Fig. 4.

Microenvironmental acidic stressors and tumor susceptibility. a Experimental scheme. Epithelial cells isolated from the regions adjacent or distant from squamocolumnar junction (SCJ). b Microscopic phenotype of three-dimensional (3D) organoids from control and Krt15-CrePR; LSL-Kras^G12D; p53^flox/flox mice. Histology of 3D organoids demonstrated that oncogenic Ras/p53 increased abnormal growth features. c–e Quantification of relative organoid formation (n = 3 independent experiments, 25 fields at high power field (HPF) per sample) and size distribution (n ≥ 150) from control and experimental mice expressing oncogenic Ras/p53 combination. f Experimental scheme. Phosphate-buffered saline (PBS) (vehicle) or PPI were daily treated by intraperitoneal (i.p.) injections (5 consecutive days per week). g Tumor incidence from Krt15-CrePR; LSL-Kras^G12D; p53^flox/flox mice, with/without PPI treatment, n = 9 animals per each. Statistical significance was determined by Fisher’s exact test; *p < 0.05. h–j Immunoblotting of Keratin5 (Krt5) and Krt6 demonstrated relatively less tumor formation in the PPI treatment group, n = 5 independent experiments. k–m Immunostaining of Krt5, Krt6, and phospho-histone H3 (ph-H3), and histology demonstrated suppressed tumor formation by daily PPI treatment in Krt15-CrePR; LSL-Kras^G12D; p53^flox/flox mice. PPI, proton pump inhibitor. Data with bar graphs are represented as mean ± SEM. Statistical significance was determined by pair-wise comparison using t test; *p < 0.05, **p < 0.005. Scale bars, 100 µm