Figure 2.

(A) H&E staining of organotypic cultures of control EPC2–hTERT–neo-zeo (panel a), EPC2–hTERT–neo-p53^R175H, (panel b) and EPC2–hTERT–EGFR–zeo (panel c), as well as EPC2–hTERT–EGFR–p53^R175H (panel d) and TE12 cancer cells (panel e) are shown. Note that there is dramatic invasion of EPC2–hTERT–EGFR–p53^R175H cells into the mesenchymal matrix in a manner that is comparable with TE12 cells, a human esophageal cancer cell line (120×). (B) Soft-agar assays demonstrated anchorage-independent growth of EPC2–hTERT–EGFR–p53^R175H cells. The histograms show the average number of colonies ± SEM; (*) P ≤ 0.002 (Student t-test; EPC2–hTERT–EGFR–p53^R175H vs. all controls); P < 0.05 is statistically significant. Assays were performed in triplicate. (C) In vivo bioluminescence imaging of control EPC2–hTERT–EGFR–puro–Luc (panel a) and EPC2–hTERT–EGFR–p53^R175H–Luc (panel b) cells implanted subcutaneously into the dorsal skin of athymic nude mice. Tumor formation occurred only in EPC2–hTERT–EGFR–p53^R175H cells but not control cells. The graph demonstrates average bioluminescence signal (photons per second per square centimeter) ± SEM for control (blue) and triple mutant (red) cells; n = 12 injection sites per cell line; (*) P < 0.02 at day 9 and P < 0.002 at day 16 after injection. (D, panel a) H&E staining of representative tumor formed in vivo by EPC2–hTERT–EGFR–p53^R175H cells revealed a squamous cell carcinoma phenotype. Overexpression of p53^R175H (panel b) and EGFR (panel c) in tumor cells was confirmed by immunohistochemistry (120×).