Characterization of the tumour microenvironment phenotypes in malignant tissues and pleural effusion from advanced osteoblastic osteosarcoma patients

Purpose Malignant pleural effusion (MPE) is an adverse prognostic factor in patients with osteoblastic osteosarcoma; however, the cellular contexts of MPE are largely unknown. Experimental design We performed single-cell RNA-sequencing (scRNA-seq) on 27 260 cells from seven MPE samples and 91 186 cells from eight osteosarcoma tissues, including one recurrent, one lung metastasis and six primary tumour (PT) samples, to characterize their tumour microenvironment. Results Thirteen main cell groups were identified in osteosarcoma tumour and MPE samples. Immune cells dominate the cellular contexts in MPE with more T/NK cells and less osteoclasts compared to PT samples. Of T/NK cells, CD8(+)GNLY(+), CD8(+)KLRC2(+) T cells and FCGR3A(+)NK cells were enriched in MPE but CD4(+)FOXP3(+) Tregs were enriched in PT samples. Naive IGHD(+) B and immune regulatory IGHA1(+) B cells were largely identified in MPE, whereas bone metabolism-related CLEC11A(+) B cells were significantly enriched in osteosarcoma PT. M2-type TAMs, including CLEC11A_TAM, C1QC_TAM and Prolif_TAMs, among myeloid cells were enriched in PT, which may suppress cytotoxicity activities of T cells through multiple ligand-receptor interactions. Mature LAMP3(+) DCs were transformed from CD1C(+) DC and CLEC9A(+) DC sub-clusters when exposure to tumour alloantigens, which may improve T cell cytotoxicity activities on tumour cells under anti-PD-L1 treatments. In further, immune cells from MPE usually present up-regulated glycolysis and down-regulated oxidative phosphorylation and riboflavin metabolism activities compared to those in PT samples. Conclusions Our study provided a novel cellular atlas of MPE and PT in patients with advanced osteosarcoma, which may provide potential therapeutic targets in the future.