A mouse LAM model by endonasal administration of human TSC2 deficient smooth muscle cells

RATIONALE: Lymphangioleiomyomatosis (LAM) is a progressive and interstitial lung disease characterized by diffuse proliferation and invasion of abnormal smooth muscle cells in involved organs, cystic degeneration of lung parenchyma, infiltration of the axial lymphatics, and renal tumors. LAM affects almost esclusively young women and mechanisms causing the disease are not yet clarified. LAM affects between 30-40% of women with tuberous sclerosis complex (TSC), a tumor suppressor gene syndrome characterized by the development of benign tumors, e.g. renal angiomyolipoma. TSC is caused by mutations in TSC1 or TSC2 genes, encoding hamartin and tuberin, respectively, which regulate mammalian target of rapamycin (mTOR). We have reported that epidermal growth factor (EGF) is necessary for proliferation of TSC2-/- smooth muscle-like cells (ASM cells) derived from the renal angiomyolipoma of a TSC2 patient. The blockade of EGF receptor (EGFR) causes progressive cell death. Developing a model to study metastasis of TSC tumor cells may help to explain how they migrate from tissue to tissue. We developed a procedure for invasion of the respiratory system by endonasally administrating TSC2-/- cells. Alveolar lung walls and lymph nodes were quickly and massively infiltrated after 8 weeks from cell administration. METHODS: TSC2-/- ASM cells (2x105), previously labelled with red dye PKH26-GL, were administered in immunodeficient female nude mice (nu/nu Hsd:athymic nude mice, 5 week old).After 4 or 26 weeks from endonasal administration anti-EGFR antibody (starting dose of 400 mg/m2 followed by subsequent dose of 250 mg/m2) and rapamycin (4mg/kg) were intraperitoneally injected twice a week for 4 weeks. RESULTS: TSC2-/- cells grow and proliferate in lung parenchyma and lymph nodes, as showed by Ki-67 staining. The average number of TSC2-/- cells in lung parenchyma was reduced from 197±58,27/mm3 to 32,17±12,74/mm3 by anti-EGFR antibody whereas to 134,18±53,94/mm3 by rapamycin. TSC2-/- cells caused progressive destruction of lung parenchyma with an emphysematous-like picture that was reversed by anti-EGFR and rapamycin treatments, with rapamycin causing hemoptysis. TSC2-/- ASM cells promoted a significant increase of LYVE-1 reactivity in lungs suggesting a possible correlation between TSC2-/- cell presence and lymphangiogenesis. LYVE-1 reactivity decreased following anti-EGFR antibody and rapamycin treatments but, while anti-EGFR antibody suppressed the excessive lymphatic vessel, rapamycin caused their collapse. Moreover, in homogenate of lungs murine VEGF levels were increased following TSC2-/- cells endonasal administration and were reduced following anti-EGFR antibody and rapamycin treatments. CONCLUSIONS: TSC2-/- ASM cells can proliferate and invade lymph nodes and lungs causing LAM-like lesions. Anti-EGFR antibody is more effective than rapamycin in promoting lung regeneration, blocking proliferation and reducing lymphangiogenesis. These data suggest a new therapeutic approach for the treatment of TSC and LAM