Reduction of Fibrosis and Immune Suppressive Cells in ErbB2-Dependent Tumorigenesis by an LXR Agonist. Reduction of Fibrosis and Immune Suppressive Cells in ErbB2-Dependent Tumorigenesis by an LXR Agonist

One of the central challenges for cancer therapy is the identification of factors in the tumor microenvironment that increase tumor progression and prevent immune surveillance. One such element associated with breast cancer is stromal fibrosis, a histopathologic criterion for invasive cancer and poor survival. Fibrosis is caused by inflammatory factors and remodeling of the extracellular matrix that elicit an immune tolerant microenvironment. To address the role of fibrosis in tumorigenesis, we developed NeuT/ATTAC transgenic mice expressing a constitutively active NeuT/erbB2 transgene, and an inducible, fat-directed caspase-8 fusion protein, which upon activation results in selective and partial ablation of mammary fat and its replacement with fibrotic tissue. Induction of fibrosis in NeuT/ATTAC mice led to more rapid tumor development and an inflammatory and fibrotic stromal environment. In an effort to explore therapeutic options that could reduce fibrosis and immune tolerance, mice were treated with the oxysterol liver X receptor (LXR) pan agonist, N,N-dimethyl-3ꞵ-hydroxy-cholenamide (DMHCA), an agent known to reduce fibrosis in non-malignant diseases. DMHCA reduced tumor progression, tumor multiplicity and fibrosis, and improved immune surveillance by reducing infiltrating myeloid-derived suppressor cells and increasing CD4 and CD8 effector T cells. These effects were associated with an LXR-dependent gene network coupled to reduction of PTGS2, CCL5, S100A9, SPP1, ANXA1 and CD14. These findings suggest that the use of DMHCA may be a potentially effective approach to reduce desmoplasia and immune tolerance and increase the efficacy of cancer therapy. Overall design: Mammary gland fibrosis was induced in female six-week-old NeuT/ATTAC mice by i.p. injection of 0.4 mg/kg AP20187 (MedChemExpress) dissolved in a vehicle (4% ethanol, 10% PEG-400 and 1.75% Tween-20 in water) three times per week, and are hereafter referred to as ‘NeuT/ATTAC+AP’ mice. AP20187 is a dimer analog of FK506 and serves as a selective FKBPv-caspase 8 dimerizer resulting in partial ablation of mammary fat and its replacement by fibrotic tissue. At eight weeks of age, NeuT/ATTAC+AP mice were administered ad libitum a diet (LabDiet 5053) supplemented with 0.05% (w/w) DMHCA (WuXi App Tec, China), which is equivalent to a dose of ~100 mg/kg. No weight loss or overt toxicity resulted from AP21087 or DMHCA treatment.

癌症治疗的核心挑战之一，在于鉴定肿瘤微环境中能够促进肿瘤进展、抑制免疫监视的各类调控因子。与乳腺癌相关的这类因子之一为基质纤维化（stromal fibrosis），它是浸润性癌的组织病理学特征，同时与不良预后密切相关。
纤维化由炎症因子及细胞外基质重塑所诱发，可形成免疫耐受的肿瘤微环境。为探究纤维化在肿瘤发生中的作用，我们构建了NeuT/ATTAC转基因小鼠：该小鼠同时表达组成型激活的NeuT/erbB2转基因，以及可诱导的脂肪靶向半胱天冬酶-8（caspase-8）融合蛋白；当该融合蛋白被激活时，可选择性且部分清除乳腺脂肪组织，并代之以纤维化组织。
在NeuT/ATTAC小鼠中诱导纤维化，可加速肿瘤发生，并形成炎症性、纤维化的基质微环境。为探索可减轻纤维化与免疫耐受的治疗方案，我们使用氧固醇类肝X受体（liver X receptor, LXR）全激动剂N,N-二甲基-3β-羟基胆烯酰胺（N,N-dimethyl-3ꞵ-hydroxy-cholenamide, DMHCA）对小鼠进行处理——该药物已被证实可在非恶性疾病中减轻纤维化。
DMHCA可减缓肿瘤进展、降低肿瘤多发率与纤维化程度，并通过减少浸润性髓系来源的抑制性细胞（myeloid-derived suppressor cells）、增加CD4与CD8效应T细胞，从而改善免疫监视功能。上述效应与依赖LXR的基因网络相关，该网络伴随PTGS2、CCL5、S100A9、SPP1、ANXA1与CD14的表达下调。
本研究结果表明，DMHCA或可成为一种潜在有效的手段，以减轻肿瘤间质增生（desmoplasia）与免疫耐受，进而提升癌症治疗的效果。
整体实验设计：对6周龄雌性NeuT/ATTAC小鼠进行腹腔注射，每周3次，注射剂量为0.4 mg/kg的AP20187（MedChemExpress公司产品，溶解于溶剂：4%乙醇、10% PEG-400与1.75%吐温-20的水溶液），以此诱导乳腺纤维化，此类小鼠后续被称为'NeuT/ATTAC+AP'小鼠。AP20187为FK506的二聚体类似物，可作为选择性FKBPv-caspase 8二聚化诱导剂，引发乳腺脂肪组织的部分清除，并代之以纤维化组织。在小鼠8周龄时，对NeuT/ATTAC+AP小鼠自由喂食添加了0.05%（w/w）DMHCA（药明康德（WuXi App Tec, 中国）生产）的纯化饲料（LabDiet 5053），该给药剂量等效于约100 mg/kg。AP21087与DMHCA处理均未引发体重下降或明显毒性。