Development of a murine model for the study of Pheochromocytoma (Pheo).

FERNANDEZ MC; VENARA MC; NOWICKI S; BARONTINI M; PENNISI PA

New York City, NY. USA

Congreso; LWPES/ESPE 8th Joint Meeting,; 2009

LWPES/ESPE , APEG, APPES, JSPE and SLEP

      We have previously shown that IGF-1R expression is increased in malignant       compared to benign pheo suggesting a potential role for the IGF/IGF-1R       system in the pathogenesis of the disease. Murine models of tumor       development are useful tools; unlike breast or colon cancer, the use of       murine models for the study of pheo has been less explored. Aim: In order       to understand the influence of IGF/IGF-1R on the biology of pheo, we aimed       to generate a murine model by using a cell line derived from pheo       occurring in heterozygous neurofibromatosis knockout mice (MPC cells) in a       no immunodeficient strain of mice. Materials and Methods: MPC cells were       grown in complete RPMI medium (5%FBS, 10%HS). Western Blot: MPC cells were       seeded in 6 well plates and allowed to reach 60% confluency, starved and       stimulated with 20nM of rhIGF-1 for 5´ to 15´. 500/50ug of protein       extracts were used for Immunoprecipitation or SDS-PAGE respectively.       Membranes were probed for IGF-1R, PY20, Total AKT, pAKT, total ERK,       p42/44. Proliferation assays: 3x105 or 8x105 cells were seeded in 24 well       plates in complete or low serum (0.5%FBS,1%HS)RPMI respectively, with or       without (w/wo) 100nM or 50nM rhIGF-1 for 7 days. Medium was replaced every       48 hs, and cells were counted on days 1, 3, 5 and 7. Eight weeks old C57B6       mice were injected sc (right flank, n=10,106cells) or iv (tail vein, n=5,       106cells) and monitored for weight, and tumor growth.On week 14 mice were       sacrificed and tumors dissected, snap frozen and fixed for IHC       analysis.Results: After 5´ incubation with rhIGF-1,IGF-1R, AKT and ERK       were phosphorylated as revealed by western blot analysis. MPC cells       proliferate in complete medium w/wo rhIGF-1. On day 5, there was an       increased number of cells upon rhIGF-1stimulation compared to basal       proliferation (2.1±0.2 vs 1.5±0.3x105,p=0.03). On Low Serum medium, MPC       cells proliferation was blunted while it was stimulated by rhIGF-1       compared to basal condition (1.5±0.4 vs 2.7±0.2x105,p=0.009). 10/10       injected sc mice developed sc tumors from week 5. On week 14 tumors had       reached 2cm diameter.3/5 iv injected mice developed hepatic tumors.       Histology was performed and in all cases pheo were confirmed. Conclusions:       MPC cells have an intact IGF-1R that is activated by rhIGF-1, stimulating       their proliferation in vitro. When injected sc or iv in normal C57B6 mice,       MPC cells grow and form tumors that have pheo histology. Therefore, we       have developed a new murine model for the study of pheochromocytoma       biology.