Changes in Global Gene Expression Associated with 3D Structure of Tumors: An Ex Vivo Matrix-Free Mesothelioma Spheroid Model

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.

[1]  D. Fennell,et al.  Bcl-2 family proteins contribute to apoptotic resistance in lung cancer multicellular spheroids. , 2009, American journal of respiratory cell and molecular biology.

[2]  S. Kjelleberg,et al.  Attenuation of Pseudomonas aeruginosa virulence by quorum sensing inhibitors , 2003, The EMBO journal.

[3]  F. Ajchenbaum‐Cymbalista,et al.  Involvement of BAFF and APRIL in the resistance to apoptosis of B-CLL through an autocrine pathway. , 2004, Blood.

[4]  K. Nagashima,et al.  The Development and Characterization of a Human Mesothelioma In Vitro 3D Model to Investigate Immunotoxin Therapy , 2011, PloS one.

[5]  T. Kipps,et al.  Nurselike cells express BAFF and APRIL, which can promote survival of chronic lymphocytic leukemia cells via a paracrine pathway distinct from that of SDF-1alpha. , 2005, Blood.

[6]  H. Kleinman,et al.  The basement membrane matrix in malignancy , 2003, The Journal of pathology.

[7]  M. Carbone,et al.  Molecular Pathways: Targeting Mechanisms of Asbestos and Erionite Carcinogenesis in Mesothelioma , 2011, Clinical Cancer Research.

[8]  Genee Y. Lee,et al.  The morphologies of breast cancer cell lines in three‐dimensional assays correlate with their profiles of gene expression , 2007, Molecular oncology.

[9]  P. Delafontaine,et al.  Intravenous hMSCs improve myocardial infarction in mice because cells embolized in lung are activated to secrete the anti-inflammatory protein TSG-6. , 2009, Cell stem cell.

[10]  Ru Wei,et al.  The M2 splice isoform of pyruvate kinase is important for cancer metabolism and tumour growth , 2008, Nature.

[11]  H. Hammad,et al.  Mesothelioma environment comprises cytokines and T-regulatory cells that suppress immune responses , 2006, European Respiratory Journal.

[12]  R K Jain,et al.  Transport of molecules, particles, and cells in solid tumors. , 1999, Annual review of biomedical engineering.

[13]  O. Warburg.  [Origin of cancer cells]. , 1956, Oncologia.

[14]  J. Glasner,et al.  Genome-wide expression profiling in Escherichia coli K-12. , 1999, Nucleic acids research.

[15]  H. Kleinman,et al.  Multiple uses of basement membrane‐like matrix (BME/Matrigel) in vitro and in vivo with cancer cells , 2011, International journal of cancer.

[16]  Mitchell Ho,et al.  In Vivo Imaging of Human Malignant Mesothelioma Grown Orthotopically in the Peritoneal Cavity of Nude Mice , 2011, Journal of Cancer.

[17]  H. Matsuzaki,et al.  Asbestos Induces Reduction of Tumor Immunity , 2011, Clinical & developmental immunology.

[18]  M. Rath,et al.  Modulation of MMP-2 and MMP-9 by cytokines, mitogens and inhibitors in lung cancer and malignant mesothelioma cell lines. , 2009, Oncology reports.

[19]  L. Bubendorf,et al.  ERK1/2 and p38 MAP kinase control MMP‐2, MT1‐MMP, and TIMP action and affect cell migration: A comparison between mesothelioma and mesothelial cells , 2006, Journal of cellular physiology.

[20]  Shuichi Takayama,et al.  High-throughput 3D spheroid culture and drug testing using a 384 hanging drop array. , 2011, The Analyst.

[21]  E. Cukierman,et al.  Physico-mechanical aspects of extracellular matrix influences on tumorigenic behaviors. , 2010, Seminars in cancer biology.

[22]  I. Tannock,et al.  Drug penetration in solid tumours , 2006, Nature Reviews Cancer.

[23]  Jeffrey R. Morgan,et al.  Mammalian Target of Rapamycin Contributes to the Acquired Apoptotic Resistance of Human Mesothelioma Multicellular Spheroids* , 2008, Journal of Biological Chemistry.

[24]  Nikolay Bazhanov,et al.  Aggregation of human mesenchymal stromal cells (MSCs) into 3D spheroids enhances their antiinflammatory properties , 2010, Proceedings of the National Academy of Sciences.

[25]  Mitchell Ho,et al.  Rapid Generation of In Vitro Multicellular Spheroids for the Study of Monoclonal Antibody Therapy , 2011, Journal of Cancer.