Considerable evidence suggests that cancer-stem-cells (CSCs) play critical roles in tumorigenesis and metastasis .Although surface marker expression or enzymatic activity of CSC has proven useful, variability of expression highlights the need for the development of high throughput functional CSC assays. One such approach utilizes the capability of CSC’s to form single-cell-derived spheres in suspension environments (functional CSCs). Microfluidic tools are suitable for handling and monitoring a large number of single cells in suspension as well as for examining the interaction of these cells with those in the tumor micro-environment including cancer associated fibroblasts (CAF’s).In order to accomplish this, we have developed a high-throughput (10,000-well), simple-to-use, vertical platform for CSC/CAF co-culture. The platform consists of a non-adherent substrate having 10,000 micro wells for single-cell-derived sphere formation and a porous trans well for CAF adherent co-culture. As predicted by Poisson’s distribution, approximately 30% of micro wells capture single cells when T47D breast cancer cells are loaded at a concentration of 200k/ml. After cell seeding, trans wells containing CAFs are placed onto the substrate. 0.4µm pores on the trans well allow for secretion-based cellular interaction while preventing direct CSC/CAF cell contact. This cell separation facilitates sample cell retrieval without cell cross contamination. Utilizing this device, we determined that co-culture of tumor cells with CAF’s doubled the frequency of sphere formation. Spheres derived from individual CSCs were then retrieved and dissociated for single-cell transcriptome analysis utilizing RNA barcoding followed by RNAseq. Molecular analysis revealed significant cellular heterogeneity among sphere derived cells. In addition, principal component analysis of single cell RNAseq, reveled significant differences in gene expression between mono-cultured and CAF co-cultured cells isolated from tumor spheres. These genes represent potential therapeutic targets for interfering with CSC stromal cell interactions in the tumor microenvironment. The vertical adhesion suspension co-culture platform provides a system to identify and evaluate novel CSC targeted therapeutics.

Citation Format: Yu-Chih Chen, Zhixiong Zhang, Yu-Heng Cheng, Euisik Yoon, Max S. Wicha. Microfluidic vertical adhesion-suspension co-culture platform for studying cancer stem cell-fibroblast interactions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5909. doi:10.1158/1538-7445.AM2017-5909