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Title:Understanding the role of interleukin-8 (IL-8) in canine osteosarcoma metastasis
Author(s):Dowling, Matthew Forrest
Advisor(s):Fan, Timothy M.
Contributor(s):Fries, Ryan; Keating, Stephanie
Department / Program:Vet Clinical Medicine
Discipline:VMS-Veterinary Clinical Medcne
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):Canine Osteosarcoma Metastasis Interleukin-8 IL-8 Cancer
Abstract:Interleukin-8 (IL-8) is a chemokine whose primary function is to regulate neutrophil chemotaxis and modulate the inflammatory response in normal physiologic conditions. Additionally, IL-8 appears to play an important role in tumorigenesis and metastasis in vitro and in vivo in human solid tumors. There is growing evidence to suggest that IL-8 can serve as a biomarker for patients with cancer, with high IL-8 levels often correlated with advanced stage disease, metastasis, and poorer overall outcome. Though IL-8 appears to be intimately associated with cancer progression and metastasis in human, the role of IL-8 in veterinary oncology is largely unexplored. Osteosarcoma (OS) is a common, naturally-occurring, highly metastatic cancer in dogs that serves as an excellent model for the disease in people. Recent comparative genomics indicate that IL-8 expression is conserved in both human and canine OS and is a negative prognostic indicator in people with this cancer. Therefore, understanding how IL-8 impacts disease progression, specifically in regards to the development of metastasis, could help us more effectively manage OS in both dogs and people. In this study, we hypothesized 1) that canine OS cells will express IL-8 and its cognate receptors, CXCR1 and CXCR2, 2) that inhibition of IL-8 signaling will attenuate proliferation and migration, 3) that inhibition of IL-8 signaling will enhance sensitivity to cytotoxic agents, and 4) that IL-8 signaling will influence the expression of genes involved necessary for angiogenesis. We investigated CXCR1 and CXCR2 gene transcription with qualitative polymerase chain reaction (PCR), and we investigated protein expression for IL-8, CXCR1, and CXCR2 with Western blotting. We evaluated OS cell production of IL-8 with an ELISA. We employed a competitive CXCR1/CXCR2 antagonist (Reparixin), as well as an IL-8 neutralizing monoclonal antibody (mAb), to mitigate IL-8 signaling in cell lines. Fluorescent cellular metabolic assays were used to determine the effects of manipulation of the IL-8 signaling axis on cellular proliferation and on chemosensitization to a platinum chemotherapeutic (carboplatin). Scratch assays were used to assess cellular migration following exposure to one of the IL-8 inhibitory agents. Quantitative PCR was again used to evaluate the gene transcription of VEGF in OS cell lines and a canine endothelial cell line following manipulation of the IL-8 signaling axis. Gene transcription of the IL-8 receptors was noted in six OS cell lines. Furthermore, we were able to demonstrate protein expression of IL-8 in all cell lines. IL-8 was produced in a cell density-dependent manner in all cell lines examined. We showed that the addition of exogenous IL-8 led to a dose-dependent increase in proliferation in an OS cell line that was a low producer of IL-8. IL-8 receptor blockade appeared to have no impact on proliferation at the doses examined. IL-8 receptor blockade also did not enhance sensitivity to carboplatin in vitro. However, receptor blockade did appear to inhibit migration in a dose-dependent manner in the low-producer of IL-8. IL-8 neutralization with a mAb had not effect on proliferation, migration, or chemosensitization. We were also able to show that addition of IL-8 increased VEGF gene transcription. These findings suggest that autocrine/paracrine IL-8 signaling may be more important in promoting a more metastatic cellular phenotype in those OS cells that are low-producers of this chemokine. Additionally, our results suggest that IL-8 likely plays a role in cultivating a pro-angiogenic tumor microenvironment.
Issue Date:2018-06-22
Rights Information:Copyright 2018 Matthew Dowling
Date Available in IDEALS:2018-09-27
Date Deposited:2018-08

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