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.