【作者】 常鑫；

【导师】 房学东；

【作者基本信息】 吉林大学 ， 外科学， 2023， 博士

【摘要】 背景:乳腺癌是世界上最常见的恶性肿瘤之一,全球最新癌症数据显示乳腺癌位居女性恶性肿瘤发病率的第一位。在所有乳腺癌病理分型中三阴性乳腺癌的恶性程度最高,预后最差。目前临床治疗三阴性乳腺癌的传统手段主要有手术、化疗及放疗,随着三阴性乳腺癌分子亚型的细化和药物的发展,已有部分患者可以接受到靶向治疗和免疫治疗。然而,手术对女性的生理和心理带来了极大的创伤,放化疗对正常细胞存在毒副作用,而小分子药物靶向治疗的使用也具有一定局限性。因此,迫切需要开发安全有效的肿瘤治疗策略,其中,肿瘤免疫治疗通过调动机体的免疫系统,增强抗肿瘤免疫力,有望彻底根治肿瘤。尽管免疫治疗作为肿瘤治疗的研究热点,展示出良好的治疗前景,但免疫原性较低和免疫抑制肿瘤微环境严重限制了免疫治疗的广泛应用。有研究表明光热治疗（Photothermal therapy,PTT）具有可诱导免疫原性死亡、激活抗肿瘤免疫等特性,为肿瘤治疗提供了新的思路。因此,在本课题中我们借助PTT诱导的免疫原性死亡与免疫治疗相结合,用于治疗恶性肿瘤,探索了更有利于治愈三阴性乳腺癌的治疗策略。PTT中使用的材料具有较高光热转换效率,可高效的将光能转化为热能从而杀伤癌细胞。PTT具有治疗部位精确可控、杀伤效率高、副作用小等优点,在临床前和临床试验中显示出一定的应用前景。本课题中,我们采用具有高光热转化效率、表面官能团丰富和良好生物相容性等特点的二维过渡金属碳（氮）化物（MXene）为光热剂,以实现高效安全的光热治疗。由肿瘤特异性内源性刺激触发的纳米酶催化治疗是一种新兴的肿瘤治疗方法,在治疗肿瘤过程中展现了较好的应用前景,具有类过氧化物酶活性的纳米酶由于能够有效产生活性氧而备受关注。已有研究表明,金纳米粒子（Au NPs）具有过氧化物酶的特点,同时具有良好的光热转换效率和化学惰性,被广泛应用于肿瘤诊疗领域中。因此,在本课题中我们制备了MXene-Au纳米复合材料,使其兼具光热效应和过氧化物酶活性,建立光热协同纳米酶催化抑瘤新策略。进一步,为了充分调动机体的免疫细胞发挥出抗肿瘤效应,我们引入抗OX40抗体,进一步增加效应性T细胞的扩增和细胞因子的分泌,实现三位一体的三阴性乳腺癌光热/催化/免疫联合治疗。目的:探究光热/纳米酶催化/免疫三元治疗策略对小鼠三阴性乳腺癌的治疗效果,为三阴性乳腺癌综合疗法的开发提供坚实的理论基础和可靠的实验依据。方法:（1）制备Ti₃C₂-MXene-Au,对其基本理化性质进行表征,检测材料的光热转换性能、化学稳定性、生物相容性及过氧化物酶样活性。（2）通过细胞摄取、细胞杀伤、活性氧检测及细胞凋亡等体外实验,考察Ti₃C₂-MXene-Au纳米复合材料通过光热联合纳米酶催化疗法对肿瘤细胞产生的杀伤作用并探究其可能的细胞死亡机制。（3）建立小鼠原位乳腺癌模型并进行体内实验,在动物水平验证Ti₃C₂-MXene-Au联合抗体OX40抑制肿瘤生长的效果、生物安全性、靶向性以及对免疫系统的激活效应。结果:（1）使用透射电镜、扫描电镜、X射线衍射、X射线光电子能谱、紫外可见分光光度计及粒径分析仪对Ti₃C₂-MXene-Au的组成、形貌、波长及电势进行表征,证明了Ti₃C₂-MXene-Au纳米复合材料的成功制备。（2）稳定性测试实验和溶血实验,结果显示Ti₃C₂-MXene-Au具有良好的化学稳定性和生物安全性。（3）Ti₃C₂-MXene-Au在808 nm红外光照射下光热转换效率高达55%,且具备浓度和功率依赖性的升温性能及良好的光热稳定性。（4）体外实验结果显示Ti₃C₂-MXene-Au具备过氧化物酶样活性可引起肿瘤细胞内活性氧水平升高以及肿瘤细胞凋亡。（5）免疫原性死亡相关分子CRT、HMGB1及ATP的水平变化提示Ti₃C₂-MXene-Au在红外光照射下可诱导4T1细胞发生免疫原性死亡。（6）小动物光声成像与热成像双成像模式显示Ti₃C₂-MXene-Au在体内肿瘤部位的蓄积,具有良好的肿瘤靶向性。（7）给予荷瘤小鼠尾静脉注射Ti₃C₂-MXene-Au联合腹腔注射抗OX40抗体,在体内展现出显著的抑瘤效果;通过流式细胞术对肿瘤细胞中的免疫细胞进行检测分析,结果显示光热、催化联合免疫治疗有效激活了树突状细胞（Dendritic Cell,DC）并刺激效应T细胞的扩增和浸润,细胞因子的分泌显著增加且髓源性抑制细胞（Myeloid-derived suppressor cells,MDSCs）比例显著下降。（8）在注射Ti₃C₂-MXene-Au的荷瘤小鼠中未观察到明显的组织脏器结构和肝肾功能异常,提示Ti₃C₂-MXene-Au具有良好的生物安全性。结论:综上,Ti₃C₂-MXene-Au纳米复合材料具备光热转换性能和过氧化物酶样特点,可引起肿瘤细胞发生免疫原性死亡和细胞凋亡,在此基础上联合免疫增强剂抗OX40抗体,抑制小鼠肿瘤生长的同时进一步促进DC及T细胞的增殖、活化,并降低MDSCs的免疫抑制活性从而逆转免疫抑制微环境,显著诱导了抗肿瘤免疫应答,是一种极具潜力的三阴性乳腺癌治疗新策略。更多还原

【Abstract】 Background:Breast cancer remains one of the most common malignant tumors worldwide,the latest global data shows that breast cancer ranks first in morbidity of female malignant tumors.Breast cancer is molecularly heterogeneous and categorized into four molecular subtypes:Luminal-A、Luminal-B、HER2 and triple-negative breast cancer.Triple-negative breast cancer has the worst prognosis among all subtypes of breast cancer,the main treatments for which are surgery,radiotherapy,chemotherapy and targeted therapy.However,surgery has brought great trauma to women’s physiology and psychology.Chemotherapy treatment regimes with