The tetraspanin CD81 regulates the metastasis-promoting function of the osteosarcoma secretome

Osteosarcoma is a highly aggressive malignancy in which pulmonary metastasis remains the leading cause of mortality. Tumor cell–derived secretome, comprising extracellular vesicles and soluble factors, has been implicated in cancer progression and metastatic dissemination; however, the mechanisms regulating its functional properties remain incompletely understood. CD81, a member of the tetraspanin family, is widely recognized as a marker of extracellular vesicles and has been associated with tumor progression and metastasis. In this study, we investigated the role of CD81 in regulating the metastasis-promoting function of the osteosarcoma-derived secretome. Secretome was collected from 143B wild-type osteosarcoma cells and 143B CD81-knockout cells and applied to in vitro and in vivo models. Secretome derived from wild-type cells significantly enhanced tumor cell proliferation, migration and invasion, whereas secretome derived from CD81-knockout cells showed markedly attenuated effects. In endothelial cells, secretome from wild-type cells promoted angiogenic activity, which was markedly attenuated in CD81-knockout cells, indicating a role in modulating tumor–endothelial interactions. In vivo, repeated administration of secretome from wild-type cells induced the accumulation of CD11b-positive and S100A8/A9-positive cells in the lung and significantly increased pulmonary metastasis following tumor cell injection. In contrast, these effects were substantially reduced in mice treated with secretome from CD81-knockout cells. These findings identify CD81 as a tumor-cell-intrinsic determinant of whether the osteosarcoma secretome itself acquires metastasis-promoting bioactivity, distinct from previously identified downstream secreted effectors, and suggest that CD81 may represent a therapeutic target acting across both tumor and microenvironmental compartments.