PO.ET06.01 · 实验与分子治疗
利用诱导铁死亡的siRNA纳米疗法靶向胆管癌
Targeting cholangiocarcinoma with ferroptosis-inducing siRNA-based nanotherapeutics
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
背景：胆管癌（CCA）是一种源自肝胆管系统上皮细胞的高度致命恶性肿瘤。目前标准治疗的疗效有限。铁死亡是一种由铁催化、不依赖caspase、由磷脂过氧化驱动的细胞死亡方式，已成为一种新型治疗靶点。我们的目标是利用基于siRNA的纳米疗法，开发一种在CCA中靶向铁死亡的肿瘤选择性治疗策略。
材料与方法：以EpCAM适配体包被并装载靶向谷胱甘肽过氧化物酶4（siGPX4-lactosomes）和铁死亡抑制蛋白1（siFSP1-lactosomes）的siRNA的乳源纳米囊泡（lactosomes），用于在小鼠CCA细胞（FAC）以及同基因肝原位CCA荷瘤小鼠中诱导铁死亡。在体外，通过基于荧光的检测、细胞活力检测和分子生物学检测评估lactosome摄取及疗效。在体内，评估靶点结合、组织分布、安全性和疗效。
结果：EpCAM（表达于CCA细胞上的上皮细胞黏附分子）包被的lactosomes以EpCAM依赖的方式被FAC细胞强力摄取。通过siRNA敲低EpCAM可降低lactosome摄取，证实了适配体的选择性。剂量依赖性的siGPX4/siFSP1-lactosome处理导致GPX4/FSP1敲低，并在72小时后诱导多达60%的FAC细胞发生铁死亡（p<0.001，相较于siNC）。在CCA荷瘤小鼠中，静脉给予siRNA lactosomes耐受性良好，未观察到肝脏或全身毒性，并且相较于非肿瘤组织显示出强力的肿瘤分布。疗效研究表明，siGPX4/siFSP1-lactosomes显著降低瘤内Gpx4和FSP1 mRNA表达达40-60%，并诱导铁死亡标志物Ptgs2和Acsl4显著上调（p<0.01，相较于siNC）。通过对磷脂过氧化标志物oxPAPC和4-HNE的免疫组化，证实了siRNA-lactosome处理肿瘤中的铁死亡。此外，在siGPX4/siFSP1-lactosome处理的肿瘤中，增殖标志物Ki67显著下调（p<0.05，相较于siNC），表明存在铁死亡介导的肿瘤抑制。重要的是，与对照小鼠相比，siGPX4/siFSP1-lactosomes降低了处理小鼠的肿瘤负荷及CCA标志物CK19/7的表达。
结论：靶向CCA的基于siRNA的纳米疗法能够在体外和体内有效且安全地诱导铁死亡，代表着一种潜在的CCA治疗策略。
查看英文原文 English abstract
Background : Cholangiocarcinoma (CCA) is a highly lethal epithelial cell malignancy of the hepatic biliary tract. Efficacy of current standard-of-care therapy is limited. Ferroptosis, an iron-catalyzed, caspase-independent cell death driven by phospholipid peroxidation, has emerged as a novel therapeutic target. Our goal is to develop a tumor-selective therapeutic approach targeting ferroptosis in CCA using siRNA-based nanotherapeutics.
Materials & Methods : Milk-derived nanovesicles (lactosomes) coated with EpCAM aptamer and packaged with siRNA against Glutathione Peroxidase 4 (siGPX4-lactosomes) and Ferroptosis Suppressor Protein 1 (siFSP1-lactosomes), were used to induce ferroptosis in murine CCA cells (FAC) and in syngeneic liver orthotopic CCA-bearing mice. In vitro , lactosome uptake and efficacy were assessed by fluorescence-based, cell-viability and molecular biology assays. In vivo , target engagement, tissue distribution, safety and efficacy were assessed.
Results : EpCAM (epithelial cell adhesion molecule expressed on CCA cells)-coated lactosomes showed robust uptake by FAC cells in a EpCAM-dependent manner. EpCAM knockdown via siRNA reduced lactosome uptake, confirming aptamer selectivity. Dose-dependent siGPX4/siFSP1-lactosome treatment led to GPX4/FSP1 knockdown and induced ferroptotic cell death in up to 60% of FAC cells after 72 hours (p<0.001 vs. siNC). In CCA-bearing mice, intravenous administration of siRNA lactosomes was well-tolerated with no liver or systemic toxicity observed and demonstrated robust tumor distribution compared to non-tumorous tissues. Efficacy studies demonstrated that siGPX4/siFSP1-lactosomes significantly reduced intratumor Gpx4 and FSP1 mRNA expression by 40-60% and induced significant upregulation of ferroptosis markers Ptgs2 and Acsl4 (p<0.01 vs. siNC). Ferroptosis in siRNA-lactosome treated tumors was confirmed by immunohistochemistry for phospholipid peroxidation markers oxPAPC and 4-HNE. Additionally, proliferation marker Ki67 was significantly downregulated in siGPX4/siFSP1-lactosome treated tumors (p<0.05 vs. siNC), indicating ferroptosis-mediated tumor suppression. Importantly, siGPX4/siFSP1-lactosomes reduced tumor burden and expression of CCA markers CK19/7 in treated mice, as compared to control mice.
Conclusions : siRNA-based nanotherapeutics targeting CCA can effectively and safely induce ferroptosis in vitro and in vivo , representing a potential therapeutic strategy for CCA treatment.
利益披露 Disclosure
P. Classon, None.