Dissertation Defense - Tang Zhewen
Dr. Ray P.S. Han, MSE, Peking University (co-advisor)
Dr. CP Wong, MSE (co-advisor)
Dr. Meilin Liu, MSE
Dr. Zou Ruqiang, MSE, Peking University
Dr. Dong Shuxiang, MSE, Peking University
Dr. Yu Haifeng, MSE, Peking University
Dr. Chang Xiaohong, People's Hospital of Peking University
Dr. Guo Mingzhou, General Hospital of PLA
Segregation, Capture and Recovery of Tumor Cells in Human Body Fluids for Microfluidic Assay & Rat Experiments with a CTC-Dialysis Animal Model
In a cancerous human body, tumor cells rapidly proliferate and metastasize in body fluids and it is this liquid manifestation that provides us with an opportunity for their segregation, capture and recovery. The liquid biopsy approach is now recognized as one of the most important tools for understanding the pathogenesis of neoplastic diseases such as cancers by studying not only the physical appearance and response of the cell but also, its molecular information contained inside it. Tumor cells in the peripheral blood are termed as Circulating Tumor Cells (CTCs) and in a pseudostatic collection of body fluid such ascitic fluid, peritoneal fluid, thoracic fluid, urine, sputum, etc. are generally termed as Dissipated Tumor Cells (DTCs). To segregate and capture tumor cells in a body fluid is most challenging because of the vast order of magnitude difference among the constituents of the fluid; take for example, in 1 ml of blood, the ratio is like 100 CTCs compared to over 10 million white blood cells (WBCs) and half a trillion red blood cells (RBCs) and these rare tumor cells are also, highly heterogeneous with diameters in the micron scale. It is because of their small size, the harvesting process is painstakingly slow for the macro volume of fluid environment that is typically encountered. Various capture methodologies have been developed; biomarker-specific (mostly EpCAM) based approach for CTC adhesion, magnetic and electric field techniques for the deflection of rare cells, centrifugation and Dean force methods for steering the rare cells in a preset trajectory, cellular size-based devices for the physical separation, etc. After harvesting, these rare cells need to be properly processed in order to accurately identify them as tumor cells. Immunofluorescence staining is most popular and a well-tested algorithm with specific antibodies to their antigen by targeting the fluorescent dyes to a specific biomolecule needs to be in place to correctly distinguish the tumor cells from normal cells, WBCs and other cellular constituents that are often inadvertently captured.
The cell capture technology adopted in my thesis research is based on the strong difference in the physical size and stiffness of tumor cells to separate them from background cells in the body fluid. This size-based method allows for the harvesting of intact CTCs and DTCs. Further, by optimizing the flow pattern and modifying the surface property of the capture chamber to reduce the adverse effects of the high shear flow environment, it is possible to attain close to 90% capture efficiency and capture purity. A comprehensive set of protocols for a stable and reliable capture and recovery of tumor cells in peripheral blood and peritoneal fluid is presented in the thesis. The results of the microfluidic assay applied in cancer research have the following outcomes: 1) the assay enumerates CTCs and their capture magnitude constitutes a measure of the severity of the pancreatic cancer in patients; 2) the assay yields high resolution images for the characterization of non-small cell lung cancer CTCs to reveal their mitochondrial autophagy; 3) the assay harvests circulating and peritoneal endometrial cells for use as a biomarker for the diagnosis of endometriosis and 4) the assay yields intact endometrial cells to provide an insight into the pathogenesis of endometriosis.
The thesis concludes by carrying out some pioneering rat experiments using a proposed CTC-dialysis animal model. Just like the hemodialysis system for the removal of body wastes in peripheral blood, the objective of the experiments is to demonstrate that a microfluidic chip can be used to remove CTCs in the peripheral blood of a rat in an attempt to inhibit cancer metastasis. The limited experiments clearly showed that a chip attached to the rat via a postcava microsurgery protocol is able to capture cancer cells that are tail-vein injected into the live animal.