1. SINGLE CELL ANALYSIS ON THE ROLE OF TGF-β1 IN AUTOPHAGY OF TUMOR CELL-INTERACTED STROMA FIBROBLASTS
Junyoung Kim1+, Hacer Ezgi Karakas2+, Yong-Jun Choi1, Devrim Gozuacik*2, Yoon-Kyoung Cho*1,3
1Department of Biomedical Engineering, Ulsan National Institute of Science and Technology (UNIST) UNIST-gil 50, Ulsan , South Korea
2Molecular Biology, Genetics and Bioengineering Program, Sabanci University, Faculty of Engineering and Natural Sciences, Orhanli-Tuzla Istanbul, Turkey.
3Center for Soft and Living Matter, Institute for Basic Science (IBS) UNIST-gil 50, Ulsan , South Korea
Motivation & Abstract
Biological Analysis
(A) (B)
(C) (D)
Autophagy activation of fibroblasts interacted by tumor cells
Control
Co-culture
Here we introduce a single cell-based screening platform consisted of PDMS porous membrane. It is used (1) to trap single tumor cells by using gravitation force and agitation, (2) to monitor the autophagy of fibroblasts after interaction, and (3) to study paracrine factors from the heterogenic tumor cells inducing autophagy activation in fibroblasts. Our study highlights the role of TGF-β1 secreted by tumor cells in the autophagy activation in fibroblast using a novel screening system.
Figure 3. Effect of coculture of tumor cells and conditioned medium with fibroblast on autophagy activation. (A) Coculture of MDA MB RFP cells with transgenic GFP-LC3 MEF cells induces autophagy in fibroblast cells. MDA MB 231 RFP cells are cultured with fibroblast cells for 48 hours. (B) Quantitative analysis of experiments (C) TGF-β1 conditioned medium secreted from tumor cells can induce autophagy activation of fibroblasts. (D) Quantitative analysis of experiments in (C) (mean±SD of independent experiments, n=5, *<0.05)
Fabrication Procedures
(A) (F)
(B)
(G)
(C)
(H)
(D)
(I)
Chip Analysis * **
(A) (B) (C)
(D) (E)
Figure 1. Fabrication procedures of PDMS membrane. (A, B) Photolithography, (C, D) Soft-lithography for PDMS master (E) RIE treatment with CHF3, (F) Inject pre-cured PDMS into the gap between PDMS master and glass, (G) Release PDMS membrane, (H, I) Assemble with PDMS reservoir and PDMS membrane BF
GFP
Control
Control_
Chip
GFP-LC3 positive cells (%)
(A) (B) (C)
(F) (E) (D)
(G) (H)
Control
Control_Chip
Figure 4. Screening platform performance (A) Image of trapped single MDA MB 231 RFP into each hole. (B) Single cell trapping efficiency of this platform with various shaking velocity (C) Screening of autophagy positive hole with single tumor cell (Red) and autophagy activated fibroblasts (Green) (D, E) Basal autophagy level of microfluidic chip which indicates the reliability for autophagy screening.
Conclusion
We fabricated a microfluidic device integrated with a porous membrane for monitoring cell-to-cell interactions unique in that it visualize the interaction of one cell with a population of other cells. In the future, we aim to utilize this platform to screen shRNA libraries of tumor cells to study mechanism between tumor cell and fibroblasts.
Acknowledgement
Figure 2. Whole procedures of fibroblasts culture and single tumor cell pairing protocol. (A) Sterilization (B, C) Surface change of the membrane (D) Fibroblasts culture on the membrane bottom part (E) Assembly of platform (F) Trap single tumor cell into each hole and Screen the autophagy positive holes (G) SEM image of the PDMS membrane (H) Image of the microfluidic screening device.
This work was supported by National Research Foundation (NRF) grant (2013R1A2A2A , 2012K2A1A , NRF-2014-Global Ph.D. Fellowship Program) and a grant from the Korean Health Technology R&D Project, Ministry of Health & Welfare (A121994) funded by the Korean government and the Scientific and Technological Research Council of Turkey (TUBITAK) 2523 grant (with 112T685 project number).