Recent studies have highlighted the AT1 receptor as a potential therapeutic target in breast cancer, while the role of the AT2 subtype in this disease has remained largely neglected. The present study describes the generation and characterization of a new cellular model of human invasive breast cancer cells (D3H2LN-AT2) stably expressing high levels of Flag-tagged human AT2 receptor (Flag-hAT2). These cells exhibit high-affinity binding sites for AngII, and total binding can be displaced by the AT2-selective antagonist PD123319 but not by the AT1-selective antagonist losartan. Of interest, high levels of expression of luciferase and green fluorescent protein make these cells suitable for bioluminescence and fluorescence studies in vitro and in vivo. We provide here a novel tool to investigate the AT2 receptor functions in breast cancer cells, independently of AT1 receptor activation. 1. Introduction Angiotensin II (AngII) is the biologically active peptide of the renin-angiotensin system, a well-known regulator of cardiovascular homeostasis and brain functions [1, 2]. Over the past ten years, a large number of studies have unraveled an additional role for the renin-angiotensin system in proliferative pathologies such as hyperplasia and cancer [3–8]. AngII binds to two major subtypes of receptors, namely, AT1 and AT2. It is generally admitted that AT1 receptor activation is responsible for most of the reported effects of AngII, whereas the AT2 receptor behaves as a negative regulator of AT1 signaling pathways [9–11]. Indeed, functional negative crosstalk between AT1 and AT2 receptors has been largely described in several physiopathological conditions including hypertension [1, 10, 11]. Activation of the AT1 receptor triggers a large number of intracellular kinases leading to modulation of cell proliferation, migration, and inflammation, three processes closely associated with tumor progression. Accordingly, several groups have shown that blocking AT1 receptors using specific receptor antagonists (ARBs) is effective in reducing tumor growth and metastasis in preclinical models [12–16]. Studies using knockout animals have further pointed out a role for stromal AT1 receptors from the host in tumor-associated macrophages infiltration and in cancer-related angiogenesis [17, 18]. A recent study [19] showing a dramatic overexpression of AT1 receptors in a subpopulation of invasive breast tumors has highlighted the potential use of ARBs as novel therapeutic agents against breast cancer. Indeed, it has been shown that losartan, an AT1 receptor blocker, was able
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