Recapitulating epithelial tumor microenvironment in vitro using three dimensional tri-culture of human epithelial, endothelial, and mesenchymal cells

Abstract

BackgroundThree-dimensional (3-D) cultures of cancer cells can potentially bridge the gap between 2-D drug screening and in vivo xenografts. The objective of this study was to characterize the cellular and extracellular matrix characteristics of spheroids composed of human lung epithelial cells (epi), pulmonary vascular endothelial (endo) cells, and human marrow-derived mesenchymal stems cells (MSCs).MethodsSpheroids composed of epi/endo/MSCs, termed herein as synthetic tumor microenvironment mimics (STEMs), were prepared by the hanging drop method. Cellular composition and distribution in the STEMs was characterized using fluorescence microscopy. Induction of reactive oxygen species and upregulation of efflux transporters was quantified using fluorometry and PCR, respectively, and phenotypic markers were qualitatively assessed using immunohistochemistry.ResultsSTEMs exhibited three unique characteristics not captured in other spheroid cultures namely, the presence of a spheroid core devoid of epithelial cells and primarily composed of MSCs, a small viable population of endothelial cells hypothesized to be closely associated with MSCs within the hypoxic core, and discrete regions with high expression for vimentin and cytokeratin-18, whose co-expression is co-related with enhanced metastasis. Although cells within STEMs show elevated levels of reactive oxygen species and mRNA for ABC-B1, an efflux transporter associated with drug resistance, they exhibited only modest resistance to paclitaxel and gemcitabine in comparison to 2-D tri-cultures.ConclusionsThe epi/endo/MSC spheroid model described herein offers a promising platform for understanding tumor biology and drug testing in vitro.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2634-1) contains supplementary material, which is available to authorized users.

Generation of 3D Soluble Signal Gradients in Cell‐Laden Hydrogels Using Passive Diffusion

“…The modular setup of 4DGG allows several degrees of freedom, namely: 1) the shape of the gradient can be varied by altering the dimensions of the channel in the ISE, 2) since cells are introduced into the channel in the ISE channel as a dispersion in a gel, several cell types – including multicellular spheroids – can be studied at various densities and simultaneously; using various reporter systems, and furthermore, the mechanical property of the gel microenvironment can be varied independently from the aforementioned variables to investigate mechanobiology paradigms, and 3) since the gelatin SSE is independently configured and compatible with a variety of soluble signals, it opens up the possibility of studying a variety of signaling scenarios. Since the 4DGG is based on passive diffusion which is more physiological than other means of transport, it eliminates shear‐related artifacts in cell behavior that could confound analysis and hence offers a valuable alternative to microfluidic devices .…”

Mesenchymal Stem Cells Relevance in Multicellular Bioengineered 3D In Vitro Tumor Models

“…However, no increased drug resistance in the 3D models was obtained when compared to standard 2D co‐cultures. In fact, only some concentrations showed a prevailing difference between models, with the 3D model presenting always an increased cellular viability in comparison to the 2D model …”

“…In vivo studies in mice demonstrated the capacity of ectopically injected BM‐MSCs of migrating toward xenografts of human pancreatic cancer, in which BM‐MSCs lead to an increase in the number of CD31+ early vascular structures, an event that directly correlates with the density of tumor vascularization, as was observed, with tumors with twice the vascularization of those in obtained in controls . Another tri‐culture was performed by Lamichane and co‐workers, 2016, in which A549 cells were co‐cultured with human pulmonary microvascular endothelial cells and BM‐MSCs . The tracking of the different cellular populations was accomplished through transfection with lentiviral particles encoding red fluorescent (RFP) and green fluorescent (GFP) transgene reporters, and followed for 15 days.…”

Modeling the Efficacy of Oncolytic Adenoviruses In Vitro and In Vivo: Current and Future Perspectives

“…3D tumor spheroids are aggregates of cells that can be grown in suspension on minimally adherent surfaces. Tumor spheroids have been reproducibly used for decades and can be composed of tumor cells only or can contain other cell types such as fibroblasts, mesenchymal stromal cells, and endothelial cells [36]. Depending on the source of cells (primary or cell line), tumors can form tight/dense spheroids, compact aggregates or loose aggregates depending on the cells' adhesive properties or epithelial vs mesenchymal origin [37].…”

“…Overall, 3D spheroid cultures mimic the physical tumor microenvironment, including cell-to-cell interactions, but fail to fully recapitulate the ECM and growth factors present in solid tumors. While tumor spheroids have been generated with other cell types such as stromal and endothelial cells [36,49], studies investigating OAds in these models have yet to be completed.…”