【作者】 周慧；

【导师】 高明远； 苗庆庆；

【作者基本信息】 苏州大学 ， 放射医学， 2023， 博士

【摘要】 研究背景:肿瘤相关成纤维细胞(cancer associated fibroblasts,CAFs)在肿瘤的发生发展和免疫抑制过程中发挥着重要的作用。为了提高抗肿瘤效果,重组CAFs和直接耗竭CAFs等CAFs靶向疗法正在被广泛地开发。然而,由于CAFs具有表型异质性、功能异质性以及生物标志物的非特异性表达等复杂的生物学行为,会影响这些系统性CAFs靶向治疗策略的效果甚至会引起严重的副作用。因此,发展CAFs的精准诊疗技术有助于提高CAFs靶向诊疗的效率和破译CAFs的底层生物学机制。然而,能够对CAFs进行精准诊疗的成像探针极少被开发出来。研究目的:为了实现对CAFs的精确监测和靶向治疗,本文开发了一种可激活的分子诊疗探针(FMP),其具有可激活的荧光、光声成像和光动力治疗(photodynamic therapy,PDT)性能,能够与肿瘤微环境中过表达的成纤维细胞活化蛋白α(fibroblast activation proteinα,FAPα)响应。研究方法:在第一部分,我们设计了两种FAPα可激活的分子诊疗探针,方法是用不同长度的自消除链将FAPα特异性识别的二肽底物与亚甲基蓝(methylene blue,MB)偶联,从而导致光活性(荧光、光声和光毒性)的猝灭。从水解速率和分子模拟两个方面,我们筛选出最佳的FAPα可激活的诊疗探针。随后,进行了一系列的体外和体内实验,以考察FAPα的反应性和肿瘤成像效果。在第二部分,激光照射与FAPα响应后的探针FMP,考察FMP的光动力性能和光热性能。随后,通过激光共聚焦成像和流式细胞术等方法研究FMP在细胞层面的光毒性作用和免疫原性细胞死亡(immunogenic cell death,ICD)作用。通过测量肿瘤体积、做病理切片、检测免疫指标等方法评估FMP的肿瘤治疗效果和免疫效应。采用血生化、血常规和病理切片等方法评估FMP的生物安全性。研究结果:第一部分工作中,在优化自消除连接链的长度后,筛选出最佳的FAPα可激活的诊疗探针FMP。体外和体内实验结果均表明,探针FMP对FAPα表现出高灵敏、高选择性的荧光和光声信号的增强效应。此外,FMP显示出良好的细胞膜渗透性,可用于监测FAPα阳性细胞和活体肿瘤的成像。第二部分工作中,首先,证明了被FAPα激活的探针FMP具有产生活性氧(reactive oxygen species,ROS)的性能。随后,细胞实验表明,FMP可靶向CAFs,并可在激光照射下产生致命水平的ROS以杀死CAFs。此外,FMP选择性地积聚在肿瘤细胞的内质网和线粒体中,并在激光刺激下产生ROS。由此产生的内质网应激和线粒体功能障碍导致钙网蛋白的外翻和高迁移率族蛋白B1的外排,触发了ICD效应。通过体内动物实验验证了FMP可在肿瘤部位被激活,将活化的PDT与aPD-L1联合使用会产生有效的CAFs耗竭和ICD效应,共同促进细胞毒性T淋巴细胞和aPD-L1进入富含CAFs的肿瘤的更深区域,从而加强FMP介导的PDT联合aPD-L1治疗的协同抗肿瘤作用,且全身副作用小。同时,获得了增强的治疗效果,实现了对原发性和远处肿瘤的长期抑制,以及对小鼠癌转移的预防。结论:本研究构建了一种FAPα可激活的诊疗探针(FMP),该探针可被肿瘤微环境中的FAPα激活,可进行荧光成像和光声成像,并可同时进行PDT介导的免疫治疗,用于杀死CAFs和肿瘤细胞。通过对探针优化发现具有较长自消除链的探针FMP对FAPα有更好的响应性。由于FMP可被CAFs和肿瘤细胞中过表达的FAPα识别激活,并恢复光毒性作用以及激活ICD效应,故可在4T1荷瘤小鼠模型中观察到原发肿瘤消退和一定程度的远端效应。通过与PD-L1检查点阻断免疫治疗相联合,有效地增强机体抗肿瘤免疫应答,不仅抑制了原发肿瘤和远端肿瘤的生长,而且高效地抑制了小鼠肺转移。总的来说,这项研究为开发用于有效CAFs靶向治疗的可激活诊疗探针提供了一种新的思路。更多还原

