Patients diagnosed with advanced osteosarcoma, often in the form of lung metastases, have abysmal five-year overall survival rates. The complexity of the osteosarcoma immune tumor microenvironment has been implicated in clinical trial failures of various immunotherapies. The purpose of this exploratory study was to spatially characterize the immune tumor microenvironment of metastatic osteosarcoma lung specimens. Knowledge of the coordinating cellular networks within these tissues could then lead to improved outcomes when utilizing immunotherapy for treatment of this disease. Importantly, various cell types, interactions, and cellular neighborhoods were associated with five-year survival status. Of note, increases in cellular interactions between T lymphocytes, positive for programmed cell death protein 1, and myeloid-derived suppressor cells were observed in the 5-year deceased cohort. Additionally, cellular neighborhood analysis identified an Immune-Cold Parenchyma cellular neighborhood, also associated with worse 5-year survival. Finally, the Osteosarcoma Spatial Score, which approximates effector immune activity in the immune tumor microenvironment through the spatial proximity of immune and tumor cells, was increased within 5-year survivors, suggesting improved effector signaling in this patient cohort. Ultimately, these data represent a robust spatial multiplexed immunofluorescence analysis of the metastatic osteosarcoma immune tumor microenvironment. Various communication networks, and their association with survival, were described. In the future, identification of these networks may suggest the use of specific, combinatory immunotherapeutic strategies for improved anti-tumor immune responses and outcomes in osteosarcoma.

被诊断患有晚期骨肉瘤（通常以肺转移的形式）的患者的五年总生存率非常低。骨肉瘤免疫肿瘤微环境的复杂性与各种免疫疗法的临床试验失败有关。这项探索性研究的目的是从空间上描述转移性骨肉瘤肺标本的免疫肿瘤微环境。了解这些组织内协调细胞网络可以在利用免疫疗法治疗这种疾病时改善结果。重要的是，不同的细胞类型、相互作用和细胞邻域与五年生存状态相关。值得注意的是，在 5 岁死亡队列中观察到程序性细胞死亡蛋白 1 呈阳性的 T 淋巴细胞与骨髓源性抑制细胞之间的细胞相互作用增加。此外，细胞邻域分析发现了免疫冷实质细胞邻域，也与较差的 5 年生存率相关。最后，骨肉瘤空间评分（通过免疫细胞和肿瘤细胞的空间接近程度来近似免疫肿瘤微环境中的效应器免疫活性）在 5 年幸存者中有所增加，表明该患者队列中的效应器信号传导有所改善。最终，这些数据代表了对转移性骨肉瘤免疫肿瘤微环境的强大空间多重免疫荧光分析。描述了各种通信网络及其与生存的关联。将来，对这些网络的识别可能会建议使用特定的组合免疫治疗策略来改善骨肉瘤的抗肿瘤免疫反应和结果。