Tumor thrombus of bone sarcomas represents a unique reservoir for various types of cancer and immune cells, however, the investigation of tumor thrombus at a single-cell level is very limited. And it is still an open question to identify the thrombus-specific tumor microenvironment that is associated with the tumor-adaptive immune response. Here, by analyzing bulk tissue and single-cell level transcriptome from the paired thrombus and primary tumor samples of osteosarcoma (OS) patients, we define the immunostimulatory microenvironment in tumor thrombus of OS with a higher proportion of tumor-associated macrophages with M1-like states (TAM-M1) and TAM-M1 with high expression of CCL4. OS tumor thrombus is found to have upregulated IFN-γ and TGF-β signalings that are related to immune surveillance of circulating tumor cells in blood circulation. Further multiplexed immunofluorescence staining of the CD3/CD4/CD8A/CD68/CCL4 markers validates the immune-activated state in the tumor thrombus samples. Our study first reports the transcriptome differences at a single-cell level between tumor thrombus and primary tumor in sarcoma.

骨肉瘤的瘤栓是各种癌症和免疫细胞的独特储库，然而，在单细胞水平上对瘤栓的研究非常有限。确定与肿瘤适应性免疫反应相关的血栓特异性肿瘤微环境仍然是一个悬而未决的问题。在这里，通过分析成对的骨肉瘤 (OS) 患者血栓和原发肿瘤样本的大块组织和单细胞水平转录组，我们定义了 OS 肿瘤血栓中的免疫刺激微环境，其中具有较高比例的 M1 样肿瘤相关巨噬细胞状态 (TAM-M1) 和 CCL4 高表达的 TAM-M1。发现 OS 肿瘤血栓具有上调的 IFN-γ 和 TGF-β 信号，这些信号与血液循环中循环肿瘤细胞的免疫监视有关。CD3/CD4/CD8A/CD68/CCL4 标记物的进一步多重免疫荧光染色验证了肿瘤血栓样本中的免疫激活状态。我们的研究首先报告了肉瘤中肿瘤血栓和原发性肿瘤在单细胞水平上的转录组差异。