Abstract 4076: Single cell dual adherent-suspension co-culture microenvironment for studying tumor-stromal interactions in breast cancer stem cells
Authors: Chen Yu-Chih , Fouladdel Shamileh , Zhang Zhixiong , Azizi Ebrahim , Yoon Euisik , Wicha Max S. 
Published: July 2016

Abstract
Considerable evidence suggests that cancer stem-like cells (CSCs) are critical in tumor pathogenesis. Though functional CSCs can be identified using dish-based tumorsphere assays, it is difficult to handle and monitor single cells in a dish-based format. Tumor-stromal interaction is another key regulator of cancer micro-environments, affecting tumorigenesis and metastasis. To enable tumorsphere assays that incorporate interaction with stromal cells, we developed a microfluidic dual adherent/suspension co-culture device. This device combined a suspension environment for single-cell tumorsphere assay and an adherent environment for co-culture of stromal cells in close proximity by selectively patterning polyHEMA in indented microwells. By minimizing dead volume and improving cell capture efficiency, the presented platform allowed the use of small samples (&lt;100 cells), affording the potential to study rare cells such as circulating tumor cells (CTCs). As a proof-of-concept, we co-cultured single T47D breast cancer cells and primary cancer associated fibroblasts (CAF) on-chip for 14 days to monitor sphere formation and growth. Compared to mono-cultured T47D cells, co-cultured T47D have significantly higher tumorigenic potential (sphere formation rate) by three times and proliferation rates (larger sphere size). In addition to the phenotypic observation, the platform allows the retrieval of mono-cultured and co-cultured spheres for downstream analyses. Multiplexed single-cell gene expression analyses using C1 and BioMark HD platforms were performed to compare the expression of 96 target genes in co-cultured and mono-cultured T47D cells. Co-cultured cells expressed significantly higher MKI67, which suggests a possible mechanism for the elevated proliferation rate. Additionally, lower BAX (pro-apoptotic marker) expression matches well with higher sphere formation rate in the co-cultured cells. Interestingly, we noticed the down-regulation of CDH1, KRT8, and KRT18 in the co-cultured cells, indicating possible epithelial-mesenchymal transition induced by CAF. Single cell resolution, while to comparing between groups, also allows examination of cellular heterogeneity within one population. While cells in the mono-cultured group were more homogeneous co-cultured cells displayed clear bimodal gene expression distribution including, ANXA3, MTOR, MKI67, BAX, and PGR. This is suggestive of variable responses to the same stimuli at the single cell level. Combining the dual adherent/suspension co-culture platform presented here with the single cell gene expression analysis, we may successfully identify functional CSCs, investigate the phenotypic and transcriptional effects induced by tumor-stromal interactions, and study cellular heterogeneity in greater detail.
Citation Format: Yu-Chih Chen, Shamileh Fouladdel, Zhixiong Zhang, Ebrahim Azizi, Euisik Yoon, Max S. Wicha. Single cell dual adherent-suspension co-culture microenvironment for studying tumor-stromal interactions in breast cancer stem cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4076.