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Neural cell 3D microtissue formation is marked by cytokines' up-regulation.

ABSTRACT: Cells cultured in three dimensional (3D) scaffolds as opposed to traditional two-dimensional (2D) substrates have been considered more physiologically relevant based on their superior ability to emulate the in vivo environment. Combined with stem cell technology, 3D cell cultures can provide a promising alternative for use in cell-based assays or biosensors in non-clinical drug discovery studies. To advance 3D culture technology, a case has been made for identifying and validating three-dimensionality biomarkers. With this goal in mind, we conducted a transcriptomic expression comparison among neural progenitor cells cultured on 2D substrates, 3D porous polystyrene scaffolds, and as 3D neurospheres (in vivo surrogate). Up-regulation of cytokines as a group in 3D and neurospheres was observed. A group of 13 cytokines were commonly up-regulated in cells cultured in polystyrene scaffolds and neurospheres, suggesting potential for any or a combination from this list to serve as three-dimensionality biomarkers. These results are supportive of further cytokine identification and validation studies with cells from non-neural tissue.

SUBMITTER: Lai Y

PROVIDER: S-EPMC3203927 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Neural cell 3D microtissue formation is marked by cytokines' up-regulation.

Lai Yinzhi Y Asthana Amish A Cheng Ke K Kisaalita William S WS

PloS one 20111028 10

Cells cultured in three dimensional (3D) scaffolds as opposed to traditional two-dimensional (2D) substrates have been considered more physiologically relevant based on their superior ability to emulate the in vivo environment. Combined with stem cell technology, 3D cell cultures can provide a promising alternative for use in cell-based assays or biosensors in non-clinical drug discovery studies. To advance 3D culture technology, a case has been made for identifying and validating three-dimensio ...[more]

PMID: 22046371

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Project description:BackgroundMulti-walled carbon nanotubes (MWCNT) have been shown to elicit the release of inflammatory and pro-fibrotic mediators, as well as histopathological changes in lungs of exposed animals. Current standards for testing MWCNTs and other nanoparticles (NPs) rely on low-throughput in vivo studies to assess acute and chronic toxicity and potential hazard to humans. Several alternative testing approaches utilizing two-dimensional (2D) in vitro assays to screen engineered NPs have reported conflicting results between in vitro and in vivo assays. Compared to conventional 2D in vitro or in vivo animal model systems, three-dimensional (3D) in vitro platforms have been shown to more closely recapitulate human physiology, providing a relevant, more efficient strategy for evaluating acute toxicity and chronic outcomes in a tiered nanomaterial toxicity testing paradigm.ResultsAs inhalation is an important route of nanomaterial exposure, human lung fibroblasts and epithelial cells were co-cultured with macrophages to form scaffold-free 3D lung microtissues. Microtissues were exposed to multi-walled carbon nanotubes, M120 carbon black nanoparticles or crocidolite asbestos fibers for 4 or 7 days, then collected for characterization of microtissue viability, tissue morphology, and expression of genes and selected proteins associated with inflammation and extracellular matrix remodeling. Our data demonstrate the utility of 3D microtissues in predicting chronic pulmonary endpoints following exposure to MWCNTs or asbestos fibers. These test nanomaterials were incorporated into 3D human lung microtissues as visualized using light microscopy. Differential expression of genes involved in acute inflammation and extracellular matrix remodeling was detected using PCR arrays and confirmed using qRT-PCR analysis and Luminex assays of selected genes and proteins.Conclusion3D lung microtissues provide an alternative testing platform for assessing nanomaterial-induced cell-matrix alterations and delineation of toxicity pathways, moving towards a more predictive and physiologically relevant approach for in vitro NP toxicity testing.

Project description:Oral squamous cell carcinoma (OSCC) ranks among the top ten most prevalent cancers worldwide. Like most head and neck squamous cell carcinomas (HNSCCs), OSCC is highly inflammatory and aggressive. However, the signaling pathways triggering the activation of its inflammatory processes remain elusive. G protein-coupled receptor signaling regulates the inflammatory response and invasiveness of cancers, but it remains unclear whether G?12 is a critical player in the inflammatory cytokine pathway during the tumorigenesis of OSCC. This study was undertaken to determine the role of G?12 signaling in the regulation of proinflammatory cytokines in their mediation of OSCC invasion. We found that both the transcription and protein levels of G?12 are up-regulated in OSCC tumors. The elevated G?12 expressions in OSCC patients also correlated with extra-capsular spread, an indicator of tumor invasiveness in HNSCCs. This clinical finding was supported by the studies of overexpression and RNAi knockdown of G?12 in OSCC cells, which demonstrated that G?12 promoted tumor cell migration and invasion. To understand how G?12 modulates OSCC invasiveness, we analyzed key biological processes in microarray data upon depletion of G?12 and found that cytokine- and other immune-related pathways were severely impaired. Importantly, the mRNA levels of IL-6 and IL-8 proinflammatory cytokines in clinical samples were found to be significantly correlated with the increased G?12 levels, suggesting a potential role of G?12 in modulating the IL-6 and IL-8 expressions. Supporting this hypothesis, overexpression or RNAi knockdown of G?12 in OSCC cell lines both showed that G?12 positively regulated the mRNA and protein levels of IL-6 and IL-8. Finally, we demonstrated that the G?12 promotion of tumor cell invasiveness was suppressed by the neutralization of IL-6 and IL-8 in OSCC cells. Together, these findings suggest that G?12 drives OSCC invasion through the up-regulation of IL-6 and IL-8 cytokines.

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Neural cell 3D microtissue formation is marked by cytokines' up-regulation.

Publications

Neural cell 3D microtissue formation is marked by cytokines' up-regulation.

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