Single cancer cell analysis on a chip

Abstract

Cancer cells in blood may represent “a real time liquid biopsy” through the interrogation of single cancer cells thereby determining the outspread of their heterogeneity and guiding therapy. In this thesis, we focused on single cancer cell analysis downstream of the isolation of cancer cells from blood. We designed and developed various microfluidic devices for genetic and phenotypic characterization of single cancer cells. The limited DNA content in a single cell requires DNA amplification to evaluate genetic information at the single cell level. We designed a microfluidic device for 8 parallel DNA amplification reactions in 20 nanoliter chambers. Button-shaped valves were integrated to enhance mixing of reagents in closed chambers. A 1,000-fold DNA amplification was achieved and 33% of the DNAs were template-specific. A 2nd microfluidic platform for DNA amplification was designed to aim for investigation of single circulating tumor cells in whole blood. By combining filtration technology for handling large number of cells with microfluidic technology for manipulating small volumes we demonstrated the feasibility of DNA amplification of single cancer cells isolated from whole blood. Spiked MCF-7 cells in blood were recovered in the self-seeding microwells and single cells were isolated by punching the bottom of the microwells containing the MCF-7 cell into the open chamber of the microfluidic device. The retrieved samples after DNA amplification were quantified, purified and validated by qPCR targeting specific genes. For isolation of single cells from a suspension, we designed and developed a new type of valve. Actuation of this v-type valve can create various heights of the structure by using different operation pressures thereby focusing the flow in the channel. Therefore, particles and cells were focused at the center of the channel and captured at the v-type valve. In addition cells could be released and isolated after microscopic identification of the desired features. To demonstrate the potential to probe sensitivity to drugs of single cancer cells we developed a device permitting the observation of drug uptake and accumulation. Cancer cells exposed to various concentration of doxorubicin were monitored in confined chambers for several hours at the single cell level.