Abstract B31: Death of endothelial cells induced by paracrine exposure to factors derived from murine melanoma cells upon interferon-beta gene therapy
Authors: Vieira Igor de Luna , Tamura Rodrigo Esaki , Strauss Bryan Eric 
Published: May 2019

Abstract
Tumor vasculature plays a central role in tumor progression, making it a therapeutic target. Inhibition of angiogenesis has the potential to slow down tumor progression and inhibit metastasis. Interferon-beta (IFN) is a cytokine that shows antitumor effects, including immune activation. In addition, IFN inhibits angiogenesis by reducing the production of proangiogenic factors and by inducing death of endothelial cells. Gene therapy is showing significant results in cancer treatment including when recombinant adenoviral vectors are used for the delivery of therapeutic genes. In our previous studies, we developed a specialized adenoviral vector for the delivery of IFN to p53-positive cells. We propose that the impact of our cancer gene therapy approach may include the inhibition of angiogenesis. Here we explore the response of endothelial cells exposed to IFN through distinct routes of delivery. The efficient transduction of tEnd cells, a murine endothelial cell line, was confirmed by detection of GFP encoded by the control vector (Ad-GFP). In addition, tEnd cells readily produced IFN, as measured by ELISA, after treatment with Ad-IFN. Unexpectedly, IFN gene transfer did not alter angiogenic characteristics, such as migration or tube formation as evidenced in wound healing and tubulogenesis assays, respectively. Our results also indicate that the gene transfer of IFN does not impact cell viability since treatment did not provoke the accumulation of hypodiploid cells, was not associated with AnnexinV staining, and did not impact clonogenesis or MTT staining. These data indicate that expression of IFN by the transduced endothelial cells may not have any effect, a surprise given IFN's well-known ability to inhibit tumor angiogenesis. We next sought to investigate the impact of IFN treatment in a model that mimics certain aspects of the tumor microenvironment. In the first set of assays, B16 (mouse melanoma, p53wt) tumor cells were transduced, washed to remove excess virus particles, and only after were they cocultivated with tEnd cells, permiting the direct contact of adenoviral transduced tumor cells and non-treated endothelial cells. In order to differentiate the two cell lines, we used tEnd cells marked with the dTomato fluorescent protein (which we call tEndTO). Strikingly, the number of tEndTO cells was drastically reduced in the presence of B16 cells treated with Ad-IFN, suggesting that treated tumor cells promote a paracrine effect that inhibits tEnd cells. This point was further explored by exposing tEnd cells to conditioned medium derived from B16 cells that had been transduced with the recombinant adenoviral vectors before analysis of the viability of the endothelial cells. We observed significantly reduced colony formation and MTT staining accompanied by greatly increased accumulation of hypodiploid cells as well as AnnexinV staining when the tEnd cells were cultivated in conditioned medium derived from Ad-IFN transduced tumor cells, but not Ad-GFP. These data suggest that the exposure of tEnd to the secretome of Ad-IFN tranduced B16 cells results in the death of the endothelial cells in a paracrine fashion. In the context of gene therapy for melanoma, the use of modified adenoviral vectors carrying IFN as the therapeutic gene may be of great interest. Our results show a remarkable paracrine effect on endothelial cells when they are exposed to factors produced by melanoma cells transduced with Ad-IFN. Currently, we are continuing our evaluation of the use of these vectors to inhibit melanoma angiogenesis to cause retardation of tumor growth, though special attention will be given to the differential response associated with the route of IFN delivery and the possibility that additional factors are required to bring about endothelial cell death.
Citation Format: Igor de Luna Vieira, Rodrigo Esaki Tamura, Bryan Eric Strauss. Death of endothelial cells induced by paracrine exposure to factors derived from murine melanoma cells upon interferon-beta gene therapy [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr B31.