*3.3. Dysplastic Areas Exhibited Impaired Polarity of Basal Keratinocytes*

As an abnormal organization of the keratinocyte basal layer was systematically observed in dysplastic areas, a particular focus was made on this epidermal compartment. In healthy skin, basal keratinocytes are oriented perpendicularly to the dermo-epidermal junction (JDE), whereas loss of polarity occurs in various pathophysiological processes including epithelial-to-mesenchymal transition (EMT). Measurements of basal nuclei orientation were performed on the xenograft sections by image analysis of stained nuclei (Figure 3). Nuclei orientations versus the JDE plane were determined (Figure 3A) and classified according to three categories: nearly perpendicular (angles between 60◦ and 90◦ ), nearly parallel (angles between 0 ◦ and 30◦ ) and oblique (angles between 30◦ and 90◦ ) (Figure 3B). In control xenografts, a majority of nuclei had a nearly perpendicular orientation, and similar data were obtained in normal areas of xenografts from irradiated keratinocyte precursors. In contrast, a marked increase of oblique and nearly horizontal nuclei orientations was detected within DAs, demonstrating a significant loss of epithelial polarity (*p* < 0.0001 versus repartition in normal areas) (Figure 3B). To further document this observation, the expression pattern of VANGL2, a membrane protein involved in the regulation of cell polarity and migration [23], was then analyzed in xenograft sections by immunofluorescence (Figure 3C,D). This protein was expressed in all basal keratinocytes in normal epidermis, whereas it was reduced or absent in dysplastic cells (Figure 3C). Semi-quantitative image analysis confirmed that the VANGL2 level was significantly reduced in DAs (*p* < 0.0001 versus control areas) (Figure 3D). In summary, a loss of basal keratinocyte polarity that spatially correlated with a perturbated expression of VANGL2 was identified as a marked characteristic of dysplastic areas.
