LAUSR

Introduction: Osteosarcoma is a primary malignant bone tumor characterized by the production of spindle cells resulting in immature bone formation. It has been revealed that metastatic cancers including osteosarcoma have a subset of cells with different phenotypic and genetic signatures conferring advantage to drive progression and drug resistance within the intra-tumorally heterogeneous population. However, the lack of a defined in vivo model that can identify the rare genetic subpopulation and recapitulate clonal evolution has been a challenge. In this study, we used an established model to track and identify the clonal subpopulations and assess the genetic signatures that confer specific advantages selecting for uncontrolled growth, adaptation and colonization in the lungs. Methods: We injected barcoded M36 PDX osteosarcoma cells intratibially in ten severe combined immunodeficiency disease (SCID) mice to track the clonal population/subpopulation of cells that metastasize the established primary tumors to the lungs. Limb amputation was performed on the tumor bearing leg upon detection of palpable tumor. Lung tissue was harvested post-amputation following death from metastasis or euthanasia. DNA and RNA from frozen tissues was extracted and analyzed by PCR, NGS, WES and RNA sequencing to map clonality, mutational and evolutionary profiles. Results: Limb amputation was performed in 7 mice with all mice surviving the surgery. The mean mouse survival following amputation was 46.9 days and the longest survival was 167 days. Four (40%) of the primary tumor bearing mice had lung metastasis. A Shannon-Wiener and Jaccard similarity index show a diversity in clonal architecture between the primary tumors of all mice injected on the same day and a diversity in clonal population in both the primary tumors and their matched lung metastatic nodules. Clonal mapping further revealed 21 unique clones shared in all primary tumors examined. However, the clonal frequencies and distribution varied within each primary tumor and across the tumor samples. Interestingly, clonal frequencies increased in the lung metastatic nodules when compared to their matched primary tumors. Four distinct clones that were present in all the primary tumors were also found in all lung metastatic samples. These unique clones, here in called “seeding clones” may play an important role in the lung metastatic process. Conclusions: We previously showed that the barcoded osteosarcoma PDX models are inter and intratumorally heterogenous both in vitro and in vivo. In the current model, we identify the multiple tumorigenic seeding clones that potentially act as drivers in the lung metastatic process. Furthermore, we show the dynamic evolutionary relationships between the clones by reconstructing a phylogenetic map of the M36 PDX tumor model. Funding: Swim Across America, Foster Foundation and the Barbara Epstein Foundation. Citation Format: Sylvester Jusu, Zhongting Zhang, Zhang Wendong, Zhaohui Xu, Michael Roth, Jonathan Gill, Richard Gorlick. Clonal mapping phylogenetic and transcriptomic analysis of an M36 PDX amputation lung metastatic osteosarcoma model. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5932.