Changes in global gene expression associated with 3D structure of tumors: an ex vivo matrix-free mesothelioma spheroid model

Our plain-English summary of Changes in global gene expression associated with 3D structure of tumors: an ex vivo matrix-free mesothelioma spheroid model, by H. Kim, Y. Phung, M. Ho (PloS One, 2012).

The study

• Authors: H. Kim, Y. Phung, M. Ho
• Journal: PloS One
• Published: 2012;7(6):e39556
• Cited by: 53 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Tumor microenvironments present significant barriers to anti-tumor agents. Molecules involved in multicellular tumor microenvironments, however, are difficult to study ex vivo. Here, we generated a matrix-free tumor spheroid model using the NCI-H226 mesothelioma cell line and compared the gene expression profiles of spheroids and monolayers using microarray analysis. Microarray analysis revealed that 142 probe sets were differentially expressed between tumor spheroids and monolayers. Gene ontology analysis revealed that upregulated genes were primarily related to immune response, wound response, lymphocyte stimulation and response to cytokine stimulation, whereas downregulated genes were primarily associated with apoptosis. Among the 142 genes, 27 are located in the membrane and related to biologic processes of cellular movement, cell-to-cell signaling, cellular growth and proliferation and morphology. Western blot analysis validated elevation of MMP2, BAFF/BLyS/TNFSF13B, RANTES/CCL5 and TNFAIP6/TSG-6 protein expression in spheroids as compared to monolayers. Thus, we have reported the first large scale comparison of the transcriptional profiles using an ex vivo matrix-free spheroid model to identify genes specific to the three-dimensional biological structure of tumors. The method described here can be used for gene expression profiling of tumors other than mesothelioma.

In plain English

This laboratory study used cells, not people, and did not test asbestos or any other exposure. The authors grew a human mesothelioma cell line, NCI-H226, originally isolated from the pleural fluid of a malignant mesothelioma patient, in two ways. One set grew as flat single layers. The other formed flattened three-dimensional clusters, called spheroids, without any added supporting matrix. The authors used microarrays to compare gene activity in three samples of each. Of 54,675 probe sets, 142 differed at least five-fold with p less than 0.05: 112 were higher and 30 lower in spheroids. Genes more active in spheroids were mainly linked to immune response, wound response, lymphocyte stimulation and response to cytokines. Less active genes were mainly linked to apoptosis (programmed cell death). Twenty-seven of the 142 were membrane-related. Protein tests found higher levels of MMP2, BAFF/BLyS/TNFSF13B, RANTES/CCL5 and TNFAIP6/TSG-6 in spheroids. The authors concluded that some of these genes could be new biomarkers or treatment targets. They noted that their five-fold cut-off was arbitrary and could miss changes, and that spheroids do not model stroma or immune cells.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Laboratory cell-culture experiment comparing gene expression between three-dimensional spheroids and flat monolayers of one mesothelioma cell line. Microarrays were run in triplicate, and Western blots checked protein levels.
• Who was studied: The human mesothelioma cell line NCI-H226, obtained from the American Type Culture Collection and originally isolated from the pleural fluid of a malignant mesothelioma patient. There were six samples: three monolayer and three spheroid.
• Exposure: No asbestos or other fiber exposure was studied. The comparison was between cells grown as matrix-free 3D spheroids (10,000 cells per spheroid) and cells grown as 2D monolayers.
• Main results:
  • After 24 hours of incubation, the spheroids formed tight disks with smooth edges.
  • The microarray covered 54,675 probe sets in total.
  • In spheroids compared with monolayers, 142 probe sets (112 upregulated and 30 downregulated) showed at least a five-fold change in expression and a p-value less than 0.05.
  • Genes upregulated in spheroids were mainly related to immune response, wound response, lymphocyte stimulation and response to cytokine stimulation. Downregulated genes were mainly associated with apoptosis.
  • Twenty-seven of the 142 differentially expressed transcripts were in the membrane-related group.
  • Western blots showed higher protein expression of MMP2, TNFAIP6/TSG-6, RANTES/CCL5 and BAFF/BLyS/TNFSF13B in spheroids than in monolayers. The authors report that the active form of MMP2 was significantly increased in spheroids, but no statistical test is given for this.
  • In spheroids, expression of PKM2, GLUT4 and GLUT1 was increased by 1.02, 3.15 and 3.46 fold, respectively.
• The authors' conclusion: The authors concluded that some of the genes identified as differing in 3D spheroids could be new biomarkers or therapeutic targets in malignant mesothelioma, and that this needs further study.
• Limitations the authors state:
  • The five-fold cut-off for selecting genes was arbitrary and may have missed important biological changes.
  • Spheroids do not model some features of malignant mesothelioma, including the influence of stroma and immune cells.

Topics

Medical terms in this study, explained in our Lexicon: Mesothelioma, Tumor Microenvironment, Cytokine, Apoptosis.

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