Adolescent bone sarcomas have distinct immune signatures and intercellular communication networks

Ewing and osteosarcoma are bone sarcomas that occur predominantly in adolescents. In the setting of metastatic or recurrent disease, overall prognosis is poor. While immunotherapy is effective for some adult soft tissue sarcoma, limited success has been achieved with immunotherapy for pediatric bone sarcomas. To better understand antitumor immunity in Ewing and osteosarcoma patients, we performed single-cell RNAseq on peripheral blood and tumor infiltrating immune populations using 10X Genomics droplet-based approach. We also included peripheral blood from healthy adolescent blood donors as a comparator. Utilizing these transcriptional profiles, we characterized tumor infiltrating CD8+ T cells and myeloid cells and identified intercellular communication networks in both primary and recurrent disease. Overall design: In total, we generated single-cell transcriptional profiles from sorted live CD45+ immune cells from paired blood and tumor samples from 4 patients with Ewing sarcoma and 4 patients with osteosarcoma. Among these, 2 patients had primary disease and 2 patients had recurrent disease from both Ewing and osteosarcoma. We also generated single-cell transcriptional profiles from PBMC obtained from 4 healthy adolescent blood donors.

尤因肉瘤（Ewing sarcoma）与骨肉瘤（osteosarcoma）均为主要累及青少年的骨源性肉瘤。当发生转移性或复发性病变时，患者总体预后较差。尽管免疫治疗对部分成人软组织肉瘤具有明确疗效，但针对儿童骨源性肉瘤的免疫治疗仅取得了有限的进展。为深入解析尤因肉瘤与骨肉瘤患者体内的抗肿瘤免疫机制，本研究采用10X Genomics液滴测序技术，对外周血及肿瘤浸润免疫细胞群开展了单细胞RNA测序（single-cell RNAseq），并纳入健康青少年献血者的外周血作为对照。依托上述转录组图谱，我们对肿瘤浸润性CD8+ T细胞与髓系细胞进行了特征分析，并阐明了原发性及复发性病变中的细胞间通讯网络。
实验设计概览：本研究共从4例尤因肉瘤患者与4例骨肉瘤患者的配对血液及肿瘤样本中，分选得到活的CD45+免疫细胞并构建了单细胞转录组图谱。其中，尤因肉瘤与骨肉瘤患者各有2例为原发性病变，2例为复发性病变。此外，我们还从4名健康青少年献血者的外周血单个核细胞（peripheral blood mononuclear cell, PBMC）中构建了单细胞转录组图谱。