去甲基化治疗联合过继性T细胞输注治疗骨肉瘤

发布时间：2018-03-08 22:19

本文选题：骨肉瘤　切入点：免疫治疗　出处：《浙江大学》2017年博士论文　论文类型：学位论文

【摘要】：骨肉瘤是最常见的原发于骨组织的恶性肿瘤,多见于青少年,具有局部侵犯和早期发生远处转移的特点。尽管采用最新的新辅助化疗+手术+术后化疗的治疗策略后患者的5年生存率能达到60-80%左右,然而一旦发生化疗耐药或复发,则预后极差。另外,骨肉瘤的一线化疗方案核心药物(甲氨蝶呤、阿霉素、顺铂,MAP方案)已延用几十年之久,临床上对化疗耐药的患者手段十分有限。因此,若能寻找一个除了常规手术、化疗、放疗之外的治疗方法,将会是对常规疗法的极大补充。免疫治疗并被誉为是除三种常规疗法之外的"第四种疗法"。过继性细胞输注治疗是重要的免疫治疗手段之一,近期在临床试验中尤其是对白血病取得了极大突破。然而相比较于白血病,绝大多数实体肿瘤都缺少特异性强、表达率高的免疫治疗靶点,而且实体肿瘤的肿瘤微环境也从多个方面阻止效应免疫细胞对肿瘤细胞的攻击,骨肉瘤亦有这些特点。因此,如何对骨肉瘤进行安全、有效的过继性细胞输注免疫治疗是时下的难点。肿瘤/睾丸抗原几乎只存在于恶性肿瘤和免疫逃逸区,是特异性很强的肿瘤特异性抗原。但其在骨肉瘤上表达率差别很大,且常常处于高甲基化状态导致表达降低乃至沉默。另外,肿瘤细胞可以通过异常的高甲基化使诸多抑癌基因沉默,并影响某些与免疫反应直接相关的蛋白质的表达。我们使用去甲基化药物地西他滨对骨肉瘤细胞系进行治疗后,发现肿瘤/睾丸抗原在骨肉瘤细胞中的表达得到显著提高,且提高的抗原表达能成功被体外培养的肿瘤/睾丸抗原特异性CD8+T细胞识别。另外,去甲基化治疗本身对骨肉瘤细胞有杀伤作用;在联合T细胞治疗后,杀伤率得到进一步提高。我们随后使用人骨肉瘤细胞系在免疫缺陷鼠建立皮下荷瘤模型,对小鼠进行去甲基化治疗和人源CD8+T细胞输注治疗,并使用小动物活体成像系统监测输注入小鼠体内的T细胞分布。我们发现只有在经过去甲基化治疗的小鼠中,肿瘤/睾丸抗原特异性CD8+T细胞才能归巢至肿瘤区域。治疗过程中测量肿瘤大小、治疗结束后肿瘤称重均提示联合治疗能显著抑制肿瘤生长。但去甲基化治疗单药治疗对在体的骨肉瘤效果不明显,只有在联合T细胞输注后才显示出抗肿瘤效果。我们同时还使用鼠骨肉瘤细胞系在免疫健全鼠建立原位模型,对小鼠进行去甲基化治疗,检测去甲基化治疗对肿瘤局部免疫反应的影响。我们发现去甲基化治疗促进了肿瘤灶的淋巴细胞浸润,并提高了肿瘤内CD8+T细胞的活性,且这些效果具有剂量依赖性。我们进一步探索去甲基化治疗促进肿瘤内淋巴细胞浸润的可能机制时,发现可能与趋化因子CXCL12的表达量有关。骨肉瘤存在着高甲基化的CXCL12,从而导致CXCL12表达量降低、T细胞归巢减弱。而去甲基化治疗能提高骨肉瘤的CXCL12表达,为T细胞归巢创造条件。综上,本研究表明去甲基化治疗能促进骨肉瘤局部的免疫反应,并能诱导肿瘤/睾丸抗原特异性CD8+T细胞趋化、识别骨肉瘤,进而有效地控制骨肉瘤生长。
[Abstract]:Osteosarcoma is the most common primary bone malignant tumor, more common in young people, occurrence of distant metastasis with local invasion and early. Despite the use of neoadjuvant chemotherapy plus surgery + patients new treatment strategies after chemotherapy in patients after 5 years survival rate can reach about 60-80%, but once the occurrence of resistance to chemotherapy or recurrence, the prognosis is poor. In addition, first-line chemotherapy for osteosarcoma core drugs (methotrexate, doxorubicin, cisplatin, MAP) has been used for decades, clinical resistance to chemotherapy in patients with very limited means. Therefore, if we can find one in addition to conventional surgery, chemotherapy, radiotherapy treatment outside, will is a great complement to conventional therapies. Immunotherapy and known as the three is in addition to conventional therapy besides "fourth therapy". Adoptive cell transfusion therapy is one of the important means of immunotherapy, recently in clinical trial In the experiment of leukemia especially made great breakthrough. However, compared to the vast majority of solid tumors, leukemia, lack of specificity, the expression of immune therapeutic target rate is high, and the solid tumor tumor microenvironment also from many aspects of blocking immune cells to tumor cells of osteosarcoma have these attacks characteristics. Therefore, how to carry out the safety of osteosarcoma, effective adoptive cell immunotherapy infusion is nowadays difficult. The cancer / testis antigens found almost exclusively in malignant tumor and immune escape, is tumor specific strong specificity antigen expression in osteosarcoma. But the rate difference is very big, and often in high methylation leads to reduced expression and silence. In addition, tumor cells can make many aberrantly hypermethylated tumor suppressor gene silencing, and the influence of some immune response is directly related to the expression of the protein. We used to go on osteosarcoma cell line were treated with demethylating agent decitabine, tumor / testis antigen expression in osteosarcoma cells was significantly increased, and the increase in the expression of antigen can be successfully cultured in vitro cancer / testis antigen specific CD8+T cell recognition. In addition, demethylation treatment itself the killing effect of osteosarcoma cells; in combined with T cells after the treatment, the killing rate can be further improved. We then use the human osteosarcoma cell lines established subcutaneous tumor model in immunodeficient mice, the mice were demethylation therapy and human CD8+T cell infusion therapy, T cell distribution and the use of small animal imaging monitoring system infused into mice. We found that only through methylation treatment in mice, cancer / testis antigen specific CD8+T cells to tumor homing to the area. During the treatment of swelling measurement Tumor size, tumor weight after treatment indicated that combined treatment could significantly inhibit tumor growth. But demethylation treatment of osteosarcoma in the single drug treatment effect was not obvious, only in combination with T cell infusion showed antitumor effect. We also use the rat osteosarcoma cell line in immunocompetent the establishment of rat orthotopic model of mice by demethylation treatment, detection of demethylation treatment of local tumor response. We found that demethylation therapy promotes tumor infiltrating lymphocytes, and increased CD8+T activity in tumor cells, and the effect is dose dependent. We further explore the demethylation treatment of possible mechanisms in promoting lymphocyte infiltration of the tumor, and may find expression of chemokine CXCL12 on osteosarcoma. There exist hypermethylation of CXCL12, resulting in the expression of CXCL12 Decreased T cell homing weakened. The demethylation treatment can improve the expression of CXCL12 in osteosarcoma, and create conditions for the homing of T cells. In conclusion, this study shows that demethylation therapy can promote the immune response of osteosarcoma locally, and can induce the cancer / testis antigen specific CD8+T cell chemotaxis, recognition of osteosarcoma, and to effectively control the growth of osteosarcoma.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R738.1

【相似文献】