【作者】 李阳；

【导师】 姜金兰；

【作者基本信息】 吉林大学 ， 外科学， 2025， 博士

【摘要】 乳腺癌是女性人群中最常见的癌症之一,其死亡率高主要是由于远处转移,而乳腺癌骨转移患者约占转移病例的75%,且5年总生存率仅为22.8%。尽管近年来针对乳腺癌的治疗取得了显著进展,但骨转移的治疗管理仍然是一个挑战。骨转移微环境的复杂性导致其治疗难度增加。传统药物往往由于靶向性不足,生物相容性差,生物利用度低,毒副作用大等原因,导致其临床治疗效果不佳。因此需要新的药物递送策略来提高其疗效。基于细胞的药物递送系统已被用于治疗各种疾病的研究。由于靶向性强、循环时间长以及本身具有生物可降解性和生物相容性,基于细胞的药物递送系统已在癌症治疗研究中得到快速发展。但活细胞易受到体内微环境的影响,存在免疫排斥以及免疫过度激活的风险;而且药物释放也受到细胞新陈代谢的影响,导致药物释放率不稳定;同时活细胞由于储存和运输条件复杂、要求苛刻,不适宜大规模生产和临床转化,导致其在临床应用中受到限制。近年来,基于低温休克细胞的药物递送系统成为有前景的解决方案。该药物递送系统利用液氮低温处理的细胞作为载体运输治疗药物,保留了基于活细胞载药系统靶向性强、生物相容性好、免疫原性低、毒副作用小的优点,同时具备载药量高、稳定性强、制备简便、成本较低的优势。癌症免疫疗法为恶性肿瘤的治疗开辟了新的篇章。免疫疗法通过促进肿瘤细胞释放抗原或通过逆转免疫抑制肿瘤微环境来增强效应T细胞对肿瘤细胞的免疫攻击,从而增强抗肿瘤免疫反应。癌症免疫疗法的优势在于它不仅可以治疗原发性癌症,还可以防止转移和复发,与化疗疗法和其他直接杀死癌细胞的药物相比,脱靶效应更少。尽管取得了这些重大进展,但只有一小部分癌症患者从免疫治疗中受益,而且肿瘤特异性的缺乏导致相当一部分接受治疗的患者出现特异性免疫毒性。控制免疫系统的调节仍然是一个关键挑战。近年来,随着对肿瘤微环境和免疫系统相互作用的深入了解,研究者们越来越关注于开发能够协同免疫系统对抗肿瘤的新策略。其中诱导肿瘤产生免疫原性死亡(Immunogenic Cell Death,ICD)的化学药物疗法就可以协同机体的抗肿瘤免疫反应来增强抗肿瘤作用。基于上述乳腺癌骨转移治疗现状的背景,我们设计了经低温休克处理的自然杀伤细胞(Cryo-Shocked Natural Killer Cells,CSNK)作为理想的药物递送载体,并通过装载可以诱导免疫原性死亡的化疗药物米托蒽醌(Mitoxantrone,MIT),成功构建了MIT@CSNK细胞药物递送系统,并研究了其在乳腺癌骨转移小鼠模型中的抗肿瘤机制。此外,我们将MIT@CSNK系统与肿瘤细胞程序性死亡配体1(Programmed Cell Death Ligand 1,PD-L1)的抑制剂联合应用,利用化学疗法和免疫疗法的协同抗肿瘤作用,来解决乳腺癌骨转移治疗的药物递送效率差以及免疫治疗反应率有限的问题。本研究分为两个部位:第一部分,我们首先制备了液氮低温快速处理的CSNK细胞,并高效装载了相关化疗药物MIT,成功构建了MIT@CSNK系统。CSNK细胞经液氮快速低温处理后失去了增殖活性,但保留了主要细胞结构和相关膜蛋白,是理想的体内药物运输载。MIT作为常规的化疗药物,杀伤肿瘤细胞的同时诱导其产生ICD,然后释放肿瘤相关抗原和损伤相关分子模式(Damage Associated Molecular Patterns,DAMPs),诱导树突状细胞(Dendritic Cells,DCs)成熟,有利于增强机体的抗肿瘤免疫反应。MIT@CSNK系统在体外显示出与MIT相似的肿瘤抑制能力和免疫调节功能。我们构建的MIT@CSNK系统具有独特优势:(1)载药能力强,免疫原性低,生物安全性高;(2)MIT和CSNK细胞可协同诱导DCs成熟进而调节机体抗肿瘤免疫反应;(3)MIT和CSNK细胞可协同调节肿瘤微环境中巨噬细胞极化;(4)易制备、运输和存储,成本低,临床应用前景好。本研究的第二部分,我们首先在乳腺癌骨转移的小鼠动物模型中评估了MIT@CSNK系统的抗肿瘤效果和相关免疫激活作用。MIT可以杀伤抑制肿瘤细胞的同时诱导其发生ICD,释放钙网蛋白(Calreticulin,CRT)和高迁移率族蛋白B1(High Mobility Group Box 1,HMGB1)等DAMPs,诱导DCs成熟。CSNK细胞由于NK细胞独特的性质,也可以诱导DCs成熟。因此MIT和CSNK细胞可以协同诱导DCs成熟,从而促进细胞毒性T细胞浸润到肿瘤部位,进而增强机体的抗肿瘤免疫反应。与此同时,MIT和CSNK细胞还可以调节肿瘤微环境中巨噬细胞的极化,进而改善抑制性肿瘤微环境,增强细胞毒性T淋巴细胞的肿瘤杀伤作用。然而MIT引起肿瘤组织中PD-L1的表达升高,这解释了化学药物治疗时肿瘤组织免疫抑制的原因。因此,我们在乳腺癌骨转移的小鼠动物模型中评估了MIT@CSNK系统协同PD-L1抑制剂(a PD-L1)的化学免疫疗法的协同治疗效果。我们发现阻断PD-1/PD-L1通路,挽救了细胞毒性T淋巴细胞耗竭和受抑制状态,增强其杀伤肿瘤的作用。因此联合治疗进一步增强了MIT@CSNK系统对肿瘤的抑制作用。综上所述,我们的研究提供了一种新的乳腺癌骨转移治疗思路和策略,即MIT@CSNK系统与a PD-L1联合应用,发挥了化学免疫疗法协同作用,展现了其抑制骨转移肿瘤生长的潜力。这一治疗策略为乳腺癌骨转移的治疗提供了新视角,为未来的临床研究奠定了基础。更多还原

【Abstract】 Breast cancer is one of the most common cancers among women,with a high mortality rate primarily attributed to distant metastasis.Approximately 75% of metastatic breast cancer cases involve bone metastasis,and the overall five-year survival rate for these patients is only 22.8%.Although significant progress has been made in the treatment of breast cancer in recent years,the management of bone metastasis remains a challenge.The complexity of the bone metastatic microenvironment contributes to the increased difficulty of treatment.Traditional drugs often exhibit poor clinical efficacy due to issues such as insufficient targeting,poor biocompatibility,low bioavailability,and significant toxicity.Consequently,there is a need for novel drug delivery strategies to enhance therapeutic effectiveness.Cell-based drug