Understanding clonal heterogeneity and mechanisms of methotrexate resistance in two osteosarcoma PDX models.
Authors: Sylvester Jusu, Giuseppe S. A. Longo-Sorbello, Zhang Zhongting, Xu Zhaohui, Michael Roth, Douglas James Harrison, Jonathan Benjamin Gill, Richard Greg Gorlick
Journal: Journal of Clinical Oncology (2024)

e23512 Background: Osteosarcoma is a primary malignant bone tumor that is known to have significant intratumoral heterogeneity with branched cancer evolution. The lung is the most frequent site of metastatic disease and relapse occurs in more than 30 % children and young adults. It has been revealed that the metastatic ability and resistance of cancers including osteosarcoma might be due to the presence of a subset of cells within the intra-tumorally heterogeneous population. We investigated the emergence of resistant clonal subpopulations following targeted methotrexate treatment in two patient derived xenograft (PDX) models in vitro and in vivo. Our aim was to not only track the subclonal population of cells that establish in the lungs but also to identify clonal drivers and the mutational signature of the genes and pathways involved in MTX resistance. Methods: We previously established 20 barcoded osteosarcoma PDX models and demonstrated that the application of methotrexate (MTX-IC95) treatment as a selective pressure in seven models produces clonal subpopulations that were more resistant to MTX. In the current work we used barcoded next generation and RNA sequencing techniques to map the clonal and transcriptomic architecture of MTX resistant single clones in M36 and OS252 PDX models. We next injected the barcoded PDX cells intratibially in twenty severe combined immunodeficiency disease (SCID) mice to track the clonal population/subpopulation of cells that metastasize to the lungs. Following primary tumor growth, limb amputation was carried out and mice were treated with methotrexate or vehicle for two weeks. Primary tumor, blood and lung samples were collected after death from metastasis or euthanasia. All samples collected were snap frozen in liquid nitrogen and stored at -80 0 C. DNA and RNA was extracted and analyzed by PCR, NGS or WES to map clonality, mutational and evolutionary profiles. Results: MTX treated single clones showed diversity and barcode abundance when compared to vehicle; barcode abundance reduced in the resistant clonal subpopulations when compared to the vehicles. Ten unique subclones were shared in both vehicle and MTX resistant clones in M36 while OS252 had only one shared subclone respectively. More than 50% of the injected mice developed primary tumors and matched lung micrometastases. We are currently analyzing both targeted and RNA sequencing results to further understand clonality, the mutational signature of the genes (DHFR, MTR, FPGS, GGH) and pathways involved in MTX resistance. Conclusions: We investigated mechanism of methotrexate resistance in two barcoded PDX models in vitro and in vivo using cytotoxicity, next generation and RNA sequencing techniques. Targeted amplicon sequencing of key genes involved in antifolate pathway will yield new information on clonal evolution and mechanisms of resistance.