The Study of Pancreatic Circulating Tumor Cells via Isolation with an Integrated Microfluidic Immunomagnetic (IMI) Device

Abstract

In 2016 alone, it is projected that over 40,000 Americans will succumb to pancreatic cancer. One avenue that holds promise in developing accurate predictive tools and effective therapeutic agents to mitigate cancer mortality lies in the analysis of circulating tumor cells (CTCs). CTCs describe a subset of tumor cells that have acquired the ability to disseminate from a primary tumor and intravasate into the circulatory system to subsequently spur new metastases. As such their detection and isolation from peripheral blood is recognized as a potent tool in the study and treatment of metastasis. CTCs are accessible via facile, non-invasive patient blood draws compared to complex surgical procedures traditionally employed to obtain tissue biopsies. However these cells are quite rare, occurring on the order of 1:1 billion among other blood cells. Therefore the need to enrich them, though challenging, is exigent. This thesis delineates the development of an integrated polymer-based microfluidic CTC sorting device that facilitates significant enrichment of CTCs in a continuous, high-throughput manner through strategic combination of size-based inertial and magnetic cell sorting. This process enables targeted isolation of as few as 10s of CTCs from billions of other blood cells with average pancreatic CTC purity of 82.5%±23.5. Detection of >13 CTCs/mL was observed in 100% of pancreatic cancer patient samples. Subsequently, the work describes molecular characterization of CTCs. Specifically, messenger RNA profiling revealed that significantly higher expression of seven mRNAs could distinguish metastatic CTC profiles from other patient profiles. Moreover, through CTC micro-RNA profiling, high expression of several miRNAs was observed for the first time. Additionally, preliminary studies on CTC heterogeneity have been reported. In one study, CTCs were sorted according to a continuum of EpCAM protein expression, a CTC surface protein of paramount interest. In another study, CTCs have been isolated based on a dual protein marker approach where a combination of two surface proteins of interest have been used to immunomagnetically enrich and probe these rare cells. Overall, sensitive CTC interrogation as described opens the doorway to identifying biological pathways and molecular targets that can be exploited in the fight against cancer.