Abstract
BackgroundNuclear factor-kappa B (NF-kappaB) signaling is an important link between inflammation and peritoneal carcinomatosis in human ovarian cancer. Our objective was to track NF-kappaB signaling during ovarian cancer progression in a syngeneic mouse model using tumor cells stably expressing an NF-kappaB reporter.MethodsID8 mouse ovarian cancer cells stably expressing an NF-kappaB-dependent GFP/luciferase (NGL) fusion reporter transgene (ID8-NGL) were generated, and injected intra-peritoneally into C57BL/6 mice. NGL reporter activity in tumors was non-invasively monitored by bioluminescence imaging and measured in luciferase assays in harvested tumors. Ascites fluid or peritoneal lavages were analyzed for inflammatory cell and macrophage content, and for mRNA expression of M1 and M2 macrophage markers by quantitative real-time RT-PCR. 2-tailed Mann-Whitney tests were used for measuring differences between groups in in vivo experiments.ResultsIn ID8-NGL cells, responsiveness of the reporter to NF-kappaB activators and inhibitors was confirmed in vitro and in vivo. ID8-NGL tumors in C57BL/6 mice bore histopathological resemblance to human high-grade serous ovarian cancer and exhibited similar peritoneal disease spread. Tumor NF-kappaB activity, measured by the NGL reporter and by western blot of nuclear p65 expression, was markedly elevated at late stages of ovarian cancer progression. In ascites fluid, macrophages were the predominant inflammatory cell population. There were elevated levels of the M2-like pro-tumor macrophage marker, mannose-receptor, during tumor progression, and reduced levels following NF-kappaB inhibition with thymoquinone.ConclusionsOur ID8-NGL reporter syngeneic model is suitable for investigating changes in tumor NF-kappaB activity during ovarian cancer progression, how NF-kappaB activity influences immune cells in the tumor microenvironment, and effects of NF-kappaB-targeted treatments in future studies.
Highlights
Nuclear factor-kappa B (NF-kappaB) signaling is an important link between inflammation and peritoneal carcinomatosis in human ovarian cancer
Generation of ID8-NF-kappaB-dependent green fluorescent protein (GFP)/luciferase (NGL) cells Mouse epithelial ovarian cancer (ID8) cells are a wellestablished cell line derived from C57BL/6 mice that are routinely used in syngeneic mouse models of ovarian cancer [4,11,27,28]
Effects of stimulation or inhibition of Nuclear factor-kappaB (NF-κB) can be quantified in ID8 mouse ovarian cancer cells stably expressing the NF-κB reporter (ID8-NGL) Mouse epithelial ovarian cancer (ID8) cells are a wellestablished cell line derived from C57BL/6 mice that are routinely used in syngeneic mouse models of ovarian cancer [4,11,27,28]
Summary
Nuclear factor-kappa B (NF-kappaB) signaling is an important link between inflammation and peritoneal carcinomatosis in human ovarian cancer. NF-κB signaling is known to play an important role in several malignancies, including ovarian cancer [9,12,13,14,15,16]. There are five known NF-κB subunits in humans: NF-κB 1 (p105/p50), NF-κB 2 (p100/p52), RelA (p65), RelB and c-Rel [17]. Activation of the classical NF-κB cascade is initiated by growth factors, microbes, cytokines and genotoxic stress, which in turn activate the IκB kinase (IKK) complex. The p50-RelA dimer translocates to the nucleus, where it binds to the promoter/enhancer regions of genes involved in the regulation of cell growth, apoptosis and inflammation [16]
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