Activation of α2-adrenergic receptors as a therapeutic strategy for immune rejection in post-surgery osteosarcoma recurrence treatment

The clinical treatment of Osteosarcoma (OS) faces major barriers due to the risk of tumor recurrence. Immunotherapy utilizing immune checkpoint blockade (ICB) antibodies has exhibited promise in inducing tumor rejection and providing clinical benefits for patients among various tumor types. However, tumors often develop resistance to immune rejection. Given the limitations of current therapies, there is an urgent need to explore novel therapeutic strategies to enhance anti-tumor immunity and prevent recurrence. This study aims to investigate the potential of α2-adrenergic receptor (α2-AR) agonists, delivered via a thermo-sensitive hydrogel (PLGA-PEG-PLGA), as a therapeutic strategy to combat immune rejection and tumor recurrence in osteosarcoma. In this study, we investigated the anti-tumor effects of α2-AR agonists using a thermo-sensitive PLGA-PEG-PLGA hydrogel as a drug delivery system. The hydrogel was loaded with the α2-AR agonist UK14,304 and evaluated for its efficacy and biosafety in vitro and in vivo . In vitro experiments included cell viability assays (CCK-8), scratch wound healing assays, and Transwell assays to assess the impact of UK14,304 on OS cell lines. For in vivo studies, a subcutaneous OS xenograft mouse model was established using BALB/c nude and immunocompetent BALB/c mice. Tumor recurrence and growth were monitored after surgical resection and treatment with the hydrogel-agonist formulation. Proteomic analysis of tumor immune microenvironment (TME), Metascape, STRING, Cytoscape, TCGA and GTEx databases were performed to elucidate the underlying mechanisms of the anti-tumor effects. We evaluated the anti-tumor effects of α2-AR agonists (specifically UK14,304) loaded into a thermo-sensitive PLGA-PEG-PLGA hydrogel both in vitro and in vivo . In vitro experiments using OS cell lines (K7M2, 143b, and Khos) showed that UK14,304 did not significantly affect cell viability, migration, or invasion, indicating minimal direct cytotoxicity. In vivo studies using immunocompetent BALB/c mice demonstrated a significant reduction in tumor recurrence and growth when treated with UK14,304-loaded hydrogels compared to controls, highlighting an immune-mediated anti-tumor effect. Proteomic analysis of TME revealed that the anti-tumor mechanism involves the activation of CD8 + T cells and TCR signaling pathways, with ITGAL identified as a central regulatory factor. Additionally, bioinformatics analysis suggested the involvement of LLPS in enhancing TCR signaling. Correlation analysis with TCGA and GTEx databases further indicated that the identified proteins (e.g., MSN, TOLLIP, ITGAL) are associated with improved clinical outcomes. Overall, our results demonstrate that α2-AR agonists may serve as a potential drug for a prospective drug delivery system in the post-surgical management of OS. This study highlights the therapeutic efficacy of α2-AR agonists in osteosarcoma treatment through a novel drug delivery system, suggesting their potential to enhance anti-tumor immune responses by modulating CD8 + T cell activity and TCR signaling, as supported by correlations with clinical outcomes in TCGA and GTEx databases. The findings of this study present a promising translational potential by demonstrating the efficacy of α2-AR agonists in enhancing anti-tumor immunity in OS, potentially paving the way for targeted immunotherapies in post-surgical management to combat tumor recurrence and resistance. A schematic procedure of this study.