Drug cytotoxicity and signaling pathway analysis with three-dimensional tumor spheroids in a microwell-based microfluidic chip for drug screening

• A great deal of uniform 3D tumor spheroids could be formed within a short period of time using the microfluidic chip.
• The microfluidic platform was easy-handling and high integration in a more physiologically relevant context.
• The in situ cytotoxicity assay of 3D tumor spheroids on microfluidic chip was comparable with a microplate reader.
• The first time to combine the drug susceptibility testing and offline drug signaling pathway in one study.

Currently, there has been a growing need for developing in vitro models to better reflect organism response to chemotherapy at tissue level. For this reason, a microfluidic platform was developed for mimicking physiological microenvironment of solid tumor with multicellular tumor spheroids (MTS) for anticancer drug screening. Importantly, the power of this system over traditional systems is that it is simple to operate and high integration in a more physiologically relevant context. As a proof of concept, long-term MTS cultures with uniform structure were realized on the microfluidic based platform. The response of doxorubicin and paclitaxel on different types of spheroids were simultaneously performed by in situ Live/Dead fluorescence stain to provide spatial distribution of dead cells as well as cytotoxicity information. In addition, the established platform combined with microplate reader was capable to determine the cytotoxicity of different sized MTS, showing a more powerful tool than cell staining examination at the end-point of assay. The HCT116 spheroids were then lysed on chip followed by signaling transduction pathway analysis. To our knowledge, the on chip drug screening study is the first to address the drug susceptibility testing and the offline detailed drug signaling pathway analysis combination on one system. Thus, this novel microfluidic platform provides a useful tool for drug screening with tumor spheroids, which is crucial for drug discovery and development.

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