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Patients with advanced head and neck squamous cell carcinoma (HNSCC) have a poor prognosis with the currently available therapy, and tumor recurrence is frequently observed.The discovery of specific membraneassociated cancer stem cell (CSC) markers is crucial for the development of novel therapeutic strategies to target these CSCs.To address this issue, we established sphere cultures to enrich CSCs and used them for plasma membrane proteomics to identify specific membrane signatures of the HNSCC spheres.