Abstract
We established co-cultures of invasive or non-invasive NSCLC cell lines and various types of fibroblasts (FBs) to more precisely characterize the molecular mechanism of tumor-stroma crosstalk in lung cancer. The HGF-MET-ERK1/2-CREB-axis was shown to contribute to the onset of the invasive phenotype of Calu-1 with HGF being secreted by FBs. Differential expression analysis of the respective mono- and co-cultures revealed an upregulation of NFκB-related genes exclusively in co-cultures with Calu-1. Cytokine Array- and ELISA-based characterization of the "cytokine fingerprints" identified CSF2 (GM-CSF), CXCL1, CXCL6, VEGF, IL6, RANTES and IL8 as being specifically upregulated in various co-cultures. Whilst CXCL6 exhibited a strictly FB-type-specific induction profile regardless of the invasiveness of the tumor cell line, CSF2 was only induced in co-cultures of invasive cell lines regardless of the partnered FB type. These cultures revealed a clear link between the induction of CSF2 and the EMT signature of the cancer cell line. The canonical NFκB signaling in FBs, but not in tumor cells, was shown to be responsible for the induced and constitutive CSF2 expression. In addition to CSF2, cytokine IL6, IL8 and IL1B, and chemokine CXCL1 and CXCL6 transcripts were also shown to be increased in co-cultured FBs. In contrast, their induction was not strictly dependent on the invasiveness of the co-cultured tumor cell. In a multi-reporter assay, additional signaling pathways (AP-1, HIF1-α, KLF4, SP-1 and ELK-1) were found to be induced in FBs co-cultured with Calu-1. Most importantly, no difference was observed in the level of inducibility of these six signaling pathways with regard to the type of FBs used. Finally, upon tumor fibroblast interaction the massive induction of chemokines such as CXCL1 and CXCL6 in FBs might be responsible for increased recruitment of a monocytic cell line (THP-1) in a transwell assay.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Cadherins / metabolism
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Carcinoma, Non-Small-Cell Lung / genetics
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Carcinoma, Non-Small-Cell Lung / metabolism*
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Carcinoma, Non-Small-Cell Lung / pathology*
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Cell Communication / drug effects
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Cell Line, Tumor
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Cytokines / metabolism
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Dermis / pathology
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Epithelial-Mesenchymal Transition / drug effects
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Epithelial-Mesenchymal Transition / genetics*
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Fibroblasts / drug effects
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Fibroblasts / metabolism*
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Gene Expression Profiling
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Gene Expression Regulation, Neoplastic / drug effects
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Gene Regulatory Networks / drug effects
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Genes, Reporter
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Granulocyte-Macrophage Colony-Stimulating Factor / metabolism*
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Hepatocyte Growth Factor / pharmacology
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Humans
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Inflammation / pathology
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Kruppel-Like Factor 4
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Lung Neoplasms / genetics
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Lung Neoplasms / metabolism
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Lung Neoplasms / pathology*
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NF-kappa B / metabolism*
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Neoplasm Invasiveness
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Phenotype
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Proto-Oncogene Proteins c-met / metabolism
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RNA, Messenger / genetics
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RNA, Messenger / metabolism
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Rats
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Signal Transduction / drug effects
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Signal Transduction / genetics
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Spheroids, Cellular / drug effects
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Spheroids, Cellular / metabolism
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Spheroids, Cellular / pathology
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Stromal Cells / metabolism
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Up-Regulation / drug effects
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Up-Regulation / genetics
Substances
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Cadherins
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Cytokines
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KLF4 protein, human
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Klf4 protein, rat
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Kruppel-Like Factor 4
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NF-kappa B
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RNA, Messenger
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Hepatocyte Growth Factor
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Granulocyte-Macrophage Colony-Stimulating Factor
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Proto-Oncogene Proteins c-met
Grants and funding
This work has been supported by the Innovative Medicines Initiative Joint undertaking under grant agreement n° 115188, resources of which are composed of financial contribution from the European Union’s Seventh Framework Programme (FP7/2007-2013) and EFPIA companies in kind contribution. Boehringer Ingelheim RCV GmbH & Co KG provided support in the form of salaries for authors AR, NH, AW, WS, MRD, LGH and AB but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.