SCREENING CHIP FOR AUTOPHAGY OF FIBROBLAST IN TUMOR
CELL ENVIRONMENT
J.Kim1, H.E. Karakaş2, C. Bathany1, D. Gözüaçık2*, and Y.-K. Cho1*
1
UNIST, Republic of Korea and 2Sabanci University, Turkey
ABSTRACT
We demonstrate a single cell based microfluidic screening system using a co-culture device assembled with an SU-8 membrane. The device is designed to trap single tumor cell on a well by using hydrodynamic force, in which the trapped tumor cell is in interaction with the fibroblasts located in the bottom.
Finally, the autophagy of fibroblasts is measured after interaction with the tumor cells. This novel microfluidic chip can be useful for the investigation of the cell to cell interaction in a single cell level and to
study the heterogeneity of tumor cells in terms of autophagy activation in fibroblast cells.
KEYWORDS: Single cell trapping, Tumor cell, Fibroblast, Autophagy
INTRODUCTION
Interactions between carcinoma-associated fibroblast (CAF) and tumor cells play a critical role in
tumor proliferation and metastasis.[1] Also, the autophagy of CAF is important in the survival, proliferation and metastasis of cancer.[2-3] However, the interaction mechanisms of autophagy activation between CAF and tumor cell have not yet fully elucidated. To study the cell to cell interactions between
CAF and tumor cells, conventional co-culture system have been used. However, it can provide only average signals and is difficult to elucidate single cell level information. In this paper, we fabricated a single
cell based microfluidic screening system integrated with a SU-8membrane between the PDMS reservoirs.
This device can trap single tumor cell on a well which bottom surface is previously loaded with fibroblast.
It can be used to study the heterogeneity of tumor cells in autophagy activation of fibroblasts.
EXPERIMENTAL
The microfluidic system is composed of two polydimethylsiloxane (PDMS) molded reservoirs
separated by a SU-8 porous membrane as shown in Figure 1. The 50 µm thick SU-8 membrane with 5000
holes with the diameter of 30 µm is patterned by photholithography (Figure 2A). The bottom reservoir is
cut out from a spin-coated PDMS layer, which has controlled height of PDMS layer to prevent trapped
tumor cells from escaping. The SU-8 membrane and the PDMS reservoirs are permanently assembled by
using (3-Aminopropyl) triethoxysilane (APTES) treatment (Figure 2B). The fibroblast cells are seeded
into the fibronectin-coated bottom reservoir via the two inlets connected in the bottom reservoir.
Figure 1: Microfluidic device for high throughput screening of autophagy in CAF resulting from the interaction
with a tumor cell (A) Cross-section view of the device depicting tumor cells trapping on the fibroblast monolayer.
Tumor cells loaded in the top PDMS reservoir are located into the holes via hydrodynamic driving force. (B) Photograph of whole chip. A chip consists of inlet/outlet, PDMS reservoirs and SU-8 membrane.
978-0-9798064-7-6/µTAS 2014/$20©14CBMS-0001
452
18th International Conference on Miniaturized
Systems for Chemistry and Life Sciences
October 26-30, 2014, San Antonio, Texas, USA
Figure 2: (A) Optical microscopic image of SU-8 membrane. It has 5000 holes of 30 µm diameter. (B) Schematic representation of APTES bonding method. An irreversible bonding was formed between SU-8 layer and PDMS layer by treatment with
1% v/v aqueous APTES solution and oxygen plasma treatment of SU-8 layer and oxygen plasma treatment of PDMS.
A transgenic mouse embryonic fibroblast (MEF) cell monolayer with GFP-LC3 protein expression
was prepared after one day culture in a 37℃ incubator with 5% CO2 (Figure 3A). Biocompatibility of
heat-treated SU-8 membrane was verified as the fibroblast cells have about 90% viability (Figure 3B).
Next, 15,000 RFP MDA-MB-231 tumor cells were loaded on the top reservoir and cells are trapped into
the pore by shaking motion and suction pressure from the siphon effect. After the cell trapping action, the
cells trapped on the pore are arrayed on a MEF monolayer.
Figure 3: Biocompatibility of SU-8 membrane. (a) Culture of GFP LC3 MEF cells on bottom reservoir after 1 day. (b) Live/dead
assay of GFP LC3 MEF cells on SU-8 membrane. MEF cells have about 90% viability heat-treated SU-8 membrane for 3days.
RESULTS AND DISCUSSION
Figure 4 shows single cell trapping results using various negative pressures by siphon effect. These
results show that the trapping efficiencies have a tendency with different suction pressures. It had an optimized tumor cells trapping yield of 29.2% ± 13.0 at suction pressures of 200 Pa, 100 rpm and 5 min
shaking (Figure 4C). In addition, we observed the cell viability of GFP-LC3 transgenic fibroblast in a
chip by live/dead assay. The fibroblast cells have almost 90% viability under a SU-8 membrane after 3
days of culture. Finally, we monitored that transgenic GFP LC3 MEF cell interacted with MDA MB 231
RFP tumor cell have upregulated autophagy activation after 1 day interaction (Figure 4D).
453
Figure 4: Trapping efficiency of MDA MB 231 tumor cells with CMTMR staining and screening of autophagy activation of
GFP-LC3 transgenic MEFs in microfluidic chip system. (A), (B) Trapping result of CMTMR stained MDA MB 231 cancer cells
with 200Pa, 300Pa negative pressure respectively. (100 rpm and 5min shaking duration) ((A) 20x magnification, (B) 40x magnification) (C) Quantitative analysis of the trapping experiment depending on the negative pressure applied, (D) GFP-LC3 transgenic MEFs with interaction of MDA MB 231 RFP tumor cells under green fluorescent light in screening chip with PDMS
membrane.
CONCLUSION
This microfluidic device can make interactions between a single tumor cell and fibroblast. Importantly, it can monitor autophagy in fibroblasts by the interaction with single tumor cell. It can open up an opportunity of investigation of the heterogeneity of tumor cells in mechanism of autophagy activation of fibroblasts.
ACKNOWLEDGEMENTS
This work was supported by National Research Foundation (NRF) grant (2013R1A2A2A05004314,
2012K2A1A2033560, 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.
REFERENCES
[1] Bremnes et al., “The Role of Tumor Stroma in Cancer Progression and Prognosis: Emphasis on Carcinoma-Associated Fibroblasts and Non-small Cell Lung Cancer,” Journal of Thoracic Oncology, 6,
209-217, 2011.
[2] Martinez-Outschoorn UE et al., “Role of Hypoxia, HIF1 Induction and NFkB Activation in the Tumor Stromal Microenvironement,” Cell Cycle, 9, 3515-33, 2010.
[3] Capparelli, C. et al., “Autophagy and Senescence in Cancer-Associated Fibroblasts Metabolically
Supports Tumor Growth and Metastasis via Glycolysis and Ketone Production,” Cell Cycle, 11,
2285-2302, 2012.
CONTACT
*Y.-K. Cho, phone: +82-52-217-2511; e-mail: [email protected]
454