Inherent tissue homeostasis of the juvenile metaphysis provides a foundation for osteosarcoma development.

Osteosarcomas preferentially arise in the metaphysis of juvenile long bones, near the growth plate, unlike most cancers, whose incidence increases with age. Here, we show that p21, a negative regulator of the cell cycle, is induced in proliferating juvenile metaphyseal osteoblasts in response to DNA replication-associated damage. Single-cell RNA sequencing defines a differentiation hierarchy from multipotent progenitors to mature osteoblasts and identifies immature osteoblasts enriched for proliferation and replication stress responses. p21-positive metaphyseal osteoblasts associate with growth plate Indian hedgehog expression and decline after growth plate maturation or Hedgehog inhibition. c-Myc induction selectively promotes juvenile osteoblast proliferation despite p53 activation, but this proliferative response remains Hedgehog-dependent and ceases after growth plate maturation. By contrast, p53 inactivation enables sustained Hedgehog-independent proliferation of c-Myc-induced osteoblasts and lung metastasis. These findings reveal juvenile metaphyseal tissue homeostasis as a potential basis for the age of onset, anatomical specificity, and mutational profile of human osteosarcomas.