Radial shock waves prevent growth retardation caused by the clinically used drug vismodegib in ex vivo cultured bones

Abstract

In childhood medulloblastoma patients, the hedgehog antagonist vismodegib is an effective anticancer treatment but unfortunately induces irreversible growth arrests and growth impairment limiting its use in skeletally immature patients. We hypothesized that radial shock wave treatment (rSWT) may protect drug-induced growth impairment owing to its osteogenic effects. Fetal rat metatarsal bones were exposed to vismodegib (day 0-5; 100 nM) and/or rSWT (single session); other bones from day 1 were continuously exposed to a Gli1 antagonist (GANT61; 10 µM) and/or rSWT (single session). Control bones were untreated. The bone length was measured at intervals; histomorphometric analysis and immunostaining for PCNA, Gli1, and Ihh were performed on the sectioned bones. Bones treated with vismodegib showed impaired bone growth, reduced height of the resting-proliferative zone and reduced hypertrophic cell size compared to control. In vismodegib treated bones, a single session of rSWT partially rescued bone growth, increased the growth velocity, hypertrophic cell size, and restored growth plate morphology. Bones exposed to GANT61 showed impaired bone growth and disorganized growth plate while when combined with rSWT these effects were partially prevented. Locally applied rSWT had a chondroprotective effect in rat metatarsal bones and suggest a novel strategy to prevent growth impairment caused by vismodegib. Hedgehog (Hh) proteins are well-known to be overexpressed in paediatric medulloblastoma 1. Mutations that occur in the family of Hh-pathway genes such as patched-1, suppressor of fuse and smoothened leads to an increased level of the glioma-associated oncogene (Gli1), a downstream transcription factor of Hh 2. In the clinic, hedgehog inhibitors are used to decrease the Hh-activity and thereby impede tumor progression 3-5. However, stable expression of the Hh-gene is essential to maintain chondrocyte proliferation and hypertrophy during bone growth 6. A recent study reported that prolonged exposure to vismodegib, a Hh-antagonist, in children with medulloblastoma resulted in irreversible growth plate fusion causing growth arrest of long bones 7,8. Preclinical studies in young mice exposed to a Hh-antagonist also showed growth arrests and bone growth defects 9. Mechanistic studies revealed that even brief exposure to a Hh-inhibitor was enough to damage the growth plates by diminishing the numbers of reserve and proliferative chondrocytes 9. These findings further imply that it may not be possible to arrive at a dose that selectively targets tumor growth with no side-effects on bone development. Therefore, in children, a protective strategy for growth plate shielding without interfering with the desired anticancer effects of vismodegib in the neural tissue is highly desired. In vitro studies using cultured rat metatarsal bones 10 and in vivo studies in rabbits 11 and humans 12 have shown that radial shock wave treatment (rSWT), a non-invasive modality used in the clinic, have stimulatory effects on bone growth 10. Furthermore...

Humanin Treatment Protects Against Venetoclax-Induced Bone Growth Retardation in Ex Vivo Cultured Rat Bones

“…Of note, we observed that co-treatment with HNG not only prevented venetoclax-induced growth impairment but also restored the histomorphology and the organization of the growth plate chondrocytes. Interestingly, in a similar organ culture system we earlier reported that shockwave treatment can prevent growth retardation and growth plate disorganization caused by vismodegib, an Indian hedgehog inhibitor [ 29 ].…”

“…The height of the resting + proliferative (R + P) zone was measured at 10 different regions of the metatarsal growth plate, and the size of hypertrophic cells was averaged based on measurements from 10 different hypertrophic cells at the proximal and distal ends. A cell was considered hypertrophic if the longitudinal height was bigger than 7 μm, as previously described [ 29 ]. Analyses were performed in a blinded manner with Image J software (National Institutes of Health).…”

Exogenous Indian hedgehog antagonist damages intervertebral discs homeostasis in adult mice

“…Therefore, the clinical application of vismodegib regulating the Ihh signaling pathway needs to be particularly cautious, especially for juvenile patients with benign diseases. For example, the systematic application of vismodegib was reported to induce early epiphyseal closure in juveniles with cancers, which has received increasing attention [ [46] , [47] , [48] ]. To the best of our knowledge, the present study is the first to observe vismodegib causing early fusion of the metaphyseal in the vertebral body, and we further elucidate the possible mechanism that vismodegib causes IDD-like changes, at least in part, via inhibiting proliferation of IVDs chondrocytes and promoting apoptosis to disrupt cartilage homeostasis.…”

Micromechanical Loading Studies in Ex Vivo Cultured Embryonic Rat Bones Enabled by a Newly Developed Portable Loading Device

“…3 ). After dissection, each bone was transferred to a 24-well plate and cultured in minimum essential medium α (MEM-α, Invitrogen, Scotland) supplemented with 0.2% endotoxin-free BSA, 1 mM β-glycerophosphate, 0.05 mg/ml ascorbic acid and 20 μg/ml gentamycin at 37 °C under a humidified atmosphere with 5% CO 2 [ 10 ]. The medium was changed every 2 to 3 days.…”

“…The effects of mechanical loading on ex vivo cultured bones have been investigated in numerous studies [ 8 ]. Ex vivo cultured fetal metatarsal bones are a well-established model for studying longitudinal bone growth and responsiveness to different growth-regulating factors, including insulin-like growth factor-I (IGF-1), glucocorticoids and radial shock waves [ 9 , 10 ]. Additionally, the fetal femur bone has a high density of growth plate chondrocytes, as visualized by histological staining, thus providing great potential for ex vivo studies of longitudinal bone growth [ 11 ].…”