Platelet-derived TGF-beta initiates a neutrophil HIF-1alpha/NETs positive feedback loop driving osteosarcoma intratumoral hypoxia and lung metastasis

Background: Osteosarcoma (OS) is the most common primary malignant bone tumor in children and adolescents, with 30%-40% of cases developing lung metastasis. Intratumoral hypoxia is a well-established risk factor for poor prognosis and lung metastasis in OS patients. Previously, hypoxia was considered a largely passive consequence of tumor growth exceeding nutrient supply. This largely passive process has traditionally been considered non-targetable. However, recent evidence reveals it can be regulated by dynamic cellular processes. We therefore investigated the mechanism driving intratumoral hypoxia in OS. Experimental design: High metastatic tumor thrombi from OS patients were collected alongside primary tumor samples (N=18). Single-cell RNA sequencing (scRNA-seq) was performed on paired tumor thrombus and primary tumor samples. The interplay between platelet-derived TGF-beta, neutrophil HIF-1alpha, and neutrophil extracellular traps (NETs) was analyzed in vitro and in vivo . Neutrophil related experiments used primary neutrophils from healthy donors and DMSO-induced neutrophil-like HL-60 (dHL-60) cells. Clinical OS data were collected for validation. Results: scRNA-seq revealed significantly increased neutrophil infiltration in tumor thrombi compared with primary tumors. Neutrophils in tumor thrombi exhibited markedly elevated TGF-beta, HIF-1alpha, and NETs signalings compared to those in primary tumors. In vivo lung colonization and metastasis models confirmed neutrophils promote OS lung metastasis. In vitro experiments demonstrated that platelet-derived TGF-beta triggers NET formation via HIF-1alpha upregulation under normoxia. During NETosis, neutrophils consumed oxygen, inducing intratumoral hypoxia and amplifying HIF-1alpha signaling. Thus, platelet-derived TGF-beta initiates a neutrophil HIF-1alpha/NETs positive feedback loop that drives OS intratumoral hypoxia under normoxia. Targeting the TGF-beta/HIF-1alpha/NETs axis significantly suppressed lung metastasis and alleviated intratumoral hypoxia in vivo . Patients with elevated HIF-1alpha and NETs expression had significantly poorer prognosis. Conclusions: Together, these findings provide novel insights into the mechanism of intratumoral hypoxia in OS, highlighting hypoxia is not a passive process but is actively driven by the TGF-beta/HIF-1alpha/NETs axis between platelets and neutrophils.