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

Abstract

BackgroundPancreatic 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.MethodsA 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.ResultsPANC-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.ConclusionsWe 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.Electronic supplementary materialThe online version of this article (10.1186/s13046-017-0654-6) contains supplementary material, which is available to authorized users.

An Organotypic Microcosm for the Pancreatic Tumor Microenvironment

“…Microfluidics is the utilization of small channels to deliver precise volumes of fluids that allows for controlled physical and chemical analyses, and this technology has been applied in the context of PDAC organoid culture [52,53]. Using a microchannel plate, Lee et al cocultured PDAC cell line-derived organoids with PSCs [52]. The plate included three compartments, one for organoids and two for PSCs, surrounded by four media channels.…”

“…The plate included three compartments, one for organoids and two for PSCs, surrounded by four media channels. This system provided ability to study paracrine activation by PSCs and subsequent growth in the number of organoids [52]. They also measured PSC-stimulated organoid migration, including the number of cells migrating, distance moved through the extracellular matrix, and expression of epithelial-mesenchymal transition markers [52].…”

“…This system provided ability to study paracrine activation by PSCs and subsequent growth in the number of organoids [52]. They also measured PSC-stimulated organoid migration, including the number of cells migrating, distance moved through the extracellular matrix, and expression of epithelial-mesenchymal transition markers [52]. While useful for detailed analyses and high-resolution imaging, microfluidic technology is limited in that organoid retrieval from microfluidic devices may be difficult, they can only hold a small number of organoids, and the polydimethylsiloxane matrix used to hold the organoids may adsorb small hydrophobic molecules, presenting a confounding variable for drug screening studies [48].…”

Cell Culture and Coculture for Oncological Research in Appropriate Microenvironments

“…The mode of coculture depends on the scientific question under investigation, since coculture can be done within the same matrix or in separate chambers. Coculture in separate chambers is reported in many publications either using an insert in a cell culture well or using microfluidics to connect distinct cell culture chambers with fluid that can be exchanged from one chamber to another (Goers, Freemont, & Polizzi, 2014;Mi et al, 2016;Lee et al, 2018). However, with matrix stiffness being important for the behavior of all cell types, many projects will also require coculturing cells in the same physical microenvironment (which of course could be replicated in separate chambers as well), enabling cells to be near each other and even make contact as they would in vivo.…”

The lipid kinase PI3Kα is required for the cohesion and survival of cancer cells disseminated in serous cavities

“…MSCs are a potential source of various cell types including tumour-associated fibroblasts (TAFs). MSC and TAFs have been shown to contribute to tumour cells' ability to form spheroids called tumourospheres under anchorage-independent culture conditions, increasing their capacity to induce tumour formation in vivo after mouse xenografts (20,21). The role of MSC in multicellular 3D culture of tumoural cells is poorly known.…”