三阴性乳腺癌具有恶性程度高、免疫应答弱、五年生存率低的特点，目前临床上手术切除后的主要术后辅助治疗方式是化疗以及放疗，手术切除原位肿瘤后预后差，易出现术后复发与转移，放化疗不仅增加了转移风险，强烈的副作用还会严重影响患者生活质量，急需更好的治疗策略支撑。

细胞焦亡是新发现的一种免疫原性的细胞程序性死亡方式，不仅可以直接杀伤肿瘤细胞还可以激活抗肿瘤免疫反应，因而在三阴性乳腺癌治疗中有着广泛的应用前景。在诱发肿瘤细胞焦亡的多种通路中，caspase-3相关通路由于其诱导细胞凋亡或细胞焦亡的双向性受到广泛关注。Caspase-3是典型的凋亡相关蛋白，当细胞内焦亡执行蛋白GSDME高表达时，胞内激活的caspase-3将切割GSDME并释放其N端结构域在细胞膜上穿孔，导致细胞肿胀、破裂并引发细胞焦亡。因此，如何激活caspase-3并提高肿瘤细胞中GSDME的表达是细胞焦亡依赖性肿瘤治疗的关键问题。本论文设计并合成了过氧化铜纳米反应器，从胞内氧化应激损伤激活caspase-3通路入手，利用临床去甲基化药物地西他滨（Decitabine，DAC/DEC）提高三阴性乳腺癌细胞中GSDME表达量，实现了细胞焦亡与肿瘤免疫应答的激活。具体研究内容及结果如下：

成功制备了可自催化产生过氧化氢（H₂O₂）的过氧化铜纳米反应器（Copper peroxide nanoreactor，CP），以高效产生活性氧（ROS）并且不受肿瘤处H₂O₂不足的制约，然后对CP性能进行了详细研究。证明CP在酸性环境/溶酶体中（pH 5.5）能够分解产生Cu²⁺和H₂O₂，进而发生类芬顿反应产生大量羟基自由基（•OH），显著提升胞内ROS水平。

通过生物信息学手段分析得出乳腺癌细胞中Gsdme高度甲基化，进一步通过实时荧光定量聚合酶链式反应（Quantitative real-time PCR，q-PCR）证明DAC有效的提高了小鼠三阴性乳腺癌细胞系4T1中Gsdme基因表达量，进而探究了DAC+CP共同给药后的细胞发生细胞焦亡且释放多种标志性因子。在此基础上，本论文利用小鼠三阴性乳腺癌细胞系4T1建立了小鼠皮下瘤模型进行抗肿瘤治疗，结果表明DAC+CP具有较好的体内抗肿瘤效果，直接杀伤肿瘤细胞的同时有效激活了小鼠在体抗肿瘤免疫反应，显著抑制了肿瘤增殖。

三阴性乳腺癌不仅治疗手段有限，手术切除后的高复发率同样威胁着患者的生存。术后复发的主要原因是微小肿瘤病灶和术后免疫抑制微环境促进残余细胞生长，因此本论文设计合成了适合术后局部缓释给药的纳米复合水凝胶CP@Gel，联合DAC在杀伤残余微小肿瘤细胞的同时逆转免疫抑制微环境，激活抗肿瘤免疫，作为三阴性乳腺癌的术后辅助治疗手段。CP@Gel是一种可注射型水凝胶，在体定点注射成胶后水凝胶缓慢解离，释放的CP纳米反应器诱导术后残余肿瘤病灶细胞的caspase-3活化，DAC提高GSDME蛋白表达，进一步触发GSDME依赖性细胞焦亡并实现抗肿瘤免疫。本论文利用带荧光素酶基因的4T1-Luc细胞系构建了小鼠肿瘤不完全切除手术模型，模拟临床中三阴性乳腺癌手术切除过程。实验结果表明DAC+CP@Gel的联合治疗可有效的抑制三阴性乳腺癌术后复发并有效地大幅度延长小鼠存活率。

综上所述，本论文成功构建了自催化型纳米反应器CP以及可注射型纳米复合水凝胶CP@Gel，设计了一种集去甲基化、自催化型纳米反应器和诱导细胞焦亡三位一体的三阴性乳腺癌治疗策略。CP作为自催化型纳米反应器，表现出良好的稳定性、水溶性以及分散性，可以在微酸环境中快速分解，发生类芬顿反应释放ROS。DAC作为临床用去甲基化药物，可以有效对三阴性乳腺癌细胞DNA去甲基化。本研究在细胞水平与动物水平上均证明了可以通过DAC+CP的双药法成功诱导乳腺癌细胞发生焦亡，有效激活小鼠的抗肿瘤免疫反应。同时，针对三阴性乳腺癌的术后复发问题，本研究设计了DAC+CP@Gel可注射型水凝胶载药体系在术后辅助治疗中表现出了良好的效果，成功抑制了小鼠三阴性乳腺癌的术后原位复发并激活了小鼠肿瘤免疫记忆。

本论文通过临床药物证明了在体水平中肿瘤细胞表观遗传的可编辑性，同时成功地利用凋亡诱导性药物引发细胞焦亡并激活机体抗肿瘤免疫反应，在三阴性乳腺癌原位肿瘤治疗与术后辅助治疗中表现出巨大的临床转化潜力，作为引发细胞焦亡的范例为其余凋亡诱导性治疗方案开辟了新的应用场景与肿瘤治疗思路。

