Development of a LAM model by using human TSC2-/- cells derived from a renal angiomyolipoma : A pharmacological evaluation

Tuberous Sclerosis Complex (TSC) is characterized by multiorgan development of tumors affecting the kidneys, brain, heart and lungs. TSC is related to lymphangioleiomyomatosis (LAM), a rare disease characterized by widespread pulmonary proliferation of abnormal smooth muscle cells leading to cystic destruction of the lung parenchyma. In LAM patients somatic mutations in TSC1 or, more frequently, TSC2 have been identified in abnormal lung tissue and renal angiomyolipomas (AML). In our laboratory human TSC2-/- smooth muscle (ASM) cells from a renal AML have been isolated. These cells are characterized by EGF-dependent proliferation, hyperphosphorylation of S6 kinase and its substrate S6 and positivity to HMB45 antibody, a marker of TSC and LAM cells. Antibodies to EGF- and IGF-1 receptors (anti-EGFR and anti-IGF-1R) cause the progressive cell death. To evaluate such a therapeutic potential and TSC2-/- ASM cell invasiveness, we developed an in vivo model of TSC and LAM complications by applying TSC2-/- ASM cells to nude mice. 5 weeks old nude mice were i.p. injected with 5x105 TSC2-/- ASM cells previously labelled with red dye PKH26-GL. A diffuse reactivity to HMB45 antibody and a strong S6 phosphorylation were observed in cervical, mediastinic and retroperitoneal lymph nodes and at lesser extent in lungs, and uteri after 30, 90 and 180 days from injection. Lungs showed an emphysematous picture with cystic degeneration. Recently we developed a procedure for the endonasal administration of TSC2-/- ASM cells, such that the respiratory system was quickly invaded. 2x105 TSC2-/- cells were nasally administrated and massively infiltrated into pulmonary alveolar walls and lymph nodes, associated with an highly diffuse S6 phosphorylation and HMB45 positivity. After nasal administration a strong progressive destruction of lung parenchyma, associated with the presence of TSC2-/- ASM cells. Interestingly, the progressive accumulation of TSC2-/- ASM cells caused an increase in LYVE-1 reactivity in pulmonary parenchyma, suggesting a powerful infiltration of lymphatic vessels. Anti-EGFR antibody treatment abolished S6 phosphorylation and reversed the HMB45 positivity in lungs and lymph nodes and killed infiltrating TSC2-/- ASM cells while rapamycin, the specific mTOR kinase inhibitor, had a rather weak effect. Both agents affected LYVE-1 reactivity, but while anti-EGFR antibody suppressed the excessive lymphatic vessel, rapamycin caused their collapse. In conclusion, human TSC2-/- ASM cells exhibit an in vivo migratory and proliferative capability and invasiveness creating LAM-like lesions. Anti-EGFR antibody was more effective than rapamycin in killing these cells and promoting lung regeneration. These data suggest a new therapeutic approach to control the abnormal smooth muscle cell growth in TSC and LAM.