Fig. 7
D2 is expressed in metastatic formations from SCC tumors and its expression in human tumors correlates with a poor diagnosis. a Representative images of metastatic lungs and lymph nodes, and H&E, D2/CXCR-4, and D2/K8 co-staining of lung sections from D2-3xFlag mice treated with DMBA/TPA for 24 weeks. Scale bars represent 200 μm. b Real-time PCR analysis of D2, D3, and K8 expressions in metastastic lungs and lymph nodes from DMBA-TPA-treated mice for 30 weeks (Met) and control, untreated mice (Normal). c Number of metastatic lesions in sD2KO, sD3KO, and control mice 20 and 30 weeks after DMBA/TPA treatment (n = 25). d Representative images showing immunohistochemical (IHC) staining for ZEB-1, E-cadherin, and D3 proteins in cutaneous squamous cell carcinoma (cSCC). Positive E-cadherin expression (a) and negative ZEB-1 expression (b) in normal tissue, and in G1 cSCC (d, e). Moderate E-cadherin expression (g) and weak ZEB-1 expression (h) in G2 cSCC. Finally, negative E-cadherin expression (j) and positive ZEB-1 expression (k) in G3 cSCC. IHC staining of D3 expression revealed positive D3 expression in normal epidermis (c), a rise in D3 expression in G1 (f) and a drastic decrease of D3 expression at the later stages G2 and G3 (i, l). IHC analysis was performed in 20 different tissues for each grading. Tumor grading was evaluated as reported in Supplementary Data File 2. e D3 and D2 mRNA levels were measured in human SCC samples at the same grading as in c. *P < 0.05, **P < 0.01, ***P < 0.001. f Kaplan–Meier plots from two independent data sets (GSE10300 and GSE 686). Blue indicates low and gray indicates high D2 or D3 expression. The number of tumors in each group is reported in parentheses. g The D2–T3–ZEB-1 axis contributes to the EMT and to the progression to invasive stages of carcinomas.
