Multifunctional siRNA/antibody nanocarriers to treat metastatic triple-negative breast cancer

项目摘要/摘要 三阴性乳腺癌(TNBC)是一种侵袭性疾病，生长迅速，转移早。 目前尚无有效的治疗转移性TNBC的方法，因此需要新的策略。我们将满足这一需求 使用我们开发的纳米颗粒(NP)来干扰Wnt信号，这是TNBC背后的关键驱动力 生长和转移。Wnt信号的主要效应者是β-catenin，当Wnt-catenin被激活时， WNT配体结合转录因子7(FZD7)受体，该受体在TNBC细胞上相对正常细胞过度表达。 活性β-连环蛋白通过扩增支持的基因表达促进肿瘤细胞生长和转移 细胞的存活、增殖、干细胞样行为、迁移和入侵。我们的中心假设是 在两个水平上抑制Wnt信号将降低TNBC细胞的转移潜力，因此我们创建了Wnt 包被FZD7抗体和β-连环蛋白小干扰RNA的抑制性NPs 这项任务。FZD7抗体使TNBC细胞结合，并阻止Wnt信号被激活 阻断WNT配体与FZD7的结合。SiRNA通过直接抑制β，进一步抑制Wnt信号转导。 通过RNA干扰实现连锁素。我们的初步研究表明，这些NPs可以进入现有的TNBC 在小鼠肺转移模型中抑制其生长。在拟议的工作中，我们将阐明 通过研究它们与TNBC细胞的体外相互作用，更详细地研究了这些纳米粒的作用机制(AIM 1)，验证它们能够穿透小鼠现有的转移瘤以阻止其生长(目标2)，并表明它们 可以安全地防止免疫能力强的荷瘤小鼠形成新的转移(目标3)。全 AIMS将比较共同传递FZD7抗体和β-连环蛋白siRNAs的NPs和传递任一试剂的NPs 以揭示它们的联合作用是相加的还是协同的。除了研究 NPS对TNBC细胞的影响，我们还将评估其对乳腺、肺和非肿瘤细胞的影响 以确认它们具有可以忽略不计的脱靶效应。我们将在目标3号进一步研究它们的安全性。 通过监测动物体重、血液化学和主要器官组织病理学。我们预计会发现这些 NPS可以抑制TNBC细胞中的Wnt信号，从而降低其转移潜能，而不影响非 癌细胞，使它们能够被用作工具来治疗现有的转移或防止形成 新的转移瘤。我们还预计，共同传递FZD7抗体和β-连环蛋白siRNA的NPs将会有更多 比单独提供任何一种代理的NP更有效。综上所述，本项目将评估WNT抑制 NPS作为治疗和/或预防TNBC转移的工具。与目前的治疗方法不同，目前的治疗方法具有不可预测的和 如果结果不足，我们的NPs将直接靶向TNBC细胞，并通过以下方式降低其生长和转移能力 抑制WNT信号。如果成功，这种疗法将提高患者的存活率和生活质量 转移性TNBC，并最终可能通过以下方式扩展到具有不同分子驱动因素的其他癌症 改变与核糖核酸一起传递的抗体和siRNA。

PROJECT SUMMARY/ABSTRACT Triple-negative breast cancer (TNBC) is an aggressive disease that grows quickly and metastasizes early. There are no effective therapies for metastatic TNBC, so new strategies are needed. We will meet this need using nanoparticles (NPs) we developed that interfere with Wnt signaling, the key driving force behind TNBC growth and metastasis. The main effector of Wnt signaling is β-catenin, and it is activated in TNBC cells when wnt ligands bind frizzled7 (FZD7) receptors that are overexpressed on TNBC cells relative to normal cells. Active β-catenin promotes TNBC growth and metastasis by amplifying the expression of genes that support cell survival, proliferation, stem-like behavior, migration, and invasion. Our central hypothesis is that suppressing Wnt signaling at two levels will reduce TNBC cells’ metastatic potential, so we have created Wnt inhibitory NPs coated with both FZD7 antibodies and β-catenin small interfering RNAs (siRNAs) to accomplish this task. The FZD7 antibodies enable TNBC cell binding and prevent Wnt signaling from being activated by blocking wnt ligands from binding FZD7. The siRNAs further suppress Wnt signaling by directly inhibiting β- catenin through RNA interference. Our preliminary studies indicate that these NPs can enter existing TNBC lung metastases in mouse models to inhibit their growth. In the proposed work we will elucidate the mechanism of action of these NPs in greater detail by studying their interaction with TNBC cells in vitro (Aim 1), validate that they can penetrate existing metastases in mice to halt their growth (Aim 2), and show that they can safely prevent the formation of new metastases in immune competent tumor-bearing mice (Aim 3). All aims will compare NPs that co-deliver FZD7 antibodies and β-catenin siRNAs to NPs that deliver either agent individually in order to reveal if their combined effects are additive or synergistic. In addition to studying the NPs’ impact on TNBC cells, we will also evaluate their effect on non-cancerous cells from breast, lung, and bone tissue in order to confirm they have negligible off-target effects. We will further study their safety in Aim 3 by monitoring animal weight, blood chemistry, and major organ histopathology. We expect to find that these NPs can inhibit Wnt signaling in TNBC cells to reduce their metastatic potential without impacting non- cancerous cells, enabling them to be used as tools to either treat existing metastases or prevent formation of new metastases. We also expect for NPs that co-deliver FZD7 antibodies and β-catenin siRNAs to be more effective than NPs that deliver either agent individually. In summary, this project will evaluate Wnt inhibitory NPs as tools to treat and/or prevent TNBC metastasis. Unlike current therapies, which have unpredictable and insufficient results, our NPs will directly target TNBC cells and reduce their ability to grow and metastasize by suppressing Wnt signaling. If successful, this therapy will improve survival and quality of life for patients with metastatic TNBC, and it may ultimately be expanded to other cancers with different molecular drivers by altering the antibodies and siRNAs that are delivered with the NPs.