Osteosarcoma (OS), which occurs most commonly in adolescents, is associated with a high degree of malignancy and poor prognosis. In order to develop an accurate treatment for OS, a deeper understanding of its complex tumor microenvironment (TME) is required. In the present study, tissues were isolated from six patients with OS, and then subjected to single-cell RNA sequencing (scRNA-seq) using a 10× Genomics platform. Multiplex immunofluorescence staining was subsequently used to validate the subsets identified by scRNA-seq. ScRNA-seq of six patients with OS was performed prior to neoadjuvant chemotherapy, and data were obtained on 29,278 cells. A total of nine major cell types were identified, and the single-cell transcriptional map of OS was subsequently revealed. Identified osteoblastic OS cells were divided into five subsets, and the subsets of those osteoblastic OS cells with significant prognostic correlation were determined using a deconvolution algorithm. Thereby, different transcription patterns in the cellular subtypes of osteoblastic OS cells were reported, and key transcription factors associated with survival prognosis were identified. Furthermore, the regulation of osteolysis by osteoblastic OS cells via receptor activator of nuclear factor kappa-B ligand was revealed. Furthermore, the role of osteoblastic OS cells in regulating angiogenesis through vascular endothelial growth factor-A was revealed. C3_TXNIP+ macrophages and C5_IFIT1+ macrophages were found to regulate regulatory T cells and participate in CD8+ T cell exhaustion, illustrating the possibility of immunotherapy that could target CD8+ T cells and macrophages. Our findings here show that the role of C1_osteoblastic OS cells in OS is to promote osteolysis and angiogenesis, and this is associated with survival prognosis. In addition, T cell depletion is an important feature of OS. More importantly, the present study provided a valuable resource for the in-depth study of the heterogeneity of the OS TME.

Background/Aims:Phytoestrogens are known to prevent tumor progression by inhibiting proliferation and inducing apoptosis in cancer cells. Formononetin is one of the main components of red clover plants, and is considered as a typical phytoestrogen. This study investigates formononetin induction of apoptosis of human osteosarcoma cell line U2OS by regulating Bcl-2 and Bax expression in vitro and in vivo. Methods: U2OS cells were treated with different concentrations of formononetin and the proliferation of the cells was measured using an MTT assay. Cell apoptosis was examined by flow cytometry. The levels of miR-375, Bax and Bcl-2 protein expression in treated cells were determined by Western blot and RT-PCR. The antitumor activity of formononetin was also evaluated in vivo in nude mice bearing orthotopic tumor implants. Results: High concentrations of formononetin significantly suppress the proliferation of U2OS cells and induce cell apoptosis. Moreover, compared to control group the expression of Bcl-2 and miR-375 decreases with formononetin in the U2OS cells, while Bax increases. Conclusion: Formononetin has inhibitory effects on the proliferation of U2SO cells, both in vitro and in vivo. This antitumor effect is directly correlated with formononetin concentration.

As compared with ACCF with plate fixation, ACDF with plate fixation showed no significant differences in terms of postoperative JOA score, fusion rate, but better improved cervical lordosis, lower complication and smaller surgical trauma. As the limitations of small sample and short follow-up in this study, it still could not be identified whether ACDF with plate fixation is more effective and safer than ACCF with plate fixation.