Metastatic osteosarcoma induced by inactivation of Rb and p53 in the osteoblast lineage

Abstract

Mutation of the RB-1 and p53 tumor suppressors is associated with the development of human osteosarcoma. With the goal of generating a mouse model of this disease, we used conditional and transgenic mouse strains to inactivate Rb and/or p53 specifically in osteoblast precursors. The resulting Rb;p53 double mutant (DKO) animals are viable but develop early onset osteosarcomas with complete penetrance. These tumors display many of the characteristics of human osteosarcomas, including being highly metastatic. We established cell lines from the DKO osteosarcomas to further investigate their properties. These immortalized cell lines are highly proliferative and they retain their tumorigenic potential, as judged by their ability to form metastatic tumors in immunocompromised mice. Moreover, they can be induced to differentiate and, depending on the inductive signal, will adopt either the osteogenic or adipogenic fate. Consistent with this multipotency, a significant portion of these tumor cells express Sca-1, a marker that is typically associated with stem cells/uncommitted progenitors. By assaying sorted cells in transplant assays, we demonstrate that the tumorigenicity of the osteosarcoma cell lines correlates with the presence of the Sca-1 marker. Finally, we show that loss of Rb and p53 in Sca-1-positive mesenchymal stem/progenitor cells is sufficient to yield transformed cells that can initiate osteosarcoma formation in vivo.

Fig. 1.

A mouse model of metastatic osteosarcoma. (A) Kaplan–Meier plot of the indicated genotypes carrying Osx1-GFP::Cre up to 12 months of age. (B–I) Analyses of osteosarcomas and associated metastases arising in DKO mice. (B) 3D reconstructed images from microComputerised Tomography are shown for a control femur (Left) versus a femur containing an osteosarcoma (Right). Central panels show 2D images at the indicated positions. Note the loss of bone cortex and the presence of bone spicules located in the tumor that has grown beyond the periosteum (arrows). (C) Histological analyses of an osteosarcoma in a femur show areas of bone cortex erosion (Left, arrow) and the presence of little mineralized bone within the tumor (Right). (D–G) Analysis of a representative snout tumor by soft x-ray image to show the typical sunburst pattern (arrow) (D), H&E staining and analysis of adjacent sections of undecalcified tumor (E) with Alizarin Red to detect calcified bone matrix (F) or Sirius Red to detect collagen (G). (H and I) Representative examples of osteosarcoma metastases (arrow), in lung (H) and liver (I) containing detectable bone matrix. (Magnification: C and E ×2; F–I ×40.)

Fig. 2.

OS cell lines can form bone tumors in immuno-compromised mice. (A and B) H&E stained section of the primary osteosarcomas 985 and 2380, respectively. Tumors derived from s.c. (C–E) or i.v. (F–H) injection of DKO-OS-985. (C and F) H&E staining. Adjacent sections were stained with either Alizarin Red (D, G) or Sirius Red (E, H) to stain calcified bone matrix and collagen, respectively. (Magnification: A–C and F ×40; D, E, G, and H ×2.)

Fig. 3.

Osteosarcoma cells lines are multipotent in vitro. DKO-OS-985 cells were induced to differentiate into the bone (Left) and fat (Right) lineages and assayed at the indicated time points (days). Mineral deposits were stained with Alizarin Red (AR) as a marker for osteogenic differentiation. Oil-Red O (ORO) was used to stain lipid droplet accumulation during adipogenic induction. Cells were pulsed with BrdU to determine the proliferative status during differentiation. Expression of differentiation markers for bone and fat was determined by qRT-PCR.

Fig. 4.

Sca-1 expression and Rb- and p53-loss are both required for efficient tumorigenesis in vivo. Sca-1 expression in DKO-OS-985 and DKO-OS-2380 cell lines versus flox MSC/MPCs (A) or flox MSC/MPC+Ad-Cre cells, in which Rb and p53 have been inactivated, versus flox MSC/MPCs (C). (B) Tumors arising in immunocompromised mice injected s.c. with 10⁵ DKO-OS-985 cells sorted for either ScaI^low/− or ScaI^high. (D and E) Tumors arising in immunocompromised mice injected s.c. with 10⁶ flox MSC/MPC+Ad-cre stained for Alp expression (D) or Alizarin Red (E). Sca-1 expression in primary DKO osteosarcomas (F) and DKO MSC/MPCs versus flox MSC/MPCs (G).