Rudisch Albin, Dewhurst Matthew Richard, Horga Luminita Gabriela, Kramer Nina, Harrer Nathalie, Dong Meng, van der Kuip Heiko, Wernitznig Andreas, Bernthaler Andreas, Dolznig Helmut, Sommergruber Wolfgang
Department of Lead Discovery, Boehringer Ingelheim RCV GmbH & Co KG, Vienna, Austria; Department of Microbiology, Immunobiology and Genetics, Center of Molecular Biology, Max F. Perutz Laboratories, University of Vienna, Vienna, Austria.
Department of Lead Discovery, Boehringer Ingelheim RCV GmbH & Co KG, Vienna, Austria.
PLoS One. 2015 Apr 28;10(4):e0124283. doi: 10.1371/journal.pone.0124283. eCollection 2015.
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.
我们建立了侵袭性或非侵袭性非小细胞肺癌（NSCLC）细胞系与各种类型成纤维细胞（FBs）的共培养体系，以更精确地表征肺癌中肿瘤-基质相互作用的分子机制。结果表明，HGF-MET-ERK1/2-CREB轴有助于Calu-1侵袭性表型的发生，其中HGF由FBs分泌。对各自单培养和共培养的差异表达分析显示，仅在与Calu-1的共培养中NFκB相关基因上调。基于细胞因子阵列和酶联免疫吸附测定（ELISA）对“细胞因子指纹”的表征确定，CSF2（GM-CSF）、CXCL1、CXCL6、血管内皮生长因子（VEGF）、白细胞介素6（IL6）、调节激活正常T细胞表达和分泌的趋化因子（RANTES）和白细胞介素8（IL8）在各种共培养中特异性上调。尽管CXCL6表现出严格的FB类型特异性诱导模式，而与肿瘤细胞系的侵袭性无关，但CSF2仅在侵袭性细胞系的共培养中被诱导，而与配对的FB类型无关。这些培养揭示了CSF2的诱导与癌细胞系的上皮-间质转化（EMT）特征之间的明确联系。结果表明，FBs而非肿瘤细胞中的经典NFκB信号传导负责诱导性和组成性CSF2表达。除CSF2外，细胞因子IL6、IL8和白细胞介素1β（IL1B）以及趋化因子CXCL1和CXCL6转录本在共培养的FBs中也显示增加。相反，它们的诱导并不严格依赖于共培养肿瘤细胞的侵袭性。在多报告基因分析中，发现与Calu-1共培养的FBs中其他信号通路（激活蛋白-1（AP-1）、缺氧诱导因子1α（HIF1-α）、 Kruppel样因子4（KLF4）、特异性蛋白1（SP-1）和 Elk1转录因子（ELK-1））被诱导。最重要的是，就所使用的FBs类型而言，未观察到这六种信号通路的诱导水平有差异。最后，在肿瘤与成纤维细胞相互作用时，FBs中大量诱导的趋化因子如CXCL1和CXCL6可能是导致在transwell实验中单核细胞系（THP-1）募集增加的原因。