poor response and high toxicity,and the use of small-molecule drug targeted therapy also has certain limitations.Therefore,it is urgent to develop safe and effective targeted tumor therapy strategies.Immunotherapy holds great promise to cure tumors by mobilizing the body’s immune system and enhancing antitumor immunity.Immunotherapy has shown good therapeutic prospects and become a hot spot in tumor research.However,the low immunogenicity and immunosuppressive tumor microenvironment（TME）severely limit the efficacy of immunotherapy.The unique properties of PTT,such as inducing immunogenic death and activating antitumor immunity,provide new insights for tumor treatment.In the present study,we take advantage of the feature that photothermal therapy can induce immunogenic death,further combine it with immunotherapy to treat malignant tumors to explore more favorable and effective treatment strategy for triple-negative breast cancer.Photothermal therapy（PTT）is a promising emerging treatment that converts nanomaterials from light to heat energy and has shown immense prospects in preclinical and clinical trials.The two-dimensional transition metal carbon（nitrogen）compound（MXene）has the distant advantages of efficient photothermal conversion efficiency,thermal conductivity,hydrophilicity,large specific surface area and abundant surface modification groups and is widely used as photothermal agents to achieve efficient treatment.Nano-enzyme catalyzed therapy triggered by tumor-specific endogenous stimulus is a new tumor treatment method,showing a high application prospect in the process of tumor treatment.Nano-enzyme with peroxide-like activity has attracted much attention due to its ability to effectively produce reactive oxygen species.The gold nanoparticles have been proved to have the characteristics of peroxidase,with good photothermal conversion efficiency and chemical inertness,and have been widely used in diagnosis,imaging and therapy.Collectively,in this topic,we choose gold nanoparticles to modify the surface of MXene,which can play the photothermal effect and also play the peroxidase activity,so as to realize the strategy of photothermal synergistic enzyme therapy of tumor cells.In addition,in order to fully mobilize the body’s immune cells to exert anti-tumor effects,we introduce anti-OX40 antibody signaling molecules on the basis of photothermal synergistic enzyme therapy to further support T cells expansion and increase cytokines secretion,thus achieving effective treatment of triple-negative breast cancer.Purpose:To verify the synergistic therapeutic effect of photothermal/catalysis/immune therapy on triple-negative breast cancer in mice and provide a solid theoretical and reliable experimental basis for developing triple-negative breast cancer therapy.Method:（1）Ti₃C₂-MXene-Au was prepared and characterized by its physical and chemical properties.The photothermal conversion efficiency,stability,biocompatibility and enzyme-like activity of Ti₃C₂-MXene-Au were analyzed.