敲除TET2增强肿瘤免疫的胸水单核细胞对骨肉瘤肺转移杀伤作用的机制研究

Osteosarcoma is the most common primary bone malignancy, and pulmonary metastasis is the most common cause of death. Currently, there is no effective treatment for pulmonary metastasis of osteosarcoma. Tumor infiltrating lymphocytes (TIL) have good therapeutic effect in metastatic melanoma. The former study indicated that lung metastases, comparing with primary osteosarcoma, had higher tumor mutational burden and more lymphocytic infiltrates. Our lab team isolated mononuclear cells from pleural effusion (PEMC), that were similar to TIL, and activated by the presentation of tumor antigen. Experiments in vitro found that the PEMCs have extremely strong cell killing effect to osteosarcoma metastases, however the specific mechanism is still unclear. Recently, it was found that the treatment effect of CAR-T was enhanced after the knockout of the immune cell activation inhibitor TET2. In summary, the applicant proposed that TET2 knockout could enhance the proliferation and anti-osteosarcoma tumor immunity of PEMC. In this study, knockout of TET2 via CRISPR/Cas9 was used to enhance the immune function of monocytes in pleural effusions, and high-throughput sequencing was used to draw the immune library to define the targeted immune mechanism for lung metastasis of osteosarcoma and obtain tumor antigen-specific T cell receptor. This project is expected to provide a reliable theoretical and experimental basis for the treatment of end-stage patients with pulmonary metastasis of osteosarcoma and the exploration of new clinical transformation of adoptive immunotherapy.

肺转移是骨肉瘤最主要的死亡原因，目前尚无针对骨肉瘤肺转移的有效治疗手段。肿瘤浸润淋巴细胞（TIL）在转移性实体瘤中具有部分疗效。申请人前期研究发现：骨肉瘤肺转移灶较原位具有更高肿瘤突变负荷，淋巴细胞浸润更多，从胸水中分离出单核细胞（PEMC），与TIL相似，均为经肿瘤抗原呈递激活，在体外实验中证实其对肺转移具有较强的细胞杀伤作用，但具体作用机制尚不明确。结合近期研究发现免疫细胞激活抑制因子TET2敲除能增强免疫治疗效果，申请人提出敲除TET2能够增强PEMC抗骨肉瘤肺转移的肿瘤免疫作用，本研究拟通过CRISPR/Cas9基因编辑TET2明确增强胸水单核细胞免疫功能，利用高通量测序绘制免疫组库，寻找骨肉瘤肺转移靶向免疫机制，获得肿瘤抗原特异性T细胞受体并验证其有效性。本项目有望为治疗骨肉瘤肺转移的终末期患者，探索新的过继性免疫治疗的临床转化提供可靠的理论及实验依据。

目前原发性恶性骨与软组织肿瘤的发病率正逐年上升的趋势，恶性程度较高的原发骨源性肿瘤多见于儿童与青少年，如骨肉瘤、尤文肉瘤。其中骨肉瘤是儿童和青少年中最常见的骨原发性恶性肿瘤，起病隐匿，恶性程度高。约70%患者出现肺转移，5年生存率仅为20%-40%，肺转移是骨肉瘤患者死亡的主要原因。目前尚无针对骨肉瘤肺转移的有效治疗手段。.本研究通过从转移性胸水中提取胸水单核淋巴细胞（PEMCs），从原位复发灶或转移灶中提取肿瘤浸润淋巴细胞（TILs），分析表型及免疫功能，验证其对肿瘤细胞的肿瘤免疫杀伤作用。进一步通过CRISPR/Cas9编辑技术增强免疫细胞的扩增及免疫功能。证明TILs对骨与软组织肿瘤过继性细胞治疗的潜在价值。研究结论主要包括：.1.针对不同的骨与软组织肿瘤肺转移伴胸腔积液，从恶性胸水中成功分离出PEMCs，通过灌续Feeder扩增的方法在体外成功扩增，并验证其对原代肿瘤具有较强的免疫杀伤作用。.2.恶性骨与软组织肿瘤肺转移病灶中的TILs多于原位病灶，同时转移灶较原位具有更多、更高的肿瘤突变负荷（TMB），更有利于提高免疫治疗的效果。.3.肺转移或胸水中的PEMCs较外周血中的单核淋巴细胞（PBMCs），高独特性CDR3的丰度及多样性下降，单个高度特性CDR3数量显著增多。在转移灶TILs较PBMCs存在TRBV6-5与TRBJ2-3的表达增高，可能与肿瘤转移、转移瘤肿瘤亲和力等相关。.4.通过CRISPR/Cas9技术，敲TILs/PEMCs中TET2、FAM49B等免疫抑制因子，可以部分增强淋巴细胞在体外的扩增及靶向杀伤能力。.综上，肿瘤浸润淋巴细胞（TILs）过继性细胞治疗可能为转移性等终末期的恶性骨与软组肿瘤患者提供新的有效治疗方法。