The HNSCC sample collection was declared to the Bioethical unit of the French Ministry of Higher Education, Research and Innovation (Declaration DC-2013-1798), and was authorized by the same authority (AC-2018-3177, approved on 22 November 2018). risk of recurrence, but also to help clinicians to choose the more appropriate therapeutic strategies. Abstract The EGFR-targeting antibody cetuximab (CTX) combined with radiotherapy is the only targeted therapy that has been proven effective for the treatment of locally advanced head and neck squamous cell carcinoma (LA-HNSCC). Recurrence arises in 50% of patients with HNSCC in the years following treatment. In clinicopathological practice, it is difficult to assign patients to classes of risk because no reliable biomarkers are available to predict the outcome of HPV-unrelated HNSCC. In the present study, we investigated the role of Caveolin-1 (Cav1) in the sensitivity of HNSCC cell lines to CTX-radiotherapy that might predict HNSCC relapse. Ctrl- and Cav-1-overexpressing HNSCC cell lines were exposed to solvent, CTX, or irradiation, or exposed to CTX before irradiation. Growth, clonogenicity, cell cycle progression, apoptosis, metabolism and signaling pathways were analyzed. Cav1 expression was ARPC4 analyzed in 173 tumor samples and correlated to locoregional recurrence and overall survival. We showed that Cav1-overexpressing cells demonstrate better survival capacities and remain proliferative and motile when exposed to CTX-radiotherapy. Resistance is mediated by the Cav1/EREG/YAP axis. Patients whose tumors overexpressed Cav1 experienced regional recurrence a few years after adjuvant radiotherapy chemotherapy. Together, our observations suggest that a high expression of Cav1 might be predictive of locoregional relapse of LA-HNSCC. = 173) were collected during the initial surgery and stored until use in the tumor Bank (Paul Strauss Cancer Center, Strasbourg, France). Informed consent was obtained from all subjects involved in the study. The collection of HNSCC samples was declared to the Bioethical unit of the French Ministry of Higher Education, Research and Innovation (Declaration DC-2013-1798), and was authorized by the same authority (AC-2018-3177, 22 November 2018). The management of patient data was declared and authorized by the French National Commission for Data Protection and Liberties (CNIL; 519013 and 601451). Patients from the northeastern region of France underwent initial surgical resection of their localized HNSCC between 2003 and 2013 at Saint Barbe Clinic (Strasbourg, France), followed by postoperative radiotherapy or chemoradiotherapy (cisplatin) at the Paul Strauss Cancer Center (Strasbourg, France) or the Civil Hospitals of Colmar or Mulhouse. Hematoxylin-eosin slides of paraffin-embedded tumor (FFPE) specimens were examined by two pathologists. All tumors were confirmed as squamous cell carcinomas. The inclusion criteria were: tumor localization (hypopharynx, oropharynx or oral cavity, HPV-negative), T3 and/or N2a with no clinical or radiographic evidence of distant metastases. The primary endpoints were metastatic disease and locoregional recurrence-free survival 3 years after surgery. Secondary endpoints included overall survival (OS), defined as the time from the surgery to the date of death or last follow-up. The recorded variables included age, Eastern Cooperative Oncology Group (ECOG) and Karnofsky Performance Score (KPS), comorbidities (Charlson comorbidity index), tumor stage, chemotherapy regimen in combination with radiotherapy, smoking and alcohol consumption, and follow-up data (survival data, biological parameters, and nutritional characteristics). For detailed patient demographics see Supplementary Data, Table S2. 2.10. Immunohistochemistry on Human Tissue Samples The expression of Chlorotrianisene Cav1 was evaluated by immunohistochemical (IHC) analysis using a Ventana Autostainer Automat (Ventana Medical Systems, Roche Tissue Diagnostics, Boulogne-Billancourt, France). Slides were prepared from formalin-fixed paraffin-embedded tumor specimens. Slides were stained for Cav1 (N-20 sc-894; Santa Cruz Biotechnology, Heidelberg, Germany; dilution 1/50) according Chlorotrianisene to the manufacturers instructions. Signals were revealed with the ultraView Universal DAB Detection Kit (Ventana Medical Systems, Roche Tissue Diagnostics, Boulogne-Billancourt, France), according to the manufacturers instructions. All images were acquired with an Olympus BX60 with 20 or 40 objectives. Contrasts were uniformly adjusted on all images with Photoshop (Adobe) software (https://www.adobe.com; access on 3 May 2021). We used two different semiquantitative analyses of the IHC staining of Cav1. In the first category, tumors were classified into 4 categories according to the percentage of Cav1-positive carcinoma cells: 0 (0%), + (1C25%), ++ (26C75%) and +++ ( 75%). In the second one, the histoscore (H-Score) was calculated Chlorotrianisene as a percentage of different positive cells for Cav1 (0, 1, 2 or 3 3) using the formula (1 (% cells 1) + 2 (% cells 2) + 3 (% cells 3))..