EGFR overexpression is associated with squamous cell carcinoma development. Altered endocytosis and polarization of receptor tyrosine kinases, including EGFR, affect migration and invasion in 3D culture. These studies have been completed via genetic sequencing, cell line or 3D in vitro and in vivo murine models. Here we describe an imaging method that allows ex-vivo examination of ligand-induced endocytosis of EGFR in non-dissociated human tumours. We analyzed sets of tumour samples from advanced cutaneous squamous cell carcinoma and Head and Neck squamous cell carcinoma, actinic keratosis, intra-epidermal carcinoma and cutaneous squamous cell carcinoma. We demonstrate that EGFR endocytosis is dysregulated in advanced SCC and correlates with anti-EGFR monoclonal antibody therapy outcomes. In actinic keratosis, intra-epidermal carcinoma and well-differentiated cutaneous squamous cell carcinoma different patterns of epidermal growth factor ligand uptake and binding were observed at the leading edge of different dysplastic lesions, suggesting that these differences in EGFR endocytosis might influence the metastatic potential of dysplastic squamous epithelium. These studies in live ex-vivo human tumours confirm that endocytosis dysregulation is a physiological event in human tumours and has therapeutic implications
