title: Tracking NF-κB activity in tumor cells during ovarian cancer progression in a syngeneic mouse model.
abstract: BACKGROUND
  Nuclear 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.
  
  
  METHODS
  ID8 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.
  
  
  RESULTS
  In 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.
  
  
  CONCLUSIONS
  Our 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.
authors.affiliation: Department of Obstetrics and Gynecology, Division of Gynecologic Oncology, Vanderbilt University Medical Center, B1100 Medical Center North, Nashville, TN 37232, USA. andrew.j.wilson@vanderbilt.edu.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Wilson
authors.forename: Whitney
authors.initials: W
authors.lastname: Barham
authors.forename: Jeanette
authors.initials: J
authors.lastname: Saskowski
authors.forename: Oleg
authors.initials: O
authors.lastname: Tikhomirov
authors.forename: Lianyi
authors.initials: L
authors.lastname: Chen
authors.forename: Hye-Jeong
authors.initials: HJ
authors.lastname: Lee
authors.forename: Fiona
authors.initials: F
authors.lastname: Yull
authors.forename: Dineo
authors.initials: D
authors.lastname: Khabele
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