delivery systems have been explored in research for the treatment of various diseases.Due to their strong targeting ability,prolonged circulation time,and inherent biodegradability and biocompatibility,these systems have seen rapid development in cancer therapy research.However,living cells are susceptible to the influence of the microenvironment in vivo,posing risks of immune rejection and excessive immune activation.Additionally,drug release is affected by cellular metabolism,leading to unstable drug release rates.Furthermore,due to the complex and stringent storage and transportation requirements,live cells are not suitable for large-scale production and clinical translation,limiting their clinical application.In recent years,drug delivery systems based on cryo-shocked cells have emerged as a promising solution.This system utilizes cells treated with liquid nitrogen as carriers for delivering therapeutic drugs.It retains the advantages of live cell-based drug delivery systems,such as strong targeting,good biocompatibility,low immunogenicity,and minimal toxicity,while also offering benefits such as high drug loading capacity,enhanced stability,simple preparation,and lower cost.Cancer immunotherapy has opened a new chapter in the treatment of malignant tumors.It enhances the immune attack of effector T cells on tumor cells by promoting the release of antigens from tumor cells or by reversing the immunosuppressive tumor microenvironment,thereby boosting the antitumor immune response.The advantage of cancer immunotherapy lies in its ability to treat not only primary cancers but also to prevent metastasis and recurrence,with fewer off-target effects compared to chemotherapy and other direct cancer cell-killing drugs.Despite these significant advancements,only a small proportion of cancer patients benefit from immunotherapy,and the lack of tumor specificity leads to immune toxicity in a considerable number of treated patients.Therefore,controlling the regulation of the immune system remains a key challenge.In recent years,with a deeper understanding of the interactions between the tumor microenvironment and the immune system,researchers have increasingly focused on developing new strategies that can synergize with the immune system to fight tumors.One such approach is chemotherapy that induces immunogenic cell death(ICD),which can enhance antitumor effects by stimulating the antitumor immune response.Based on the current treatment landscape of bone metastasis in breast cancer,we designed cryo-shocked natural killer cells(CSNK)as an ideal drug delivery carrier.By loading them with mitoxantrone(MIT),a chemotherapy drug capable of inducing immunogenic cell death,we successfully constructed the MIT@CSNK cell drug delivery system.We then investigated its antitumor mechanisms in a breast cancer bone metastasis mouse model.Additionally,we combined the MIT@CSNK system with a programmed death ligand-1(PD-L1)inhibitor,aiming to leverage the synergistic antitumor effects of chemotherapy and immunotherapy to address the challenges of poor drug delivery efficiency and limited immunotherapy response in the