High-Throughput Selection, Enumeration, Electrokinetic Manipulation, and Molecular Profiling of Low-Abundance Circulating Tumor Cells Using a Microfluidic System

摘要

A circulating tumor cell (CTC) selection microfluidicndevice was integrated to an electrokinetic enrichmentndevice for preconcentrating CTCs directly from whole blood tonallow for the detection of mutations contained within thengenomic DNA of the CTCs. Molecular profiling of CTCs cannprovide important clinical information that cannot be garnerednsimply by enumerating the selected CTCs. We evaluated ournapproach using SW620 and HT29 cells (colorectal cancer cellnlines) seeded into whole blood as a model system. BecausenSW620 and HT29 cells overexpress the integral membrane protein EpCAM, they could be immunospecifically selected using anmicrofluidic device containing anti-EpCAM antibodies immobilized to the walls of a selection bed. The microfluidic device wasnoperated at an optimized flow rate of 2 mm s-1, which allowed for the ability to process 1 mL of whole blood in <40 min. Thenselected CTCs were then enzymatically released from the antibody selection surface and hydrodynamically transported through anpair of Pt electrodes for conductivity-based enumeration. The efficiency of CTC selection was found to be 96% ( 4%. Followingnenumeration, the CTCs were hydrodynamically transported at a flow rate of 1 μL min-1 to an on-chip electromanipulation unit,nwhere they were electrophoretically withdrawn from the bulk hydrodynamic flow and directed into a receiving reservoir. Using annelectric field of 100 V cm-1, the negatively charged CTCs were enriched into an anodic receiving reservoir to a final volume of 2 μL,nproviding an enrichment factor of 500. The collected CTCs could then be searched for point mutations using a PCR/LDR/capillarynelectrophoresis assay. The DNA extracted from the CTCs was subjected to a primary polymerase chain reaction (PCR) with thenamplicons used for a ligase detection reaction (LDR) to probe for KRAS oncogenic point mutations. Point mutations in codon 12 ofnthe KRAS gene were successfully detected in the SW620 CTCs for samples containing <10 CTCs in 1mL of whole blood.However,nthe HT29 cells did not contain these mutations, consistent with their known genotype.