The online version of this article (https://doi.org/10.1186/s13046-017-0654-6) contains supplementary material, which is available to authorized users.
Pancreatic stellate cells (PSCs), a major component of the tumor microenvironment in pancreatic cancer, play roles in cancer progression as well as drug resistance. Culturing various cells in microfluidic (microchannel) devices has proven to be a useful in studying cellular interactions and drug sensitivity. Here we present a microchannel plate-based co-culture model that integrates tumor spheroids with PSCs in a three-dimensional (3D) collagen matrix to mimic the tumor microenvironment in vivo by recapitulating epithelial-mesenchymal transition and chemoresistance.
A 7-channel microchannel plate was prepared using poly-dimethylsiloxane (PDMS) via soft lithography. PANC-1, a human pancreatic cancer cell line, and PSCs, each within a designated channel of the microchannel plate, were cultured embedded in type I collagen. Expression of EMT-related markers and factors was analyzed using immunofluorescent staining or Proteome analysis. Changes in viability following exposure to gemcitabine and paclitaxel were measured using Live/Dead assay.
PANC-1 cells formed 3D tumor spheroids within 5 days and the number of spheroids increased when co-cultured with PSCs. Culture conditions were optimized for PANC-1 cells and PSCs, and their appropriate interaction was confirmed by reciprocal activation shown as increased cell motility. PSCs under co-culture showed an increased expression of α-SMA. Expression of EMT-related markers, such as vimentin and TGF-β, was higher in co-cultured PANC-1 spheroids compared to that in mono-cultured spheroids; as was the expression of many other EMT-related factors including TIMP1 and IL-8. Following gemcitabine exposure, no significant changes in survival were observed. When paclitaxel was combined with gemcitabine, a growth inhibitory advantage was prominent in tumor spheroids, which was accompanied by significant cytotoxicity in PSCs.
We demonstrated that cancer cells grown as tumor spheroids in a 3D collagen matrix and PSCs co-cultured in sub-millimeter proximity participate in mutual interactions that induce EMT and drug resistance in a microchannel plate. Microfluidic co-culture of pancreatic tumor spheroids with PSCs may serve as a useful model for studying EMT and drug resistance in a clinically relevant manner.
Additional file 1: Figure S1. Differential sensitivity of PANC-1 tumor spheroids and PSCs to gemcitabine and oxaliplatin. Cells were grown for 5 days and stained with calcein AM / PI after 72 h exposure to oxaliplatin (a, b) and gemcitabine (c) under mono- or co-culture condition. Optical sections were acquired at 6 μm intervals and stacked into a z-projection from which fluorescence intensity was calculated. Data are expressed as the mean ± SE of 3 independent experiments. Scale bars 100 μm. Data showed no significance from Student’s t-test. No statistically significantdifferences were observed. PSCs, pancreatic stellate cells; TS, tumor spheroids. (TIFF 6300 kb)13046_2017_654_MOESM1_ESM.tif
Additional file 2: Figure S2. Expression of cytokines in PSCs. (a) PSCs were grown for 5 days with or without PANC-1 spheroids in microchannel plate and harvested for proteome analysis using Proteome Profiler™. PSCs, pancreatic stellate cells; TS, tumor spheroids. (TIFF 2828 kb)13046_2017_654_MOESM2_ESM.tif
Additional file 3: Figure S3. Differential expression of EMT-related markers in different tumor cell spheroids. Immunofluorescence staining of E-cadherin and vimentin was performed in PANC-1 and HT-29 spheroids cultured for 5 days in microfluidic channels, and on paraffin sections of Huh-7 spheroids cultured for 5 days in ULA 96 well plates. For PANC-1 and HT-29 spheroids (red), confocal optical sections were acquired at 2 μm intervals and stacked into a z-projection (see Methods for details). Counter stain, DAPI (blue). Scale bars, 20 μm and 100 μm. EMT, epithelial-mesenchymal transition; TS, tumor spheroids. (TIF 667 kb)
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- Microfluidic co-culture of pancreatic tumor spheroids with stellate cells as a novel 3D model for investigation of stroma-mediated cell motility and drug resistance
Iftikhar Ali Khawar
- BioMed Central
Journal of Experimental & Clinical Cancer Research
Elektronische ISSN: 1756-9966
Neu im Fachgebiet Onkologie
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