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

FERNANDEZ MARIA CELIA; VENARA MARCELA; NOWICKI SUSANA; BARONTINI MARTA; PENNISI PATRICIA

new york city USA

Congreso; LWPES/ESPE joint meeting; 2009

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.