-step fixation with MNase digestion, and lastly, no fixation with MNase digestion. In the second study, I probed dynamic epigenomic changes during tumorigenesis using mice often require profiling epigenomes using a tiny quantity of tissue samples. Conventional epigenomic tests do not support such analysis due to the large amount of materials required by these assays. In this study, I developed an ultrasensitive microfluidics-based methylated DNA immunoprecipitation followed by next-generation sequencing (MeDIP-seq) technology for profiling methylomes using as little as 0.5 ng DNA (or ~100 cells) with 1.5 h on-chip process for immunoprecipitation. This technology enabled me to examine genome-wide DNA methylation in a C3(1)/SV40 T-antigen transgenic mouse model during different stages of mammary cancer development. Using this data, I identified differentially methylated regions and their associated genes in different periods of cancer development. Interestingly, the results showed that methylomic features are dynamic and change with tumor developmental stage. In the last study, I developed a negative enrichment of CTCs followed by ultrasensitive microfluidic ChIP-seq technology for profiling histone modification (H3K4Me3) of CTCs to resolve the technical challenges associated with CTC isolation and difficulties related with tools for profiling whole genome histone modification on tiny cell samples. / Ph. D.