Abstract

Identification of Different Subpopulations of Circulating Tumor Cells and Extracellular Vesicles in the Blood of Cancer Patients using Microfluidics

Steve Soper, Foundation Distinguished Professor; Director, Center of BioModular Multi-scale System for Precision Medicine, Adjunct Professor, Ulsan National Institute of Science & Technology, The University of Kansas

Liquid biopsies are an attractive source of biomarkers that can be used to manage a variety of cancer-related diseases. Two liquid biopsy markers found in blood are circulating tumor cells (CTCs) and extracellular vesicles (EVs). The challenge associated with using CTCs or EVs as biomarkers has been that affinity selection using anti-EpCAM antibodies only does not recapitulate the tumor microenvironment entirely providing limited predictive power of the assay. For example, CTCs expressing invasive phenotypes down-regulate epithelial antigens, such as the epithelial cell adhesion molecule – EpCAM. We developed a liquid biopsy selection strategy that employs plastic microfluidic chips modified with antibodies to select two different liquid biopsy marker subpopulations. In addition to the common marker used for positive selection (EpCAM), Fibroblast Activation Protein alpha (FAPa) expressing liquid biopsy markers were also selected. Using the dual selection strategy, both subpopulations are detected from patients with results showing better predictive value compared to selection in which only EpCAM is used. In this presentation, we will show two examples of EpCAM/FAPa selection to monitor response to therapy using CTCs in pancreatic cancer, and determining the best treatment option for breast cancer patients using EVs. The CTCs and EVs were selected using specially designed microfluidic chips that could be operated by a fluid handling robot to support large-scale clinical trials. In the case of CTCs, response to a PARPi therapy was serially monitored to determine treatment efficacy. Results indicated that CTCs were better in monitoring response to the PARPi compared to the serum marker, CA19-9, but only when using both CTC subpopulations. In addition, the CTCs could be sequenced using NGS without the need for single-cell picking. Both EpCAM and FAPa expressing EVs and their mRNA cargo were used to perform molecular subtyping of breast cancer patients. We found the four molecular subtypes (luminal A/B, HER2 enriched, and basal-like) could be successfully identified in patients only when using both EV subpopulations. The EV mRNA copy numbers were analyzed using the nCounter assay without requiring the need for an amplification step.