Abstract 3794: Comprehensive clonal and molecular profiling of primary tumors and distant lung metastases in an amputation model of osteosarcoma
Authors: Jusu Sylvester , Kannan Sankaranarayanan , Seth Sahil , Peoples Michael D. , Zhang Zhongting , Zhang Wendong , Xu Zhaohui , Wang Yifei , Zhou Xin , Tu Yizheng , Longo Giuseppe , Roth Michael , Gill Jonathan B. , Gorlick Richard 
Published: June 2022

An important feature of osteosarcoma is relapse which occurs in more than 30% of patients and metastasis to the lungs. It has been revealed that the metastatic ability and resistance to chemotherapy might be due to the presence of a subset of cells within the intra-tumorally heterogeneous population. However, tracking the rare genetic subpopulations that play a critical role in cancer recurrence, metastasis and resistance and the lack of a defined in vivo model to recapitulate clonal evolution has been a challenge.

To further understand clonal dynamics and intra-tumoral heterogeneity a new barcoded PDX (OS17) derived cell line was established by stably transducing the cell line with a lentiviral vector-based system. The highly complex barcode library of ten million enabled labelling each of the cells with a unique molecular identifier and clonally tracking more than one million cells in vitro. Following seventeen passages in vitro and transplant for three generations in vivo, flow cytometry, nested PCR and next generation sequencing showed that the barcoded cells retained the sequence in vitro and in vivo suggesting that it was possible to track cell populations and their clonal lineages both at the genomic DNA and RNA level.

The barcoded M17 PDX cells were injected in 25 SCID and NSG mice intratibially to monitor tumor growth and clonally track the cells in both the primary tumors and the lungs. Seventeen (68%) of the mice developed primary tumors. Tumor latency and growth was 61.82 ±34.5 and 51.24 ± 23 days respectively. Seven (35%) of the primary tumor bearing mice had lung metastasis. Limb amputations were performed in 5 mice with 2 surviving the surgery.

The results not only show a diversity in clonal subpopulations between the mice injected on different dates but also the primary tumor and matched lung metastatic samples. An in-depth clonal characterization further revealed a high degree of similarity in the subclonal populations between the lung nodules and the primary tumor samples. In vitro, a proportion of the barcodes were very reduced or lost in the early six passages however, these slowly expanded in the late passages suggesting that in a more favorable environment in vivo, these clones can maintain tumorigenic potential.

These results suggest a clonal evolutionary dynamic model where pre-existing clones have a fitness for persistence and dissemination. This advantage might include long term tumorigenic and proliferative potential and the ability to grow in different micro-environments. Conclusion: It has been demonstrated that the barcoded PDX models have inter and intra-tumoral heterogeneity of osteosarcoma both in vitro and in vivo and can potentially be used in tracking tumor colonization at the primary and metastatic sites. Using this model will therefore advance our understanding of osteosarcoma lung metastasis which is the critical clinical challenge for these patients.

Citation Format: Sylvester Jusu, Sankaranarayanan Kannan, Sahil Seth, Michael D. Peoples, Zhongting Zhang, Wendong Zhang, Zhaohui Xu, Yifei Wang, Xin Zhou, Yizheng Tu, Giuseppe Longo, Michael Roth, Jonathan B. Gill, Richard Gorlick. Comprehensive clonal and molecular profiling of primary tumors and distant lung metastases in an amputation model of osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3794.