A mouse model of disseminated mantle cell lymphoma highlights a lack of activity of estrogen receptor β agonists toward tumor burden

Mantle B lymphoma (MCL) is a rare, still incurable, aggressive malignant hemopathy of mature B cells [1]. In the bone marrow and secondary lymphoid organs, tumor cells are connected with their environment (stromal cells, endothelial cells, osteoblasts, osteoclasts, immune cells, etc.) by cytokines, adhesion proteins, and their respective receptors [2]. These interactions are responsible for a mechanism known as cell adhesion-mediated drug resistance (CAM-DR) [3]. This is why current therapies attempt to target both tumor cells and the tumor microenvironment (TME). Estrogens regulate numerous physiological processes through their binding to estrogen receptors (ER) of a and b types. However, while activated ERa enhances cell proliferation, activated ERb inhibits cell proliferation and induces apoptosis. Normal and malignant immune cells express both types of ER but predominantly ERb [4]. This property has been successfully used to selectively inhibit growth and induce apoptosis in an in vivo model of lymphoma [5]. In this study, the Granta-519 mantle cell lymphoma (MCL) cell line was engrafted subcutaneously (s.c.) into nude mice. Treatment of mice with diarylpropionitrile (DPN) led to the inhibition of tumor cell growth. The biological effects of DPN were mediated by the inhibition of B-cell activating factor (BAFF) and growth factor receptor-bound protein 7(GBR7) survival factors, and vascular endothelial growth factor (VEGF), an angiogenic factor. According to the dependency of MCL cells with their TME, we suspected that MCL tumor cells-stroma interactions would not be the same for MCL cells engrafted s.c. or in their physiological niche. We engineered four cell lines: JeKo1-mCherry-Luc, Z138-Luc, REC1-GFP-Luc, and Granta519-GFP from their respective parental cell lines. Cells were then injected intravenously (i.v.) into NODSCID IL2Rcnull (NSG) mice (n1⁄4 6). All animals recapitulated bona fide MCL disease. Tumor cells having luciferase activity were detected by bioluminescence imaging (BLI) with a kinetics dependent of each cell line: JeKo1 being the most aggressive and REC1 the less aggressive cells (Supplementary Figure S1). BLI examination indicated that tumor cells housed in hematopoietic organs (bone marrow, and spleen) and, in some cases, in nonhematopoietic organs (liver, lung, and kidney). The identity of tumor cells was verified at the time of mice euthanasia by cytometry analysis of CD20þ cells purified from infiltrated organs (Table 1). We analyzed the expression of ERa and b on a panel of MCL cells both at the mRNA and protein levels. ERa mRNAs were absent or very low in all cell lines (data not shown) according to previous data [5]. In contrast, ERb was expressed in all MCL cell lines (including GFP/mCherry/Luc expressing JeKo1, Granta519, REC1, and Z138 cells) tested although at various levels (Figure 1(A)). We next assessed in vivo the effects of both DPN and ERB-041 (or 2-(3-Fluoro-4-hydroxyphenyl)-7)vinyl-1,3-benzoxazol-5-ol), another selective ERb agonist [6], on MCL dissemination and growth. Z138-Luc or REC1-GFP-Luc cells were injected in NSG mice. The same day, mice were separated into three groups (n1⁄4 3) receiving: vehicle (0.01% DMSO), DPN, or ERB-041 both at the concentration of 1mg/kg. Drugs and vehicle were injected intraperitoneally (i.p.) five days a week. BLI was recorded each week starting at day 7. Mice were euthanized at day 31 (Z138-Luc) or day 41 (REC1-GFP-Luc). As shown in Figure 1(B), DPN or ERB-041 treatments did not modify