骨巨细胞瘤的免疫微环境及其对地舒单抗治疗的提示  

The immune microenvironment of giant cell tumor of bone and its role as an indicator to denosumab administration

作　　者：李冰皓[1] 严晓波[1] 柳萌[1] 潘伟波[1] 黄鑫[1] 林秾[1] 叶招明[1] Li Binghao;Yan Xiaobo;Liu Meng;Pan Weibo;Huang Xin;Lin Nong;Ye Zhaoming(Department of Orthopaedics,The Second Affiliated Hospital of Zhejiang University School of Medicine,Hangzhou 310009,China)

机构地区：[1]浙江大学医学院附属第二医院骨科,杭州310009

出　　处：《中华骨科杂志》2021年第16期1081-1089,共9页Chinese Journal of Orthopaedics

基　　金：国家自然科学基金(81802684,81872173)。

摘　　要：目的从免疫细胞角度分析骨巨细胞瘤的免疫微环境,明确骨巨细胞瘤微环境的免疫细胞组成,在单细胞水平探索地舒单抗治疗前后骨巨细胞瘤的免疫改变。方法自2018年11月至2020年5月收治的初发骨巨细胞瘤及地舒单抗治疗后骨巨细胞瘤的手术病例中收取新鲜组织样本,送质谱流式细胞术分析,使用t-distributed stochastic neighbor embedding(TSNE)降维分析将质谱流式高维数据可视化并定量,比较地舒单抗治疗前后骨巨细胞瘤的免疫细胞组成。选取部分未经地舒单抗治疗及经过地舒单抗治疗的组织进行原代体外培养并获得培养上清液,用于分析上清液对各类细胞系生长的影响。结果共15例初发骨巨细胞瘤及3例地舒单抗治疗后骨巨细胞瘤经质检合格后完成质谱流式及多色流式分析,其中男7例,女11例。骨巨细胞瘤富含T细胞及多种巨噬细胞样表型的髓系细胞,经地舒单抗治疗后巨噬细胞样表型髓系细胞比例大幅度降低而T细胞比例上升,且巨噬细胞样表型髓系细胞比例与地舒单抗治疗时间相关。骨巨细胞瘤中疑似多核破骨样巨细胞的免疫细胞亚型以γδTCR+为特征,且表达多种趋化因子受体。肿瘤中的CD8+T细胞绝大多数为活化的PD-1hiTIM-3+CD69+的T细胞。未经地舒单抗治疗直接手术的骨巨细胞瘤组织体外培养上清液能促进多种细胞系增殖,而地舒单抗治疗后肿瘤组织上清液则无该功能。结论绘制骨巨细胞瘤的免疫细胞谱图,探索骨巨细胞瘤内免疫细胞对肿瘤生长的潜在作用,为无法切除病例长时间使用地舒单抗提供了部分理论依据。Objective To investigate the alteration of immune microenvironment in giant cell tumor of bone(GCTB)after denosumab treatment from the aspect of immune cellsat single-cell level.Methods During Nov 2018 and May 2020,fresh tumor excision tissues from GCTB cases were collected and received CyTOF analyses.CyTOF datasets were analyzed and visualized by t-distributed stochastic neighbor embedding(TSNE)method of reduction dimension.The compositions of immune cells in GCTB with or without denosumab treatment were compared.The supernatant of culture medium of ex vivo inoculated primary tumor tissues was harvested to clarify if the culturing supernatant could affect cell growth.Results A total of 15 primary GCTB cases and three denosumab-treated samples were included in this study and were sent for CyTOF and multicolor FACS assay.GCTB was featured of T-cell and macrophage-like myeloid cell-dominant immune microenvironment.After denosumab treatment,the percentage of T-cells was significantly elevated,while the number of macrophage-like myeloid cells were reduced.Furthermore,the ratio of macrophage-like myeloid cells in total live cells was associated with the treatment period of denosumab.The multinuclear osteoclast like giant cells were characterized by the expression of γδTCR,while most of the intratumoral CD8+T-cells were activated PD-1+CD69+T-cells.The culturing supernatant of denosumab treatment-free GCTB tissues reinforced cell proliferation in vitro,while this phenomenon was not seen when using denosumab treated tissues.Conclusion Illustrated the immune cell atlas of GCTB,and preliminarily investigated the potential effects of immune cells on tumor progression in GCTB,providing some theoretical clues for prolonged use of denosumab in unresectable GCTB cases.

关 键 词：骨巨细胞瘤 狄诺塞麦 细胞微环境 免疫组织化学 对比研究 

分 类 号：R738.1[医药卫生—肿瘤]