Autophagy analysis of tumor-fibroblasts crosstalk on biochips
Karakaş Schüller, Hacer Ezgi and Kim, Junyoung and Park, Juhee and Min Oh, Jung and Choi, Yongjun and Gözüaçık, Devrim and Cho, Yoon-Kyoung (2017) Autophagy analysis of tumor-fibroblasts crosstalk on biochips. In: VI. International Congress of Molecular Medicine, Istanbul, Turkey
Background/Aim: Autophagy is an evolutionary conserved stress response mechanism which creates alternative sources of building blocks and energy to cells. Although induced autophagy in fibroblasts, major component of stroma, has an important role for the proliferation and metastasis of cancer, initial signal(s) of induced autophagy in fibroblasts during their crosstalk with cancer cells is not clearly understood, yet. Here, we present a biochip system to mimick a simple tumor microenvironment and to monitor communication between can-cer-fibroblasts through modulation of autophagy. Materials& Methods: In this study, transgenic GFP-LC3 mouse embryonic fibroblast (MEF) cells and stably RFP expressing MDA-MB-231 (MDA) breast cancer cells were used. For autophagy analysis under microscope, over 20 GFP-LC3 dots per MEFs was called as GFP-LC3 dot positive cells. Biocompatibility and autopha-gy analysis of PDMS membrane in biochip was performed with live/death assay and autophagy analysis methods. 1.0×104 MDA cells, infected with shGFP (CNT) or shTGFβ1 viruses, were loaded on the top reservoir of biochip and cells were trapped into the pore by shaking motion and suction pressure. Trapped MDA cells were arrayed on a MEF monolayer and autopha-gy analysis was performed using Nikon A1R microscope. Re-sults&Discussion: Transforming growth factor (TGFβ1) was tested as a communication signal between cancer-fibroblasts. First, effect of TGFβ1 on autophagy in MEFs was analyzed using microscope and immunoblot analysis. According to our results, autophagy in fibroblasts was induced in the presence of TGFβ1 and was kept basal level in the absence of TGFβ1. Next, autophagy in MEFs around single MDA-trapped or empty holes in biochip systems was analyzed. Captured single MDA cells in-duce autophagy in fibroblast cells near to the holes. To test the biochip system for screening, the effect of mixed CNT-MDA and shTGFβ1-MDA cancer cells on autophagy in fibroblast cells was tested in biochip system. CNT-MDA cells induced autophagy in fibroblast cells around the holes upto 40%. shTGFβ1-MDA cells have caused basal autophagy level in surrounding fibro-blast cells similar to empty hole result. Genomic DNA analysis confirmed the unbiased screening result with 81% accuracy. These results showed that specificity of TGFβ1-induced auto-phagy was 90%. Due to allowing cancer cells retrieval back from chip system, this biochip system is suitable for large-scale un-biased omics such as single cell genomic, transcriptomics and even metabolomics analysis. Conclusion: We propose that our biochip platform can be used as a promising tool for autophagy quantification during tumor-stroma interaction, especially for high-throughput screening of paracrine factors that are secret-ed from heterogeneous tumor cell populations.
Repository Staff Only: item control page