【Abstract】 Background:Cancer associated fibroblasts(CAFs)play an important role in tumor progression and immunosuppression.To improve the anti-cancer efficacy,numerous CAFs-targeted therapies such as reprogramming CAFs and direct CAFs-depletion strategies have been brought onto the agenda.However,due to the complex biologic behavior of CAFs including phenotypic heterogeneity,multifunctional malleability,and non-exclusive expression of biomarkers by CAFs,these systemic methods cause conflicting therapeutic results and significant side effects.Therefore,precise theranostics of CAFs are beneficial to CAFs-targeted therapies and deciphering CAFs biology.However,imaging agents enabling precise theranostics of CAFs have been rarely exploited.Objective:In order to achieve accurate monitoring and targeted treatment of CAFs,we have developed a molecular pro-theranostic probe(FMP)with activatable fluorescence(FL),photoacoustic(PA)imaging,and photodynamic therapy(PDT)in response to fibroblast activation proteinα(FAPα)overexpressed in more than 90%types of CAFs and some tumor cells.Methods:In the first part,we designed two types of FAPα-activatable molecular pro-diagnostic probes by coupling the FAPα-sensitive dipeptide substrates with methylene blue(MB)through different lengths of the self-immolative linker,resulting in the quenching of photoactivity(FL,PA and phototoxicity).The best FAPα-responsive pro-diagnostic probe was screened out through hydrolytic efficiency and molecular simulation.Subsequently,a series of in vitro and in vivo experiments were carried out to investigate the reactivity and tumor imaging effect of FAPα.In the second part,laser-irradiated FMP activated by FAPαto investigate the photodynamic and photothermal properties of FMP.Subsequently,the phototoxicity and immunogenic cell death(ICD)effect of FMP at the cellular level were studied by laser confocal imaging and flow cytometry.The tumor therapeutic effect and immune effect of FMP were evaluated by tumor volume,immunohistochemistry and immune index detection.The biological safety of FMP was evaluated by blood biochemistry,blood routine and pathological sections.Results:In the first part of the work,the best FAPα-activatable pro-theranostic probe(FMP)was selected after optimizing the length of the self-immolative linker.The results of in vitro and in vivo experiments showed that FMP showed highly sensitive,selective FL and PA enhancement of FAPα.In addition,FMP shows good cellular absorption and membrane permeability,which can be used for imaging of FAPα-positive cells and tumors.In the second part,firstly,it is proved that FMP activated by FAPαhas the property of generating reactive oxygen species(ROS).Subsequently,cell test show that FMP targets CAFs and produces lethal ROS levels under laser irradiation to kill CAFs.FMP selectively accumulates in endoplasmic reticulum(ER)and mitochondria of tumor cells and generates ROS under laser stimulation.The resulting ER stress and mitochondrial dysfunction produce effective ICD effects on tumor cells.By combining the activated PDT with aPD-L1,the effective CAF depletion and ICD effect jointly promote cytotoxic T lymphocyte and aPD-L1 to enter the deeper region of CAF-rich tumors,thus strengthening the synergistic anti-tumor effect of FMP-mediated PDT and aPD-L1-mediated immunotherapy.Meanwhile,enhanced treatment efficacy has been obtained including long-lasting tumor suppression of both primary and distant tumors as well as prevention of cancer metastasis in living mice.Conclusion:In this study,we designed a FAPα-activatable pro-theranostic probe(FMP)with turn-on FL and PA imaging-guided PDT immunotherapy for the simultaneous killing of CAFs and cancer cells.Attributing to efficient activatable photo-toxicity towards CAFs and tumor cells together with activated ICD,complete tumor regression of primary tumors and abscopal effect of distant tumors are observed on the 4T1-tumor-bearing mice model.By integration with PD-L1 checkpoint blockade immunotherapy,enhanced systemic immune responses have been evoked to obtain long-lasting tumor suppression of both primary and distant tumors as well as arrest systemic cancer metastasis in living mice.Overall,this study provides a universal method for the development of an activatable pro-theranostic probe for effective CAFs-targeted therapies.更多还原