Microfluidics for Functional Analysis of Circulating Tumor Cells

Abstract

Microfluidic technologies use micrometer-sized channel networks to handle and manipulate fluids and biological samples. This has made it possible to manipulate and analyze individual cells and led to a huge improvement in our understanding of cell-to-cell heterogeneities. Single-cell analysis is especially important in cancer research, as individual circulating tumor cells (CTCs) promote metastasis and a single surviving cell can cause relapse after treatment. A variety of microfluidic devices has been developed to compartmentalize individual cells for subsequent analysis. These devices have proven extremely useful to elucidate cell behavior and helped to answer many fundamental biological questions. However, their diagnostic use for analysis of CTCs is limited as these platforms do not match the high capture efficiency, selectivity, and throughput demands for isolation of CTCs from whole blood samples. In contrast, platforms that were developed for efficient CTCs isolation from whole blood do not provide the means for single-cell compartmentalization necessary for functional studies. To advance our understanding of cancer progression, there is a major demand for a platform that performs efficient CTCs isolation in a format compatible with functional single-cell analysis. To address this unmet analytical need, a microfluidic platform for the extraction and functional analysis of CTCs from whole blood was developed. This thesis is describing the gradual improvement, which started from an existing microchamber technology and finally resulted in a highly efficient and versatile platform for CTC analysis. At the beginning, a microfluidic device for single cell drug response testing with high throughput is described and evaluated. The device is capable of performing more than 600 single-cell experiments and the results are similar to traditional bulk assays in well plate format. This platform was adapted for multiplexed analysis of three different intracellular proteins on multiple cancer cell lines through the use of magnetic forces and barcoded beads. By combining magnetic magnetic bead traps with high throughput single-cell isolation, we were finally able to realize a device for functional analysis of CTCs from blood samples. The platform was characterized with CTCs from a mouse model to determine G-CSF secretion levels together with immunostaining for HER2, EpCAM, and CD45. We additionally tested our device with samples from several late stage breast cancer patients, where we found that current culturing conditions are insufficient for functional tests on patient-derived CTCs. Furthermore, a compact system that allows fluorescence analysis of many microfluidic chips with a smartphone to enable point-of-care testing is presented. Currently, research groups around the world are working on improved CTC culture conditions. Thus, direct functional tests on CTCs using devices such as the presented platform will become commonplace in the near future. It promises to offer unique insight to cancer biology and opportunities in personalized medicine.