（2）The killing effect of Ti₃C₂-MXene-Au nanocomposites on tumor cells by photothermal combined catalytic therapy and possible mechanisms were explored through experiments such as cell uptake,cell killing,reactive oxygen species and cell apoptosis.（3）The effect of Ti₃C₂-MXene-Au combined with antibody OX40 on tumor growth,biocompatibility,biological safety,targeting ability and the immune system was verified through a mouse orthotopic breast cancer model in vivo.Results:（1）The morphology,structure and zeta potential of Ti₃C₂-MXene-Au were characterized by TEM,SEM,UV-Vis and Zeta Sizer,which proved the successful preparation of Ti₃C₂-MXene-Au nanocomposites.（2）Stability test and hemolysis experiments showed that Ti₃C₂-MXene-Au had excellent stability,biocompatibility and safety.（3）Ti₃C₂-MXene-Au was verified as a photothermal agent for its high photothermal conversion efficiency of up to 55%,combined with concentration and power-dependent heating performance and photothermal stability.（4）Enzyme catalysis experiments confirmed that Ti₃C₂-MXene-Au had peroxidase-like characteristics,which could increase reactive oxygen species and apoptosis of tumor cells.（5）The ability of Ti₃C₂-MXene-Au to induce immunogenic death of 4T1 cells under 808 nm irradiation was confirmed by detecting immunogenic death-related molecules CRT,HMGB1 and ATP.（6）The great tumor-targeting ability of Ti₃C₂-MXene-Au was confirmed by photoacoustic imaging and photothermal imaging in vivo experiments.（7）The combination of photothermal,enzyme catalysis and immune therapy effectively inhibited tumor growth,activated antigen-presenting cells,stimulated the expansion and infiltration of effective T cells,and reduced the proportion of MDSCs through flow cytometry analysis after injection of Ti₃C₂-MXene-Au and OX40antibody in intraperitoneal.（8）H&E staining was used to detect the organ damage of the heart,liver,spleen,lung and kidney through venous blood collected from tumor-bearing mice,which verified that Ti₃C₂-MXene-Au has excellent biological safety.Conclusion:In summary,Ti₃C₂-MXene-Au has photothermal conversion properties and peroxidase-like characteristics,leading to immunogenic death and apoptosis of tumor cells.On this basis,the combination of OX40 antibody further strengthened the proliferation and activation of DC and T cells and reduced the myeloid-derived suppressor cells（MDSCs）,additionally reversed the immunosuppressive microenvironment and induced a significant anti-tumor immune response,which is a potential therapeutic strategy for triple-negative breast cancer.更多还原