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    2020-08-18

    r> Methods: MDA-MB-231 cells, control or overexpressing Cx43, were used in this study. Proliferation and invasion assays were performed. Quantitative PCR, ELISA and western blotting were performed to assess the regulation of inflammatory mediators and other factors upon Av treatment. Immunofluorescence was performed to document the translocation of Nuclear Factor-kappa B p65.
    Results: Breast cancer tissues had elevated transcriptional levels of inflammatory mediators. Av treatment in-creased expression levels of inflammatory mediators and metastatic factors in vitro and in vivo. Interestingly, overexpressing Cx43 in MDA-MB-231 Galactose1phosphate alleviated the inflammatory effects induced by Av treatment. Conclusion: This study attributes Av refractoriness to the Av therapy-induced inflammatory microenvironment.
    1. Introduction
    Triple negative breast cancer (TNBC), high-grade invasive ductal carcinoma, is characterized by the lack of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor re-ceptor 2 (HER2) [1–3] rendering it refractory to targeted therapies [1,2,4–6]. TNBCs, though heterogeneous, are mostly high-grade in-vasive ductal carcinomas that frequently affect younger patients and follow an aggressive treatment course [1,2,4–6]. TNBC is refractory to the effective targeted therapy used in luminal and HER2-positive breast
    Massive data support a central role for angiogenesis in breast cancer growth and metastasis. Vascular endothelial growth factor (VEGF) is a major regulator of angiogenesis. Avastin® (Av) is an anti-VEGF agent that has been FDA approved for the treatment of various cancers in-cluding breast cancer [8,9]. Despite being an effective treatment at early stages, Av does not improve overall survival since patients de-velop resistance [10,11]. It is crucial to understand the mechanism of Av refractoriness to improve its clinical outcomes. Tumors bypass an-tiangiogenic therapies, such as Av therapy, via a variety of mechanisms
    Abbreviations: Cx43, connexin 43; VEGF, vascular endothelial growth factor; RAGE, receptor of advanced glycation end products; IL-1β, interleukin-1β; TNF-α, tumor necrosis factor-α; NF-κB, nuclear factor-κB; MMP, matrix metalloproteinase; CD44, cluster Galactose1phosphate of differentiation 44; GFP, green fluorescent protein; FFPE, for-malin-fixed paraffin-embedded; IκB-α, inhibitor of kappa B; PVDF, polyvinylidene fluoride; PBS, phosphate buffered saline
    Corresponding author.
    2 Current address: Department of Biochemistry/Goodman Cancer Research Centre, McGill University, Montréal, Québec, Canada H3G 1Y6.
    [12]. Av induces extracellular matrix (ECM) remodeling due to induced hypoxia [13]. It also increases inflammation in diabetic mice [14]. Hypoxia and inflammation are major culprits during cancer progression and are intimately linked [15–17]. In inflammatory breast cancer (IBC), the most aggressive form of breast cancer, intermittent hypoxia acti-vating nuclear factor kappa B (NF-κB) pathway is behind its high me-tastatic potential [18]. The inflammatory mediators in the tumor mi-croenvironment promote growth and metastasis by inducing epithelial-to-mesenchymal transition (EMT) of non-transformed breast epithelial cells [19]. Cancer cells communicate with endothelial cells via para-crine and heterocellular communication, making both VEGF and gap junctions (GJs) crucial in extravasation and metastasis [20,21]. GJs are channels allowing the exchange of metabolites and electric signals be-tween adjacent cells. They are made of connexin proteins which can form non-junctional hemichannels that allow the paracrine interaction with the extracellular milieu [22–24]. VEGF was found to mediate autocrine regulation of myocyte Cx43 [25]. Moreover, hypoxia dysre-gulates Cx43 by modulating NF-κB activity [26]. In fact, over-expres-sion of Cx43 in breast cancer cells reversed their malignant properties 
    and suppressed tumor growth and angiogenesis in vivo [27–29].
    The aim of this study was to investigate the role of the inflammatory milieu in refractoriness to Av and to evaluate if Cx43 overexpression in MDA-MB-231, a highly metastatic breast cancer cell line, would at-tenuate the inflammatory effects of Av in vitro and in vivo.
    2. Materials and methods
    MDA-MB-231, a highly metastatic triple-negative mammary ade-nocarcinoma cell line, MDA-MB-231 and Cx43-Dendra (MDA-Cx43D, overexpressing Cx43) were used in this study [30].
    2.2. Lentiviral transduction and cell sorting
    MDA-Cx43D cells are MDA-MB-231 cells overexpressing Cx43 in fusion with Dendra, a photo-convertible fluorescent protein from green to red. To generate MDA-Cx43D cells, a pCSCW-Cx43-Dendra2 lentiviral