Fig 1.
Characterisation of the antibodies used in this study.
(a) E-cadherin, (b) EMP1, (c) 5T4, (d) N-cadherin and (e) CD44 expression assessed using (i) western blot and (ii) immunofluorescence microscopy analysis. Anti-E-cadherin antibody 610181 (BD Biosiences, UK) assessed using A549 cell lysates and normal oral epithelium biopsy; Anti-EMP1 antibody ab173224 (Abcam Plc, UK) assessed using MCF7 cell lysates and normal oral epithelium biopsy; Anti-5T4 antibody ab134162 (Abcam Plc, UK) was assessed using BicR56 cell lysates and normal oral epithelium biopsy; Anti-N-cadherin antibody ab76057 (Abcam Plc, UK) was assessed using A549 cell lysates and T4 OSCC biopsy; Anti-CD44 antibody ab40983 (Abcam Plc, UK) was assessed using MB-MDA-231 cell lysates and normal oral epithelium biopsy. Insets show appropriate negative control Ab staining. Green–antigen staining; Blue shows DAPI nuclear stain.
Fig 2.
Determination of cellular localisation and fluorescence intensity of marker expression in oral epithelium biopsies.
Cell surface localisation (a), cell surface and cytoplasmic localisation (b), cytoplasmic localisation (c), nuclear localisation (d), peri-nuclear localisation (e) and lack of expression (f) of individual protein markers was determined using (i) immunofluorescence microscopy analysis and (ii) cell localisation confirmed using the ‘Plot Profile’ application in ImageJ software. White lines on the images in (i) represent the region used for the Plot Profile analysis shown in (ii). (g) (i) Example of membrane localisation of marker expression in ImageJ software and (ii) quantification of fluorescence intensity using the ‘Find Maxima’ application in ImageJ software.
Table 1.
Marker expression and fluorescence intensity in oral epithelium biopsies.
(a) Cell surface expression of E-cadherin, EMP1, 5T4, N-cadherin and CD44 in the biopsies used in this study. (b) Fluorescence intensity readings from immunofluorescence microscopy analysis in NT, FEP, LGD, HGD, T1 OSCC and T4 OSCC and their expression within these biopsies (one-way ANOVA).
Table 2.
Comparison of marker expression in abnormal tissue compared to normal epithelium.
(a) Expression of E-cadherin, EMP1, 5T4 and N-cadherin in FEP, LGD, HGD, T1 OSCC and T4 OSCC biopsies compared to normal tissue (NT). (b) Expression of E-cadherin, EMP1, 5T4 and N-cadherin in FEP, LGD, HGD, T1 OSCC and T4 OSCC biopsies compared to an assumed linear relationship of FEP>LGD>HGD>T1 OSCC>T4 OSCC. (c) Hypothesis testing of an assumed linear NT>LGD>HGD relationship and putative linear HGD>T1 OSCC>T4 OSCC transition. Grey boxes show statistically significant results.
Table 3.
Marker expression in the surgical safety margin of LGD, HGD and T1 OSCC.
(a) Cell surface expression of E-cadherin, EMP1, 5T4 and N-cadherin in the surgical safety margins of LGD, HGD and T1 OSCC biopsies. (b) Marker expression in LGD, HGD and T1 OSCC surgical safety margins compared to normal tissue. (c) Marker expression in LGD, HGD and T1 OSCC surgical safety margins compared to their respective biopsy samples. (d) Statistical significance of low-grade dysplasia (LGD), high-grade dysplasia (HGD) and T1 OSCC when using healthy oral epithelium (NT), fibroepithelial polyp or the respective surgical margins as a healthy control. Grey boxes show statistically significant results.
Table 4.
Summary of marker expression in healthy, abnormal and diseased epithelium.
Marker expression used to classify normal tissue (NT), fibroepithelial polyp (FEP), low-grade dysplasia (LGD), high-grade dysplasia (HGD), T1 OSCC and T4 OSCC.