Triple-negative breast cancer (TNBC) has a high degree of malignancy, weak immune response, and low five-year survival rate. Currently, the main postoperative adjuvant treatment methods are chemotherapy and radiotherapy. Chemotherapy and radiotherapy not only increase the risk of metastasis, but also has strong side effects that seriously affect the life quality of patients. Surgical resection of tumor in situ has a poor prognosis and is prone to postoperative recurrence and metastasis. Better treatment strategies are urgently needed. Pyroptosis is a newly discovered immunogenic programmed cell death mode, which has broad application prospects in the treatment of TNBC. Among the various pathways that induce tumor cell pyroptosis, caspase-3-related pathways have received extensive attention due to their duality in inducing apoptosis or pyroptosis. Caspase-3 is a typical apoptosis-related protein. When the pyroptosis executive protein GSDME is highly expressed in the cell, the activated caspase-3 in the cell will cut GSDME and release its N-terminal domain to perforate the cell membrane, resulting in cell swelling and rupture and trigger cell death. Therefore, how to activate caspase-3 and increase the expression of GSDME in tumor cells is a key issue in the pyroptosis-dependent tumor treatment. In this project, a copper peroxide nanoreactor was designed and synthesized, starting from the activation of the caspase-3 pathway by intracellular oxidative stress damage, and using the clinical demethylation drug Decitabine (DAC/DEC) to improve TNBC The expression of GSDME in the cells realizes the activation of pyroptosis and tumor immune response.

 

(1) Successfully prepared copper peroxide nanoparticles (CP) that can autocatalyze the generation of hydrogen peroxide (H₂O₂) to efficiently generate reactive oxygen species (ROS) and not be affected by the lack of H₂O₂ in the tumor constraints, and then the properties of CP are studied in detail. CP can be decomposed to produce Cu²⁺ and H₂O₂ in an acidic environment/lysosome (pH 5.5) by •OH detection and immunofluorescent staining at the cellular level, and then a Fenton-like reaction between Cu²⁺ and H₂O₂ generates hydroxyl radicals (•OH), significantly increased the level of intracellular ROS, thereby efficiently activating the caspase-3 signaling pathway.

 

(2) Through bioinformatics analysis, it was found that Gsdme was highly methylated in breast cancer cells, and further q-PCR experiment proved that the methylation of Gsdme in the cancer cell line 4T1 is severe, and the expression of GSDME can be effectively increased by using DAC. After co-administration of DAC+CP, the cells undergo pyroptosis and release LDH, ATP, IL-1β, HMGB1 and other marker factors. Based on this, we used the mouse TNBC cell line 4T1 to establish a mouse subcutaneous tumor model for anti-tumor treatment. The results showed that DAC+CP has a good anti-tumor effect in vivo, directly killing tumor cells and at the same time the anti-tumor immune response in mice was effectively activated, and tumor proliferation was significantly inhibited.

 

(3) TNBC not only has limited treatment options, but also a high recurrence rate after surgical resection that threatens the survival of patients. The main reason for postoperative recurrence is that small tumor lesions and postoperative immunosuppressive microenvironment promote the growth of residual cells. Therefore, we designed and synthesized a nanocomposite hydrogel CP@Gel that suitable for postoperative local sustained drug release, combined with DAC to kill residual cells and simultaneously reverse the immunosuppressive microenvironment and activate anti-tumor immunity as a postoperative adjuvant therapy for TNBC. CP@Gel is an injectable hydrogel, which slowly dissociates after in vivo injection, and the released CP nanoparticles induce the activation of caspase-3 in postoperative residual tumor lesion cells, and DAC increases the expression of GSDME, further triggering GSDME-dependent pyroptosis to achieve anti-tumor immunity. We used the 4T1-Luc cell line with the luciferase gene to construct a mouse model of incomplete tumor resection, simulating the surgical resection process of TNBC in clinic. The experimental results showed that the combined treatment of DAC+CP@Gel can effectively inhibit the postoperative recurrence of TNBC and effectively prolong the survival rate of mice.

 

In summary, this work successfully constructed the autocatalytic nanoreactor CP and the injectable nanocomposite hydrogel CP@Gel, and designed a combination of demethylation, self-catalytic nanoreactor and induction of pyroptosis. An integrated treatment strategy for TNBC. As a self-catalytic nanoreactor, CP exhibits good stability, water solubility, dispersibility, and biological safety. It can be rapidly decomposed in a slightly acidic environment, and a Fenton-like reaction occurs to release ROS. As a demethylation drug for clinical use, DAC can effectively demethylate DNA in TNBC cells. This study proved that the double-drug method of DAC+CP can successfully induce pyroptosis in breast cancer cells, release a large amount of cellular content and immune signaling factors, and effectively activate the anti-tumor immune response at both the cellular level and the animal level. At the same time, for the postoperative recurrence of TNBC, we designed the DAC+CP@Gel injectable hydrogel drug-loading system, which successfully inhibited TNBC postoperative orthotopic recurrence and activated tumor immune memory in mice.

 

This study proved the editability of tumor cell epigenetics in vivo through clinical drugs, and used apoptosis-inducing drugs to trigger pyroptosis and activate the body's anti-tumor immune response successfully, which have shown great potential for clinical transformation in situ TNBC treatment and postoperative adjuvant therapy. As a paradigm of pyroptosis induction, it has opened new application scenarios and tumor treatment strategies for other apoptosis-inducing therapies.

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