treatment of breast cancer bone metastasis.The present study was divided into two parts:(1)We first prepared CSNK cells treated with liquid nitrogen and efficiently loaded them with the chemotherapy drug MIT,successfully constructing the MIT@CSNK system.CSNK cells lost proliferative activity after treated with liquid nitrogen,but retained the main cellular structure and related membrane proteins,making them ideal carriers for drug delivery in vivo.MIT,as a conventional chemotherapy drug,not only killed tumor cells but also induced ICD,releasing tumor-associated antigens and damage-associated molecular patterns.This process promoted the maturation of dendritic cells(DCs),which would enhance the body’s antitumor immune response.The MIT@CSNK system demonstrated tumorsuppressive and immunoregulatory functions similar to MIT in vitro.The MIT@CSNK system we constructed has unique advantages:(1)high drug-loading capacity,low immunogenicity,and high biosafety;(2)MIT and CSNK cells can synergistically promote DC maturation and modulate the body’s antitumor immune response;(3)MIT and CSNK cells can jointly regulate macrophage polarization in the tumor microenvironment;(4)it is easy to prepare,transport,and store,low in cost,and holds promising clinical application prospects.In the second part,we first evaluated the anti-tumor efficacy and associated immune activation of MIT@CSNK system in a mouse model of breast cancer bone metastasis.MIT killed tumor cells directly at the tumor site,while inducing ICD in tumor cells,which released DAMPs such as CRT and HMGB1,and induced maturation of DCs.CSNK cells also induced maturation of DCs due to the unique properties of NK cells.Together,MIT and CSNK cells synergistically induce DC maturation,promoting cytotoxic T cell infiltration into tumor sites and enhancing the body’s antitumor immune response.Additionally,MIT and CSNK cells regulate macrophage polarization in the tumor microenvironment,improving suppressive tumor microenvironment and boosting the tumor-killing activity of cytotoxic T lymphocytes.However,MIT application caused elevated PD-L1 expression in tumor tissues,explaining the immunosuppression of tumor tissues during chemotherapy.Therefore,we evaluated the synergistic therapeutic effect of chemoimmunotherapy with MIT@CSNK system synergized with a PD-L1 inhibitor(a PD-L1)in a mouse model of breast cancer bone metastasis.We found that blocking the PD-1/PD-L1 pathway rescued the exhausted and suppressed state of cytotoxic T-lymphocytes,enhancing their tumor-killing activity.Consequently,the combination therapy further amplified the tumor-inhibitory effects of MIT@CSNK system.In summary,our study presents a novel therapeutic approach and strategy for treating breast cancer bone metastasis,using the MIT@CSNK system in combination with a PD-L1.This combination therapy harnesses the synergistic effects of chemotherapy and immunotherapy,demonstrating potential in inhibiting bone metastatic tumor growth.This treatment strategy offers a new perspective for the management of breast cancer bone metastasis and lays the foundation for future clinical research.更多还原