Abstract 139: Modeling and genomic profiling of osteosarcoma development in murine chondrocyte-origin models

Abstract Osteosarcoma (OS) manifests during puberty, coinciding with rapid bone growth, a feature requiring exploration. We selectively deleted p53 or both p53 and Rb1 alleles in growth-dependent chondrocyte-origin models, utilizing Col10a1-Cre or Agc-CreERT2, incorporating with ROSA-Tomato for tracing and isolating the mutant cell collection. Col10p53flfl(tm) and Col10p53flflRbfl+(tm) mutants exhibited 100% penetrance in osteoblastic OS, with the latter displaying shortened latencies (mean: 335 vs 265 days) and increased metastases. Tamoxifen-induced AgcCreERT2 models highlighted growth stage-dependent OS development, correlating penetrance inversely and latencies positively with mouse age at tamoxifen treatment. Tumors exclusively occurred in endochondral bones, near growth plates. Whole-genome profiling of tm+ tumor cells revealed genetic variations, including SNPs, Indels, CNVs, and SVs. CNVs involved genes altered in human OS, emphasizing losses in Dmd and gains in Myc, Vegfa, Foxm1, Akt1, Aurkb, and Ccen1. A common CNV (losses, Chr. 10: 34,158,531-34,443,999, Log ratio ←0.05) occurred in all sequenced primary and metastatic samples, with male specimens sharing a loss in the Y chr. p region (approximately 725,200-799,409). Orthotopic transplantation of tm+ p53− stromal cells from 4-month-old Col10p53flfltm mice gave rise to bone tumors and lung migration in wild-type recipients, explicitly establishing the transplanted cells as the direct origin of the cancer precursors within early tm+ p53− stromal populations. Whole-genome sequencing unveiled a discernible progression of genomic alterations from 1 to 4-month-old tm+ p53− stromal cells, culminating in donor-derived tumors. Notably, only these specific donor-derived tumors, and not the early tm+ p53− stromal cells, exhibited the Chr. 10 CNV observed in autochthonous malignancies. Micro-metastases, identified by fluorescence imaging, were prevalent, alongside macro-metastases (~10% in Col10p53flflRbfl+tm and ~25% in AgcCreERT2p53flflRbfl+tm cohorts). Notably, amputation of primary OS sites in mice surviving longer revealed larger and more abundant tm+ cell clusters. Some micro-metastases not only expressed signature genes typical of macro-lung metastases but also shared genomic variations with corresponding primaries, including the prevalent loss in Chr. 10. Enhanced immune cell infiltration was evident in lungs hosting micro-metastases, suggesting a potential immune response triggered by pre-tumor early seeding cells. Murine OS models faithfully replicate human OS features, underscoring chondrocytes as significant contributors to OS cell sources. The inclusion of a tomato reporter enables precise tracking, rendering these models invaluable for studying OS etiology, particularly in unraveling pre-tumor molecular events in both primary and metastatic settings. Citation Format: Xin Zhou, Hannah Beird, Yanhua Yi, Zhaohui Xu, Zhongting Zhang, Harjeet Singh, Wendong Zhang, Yifei Wang, Sylvester Jusu, Michael Roth, Jonatha Gill, Richard Gorlick. Modeling and genomic profiling of osteosarcoma development in murine chondrocyte-origin models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 139.