Integrated analysis of single-cell and bulk RNA sequencing data reveals an immunostimulatory microenvironment in tumor thrombus of osteosarcoma

Abstract Background Tumor thrombus is a devastating complication of cancer. It has long been known that the tumor thrombus of bone sarcomas represents a unique reservoir for various types of cancer and immune cells, however, the systematic 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 (TME) that is associated with the tumor-adaptive immune response, which may directly contribute to tumor progression, metastases, and drug response. Methods We seek to define distinct features of the thrombus microenvironment by analyzing bulk tissue and single-cell level transcriptome from the paired thrombus and primary tumor samples of osteosarcoma (OS) patients. Additional osteosarcoma tissue specimens were analyzed by Multiplexed immunofluorescence staining (mIF) and immune markers were digitally quantified. Results OS tumor thrombus was found to have upregulated IFN-γ and TGF-β signalings that were related to immune surveillance of circulating tumor cells in blood circulation. The elevated IFN-γ signaling suggested that circulating OS cells could be recognized and activate the immune system since IFN-γ is vital for cellular immunity and initiation of the anti-tumor immune response. The single-cell RNA-sequencing (scRNA-seq) revealed that tumor thrombus had a higher proportion of tumor-associated macrophages with M1-like states (TAM-M1) and TAM-M1 with high expression of CCL4, indicating an immunostimulatory TME in OS tumor thrombus. Further mIF staining of the CD3/CD4/CD8A/CD68/CCL4 markers validated the immunostimulatory state in the tumor thrombus samples. Conclusions The current study first reported the transcriptome differences at a single-cell level between tumor thrombus and primary tumor in sarcoma. We observed an immunostimulatory microenvironment in OS tumor thrombus with upregulated IFN-γ and TGF-β signalings. This study depicted the immune microenvironment in the early stages of OS metastasis and provided deeper insights into the TME alterations during metastasis.