Tumor volume and number of lung metastases in mice bearing 4T1 tumors treated with calcitriol, PRI-2191, and PRI-2205. (A) Tumor volume measured on day 21, 28 and 33 of experiment. (B) Number of lung metastases counted in lungs from two independent experiments. (C) Score for metastases in H&E stained lungs, days 14, 28 and 33: 0, no metastasis detected; +, 1–3 metastatic foci; ++, 4–7 foci; +++, 8–10 foci; ++++, >10 metastatic foci in lungs. (D) An example of H&E stained lungs on various stages of tumor progression from mice treated with calcitriol. Black arrows indicated tumor nodules. Magnification, ×20, scale bar, 100 µm. Mice were inoculated orthotopically with 4T1 cells on day 0. From day 7 (7 days after tumor inoculation), vitamin D analogs were administered subcutaneously (s.c.) thrice a week. The single dose of compounds were as follows: calcitriol, 0.5 µg/kg; PRI-2191, 1.0 µg/kg; and PRI-2205, 10.0 µg/kg. Number of mice were 9–13 per group. Data were collected from two independent experiments. Data for individual animals are presented with median lines. Statistical analysis: Kruskal-Wallis multiple comparison test. *P<0.05.