Abstract 3537: A novel orthotopic xenograft model of spontaneous metastasis for rhabdomyosarcoma using lingual intramuscular injection
Authors: Hebron Katie E. , Isanogle Kristine , James Amy , Difilippiantonio Simone , Yohe Marielle E. 
Published: April 2023

Abstract
Rhabdomyosarcoma (RMS), a cancer characterized by the defective differentiation of skeletal muscle precursor cells, is the most common pediatric soft tissue sarcoma. Despite significant efforts to characterize the transcriptomic and genetic abnormalities of the disease, the prognosis for patients with advanced or refractory disease has remained unchanged for more than four decades. Preclinically, studies have been hampered by insufficient animal models. For example, the hindlimb orthotopic model, an injection into the gastrocnemius muscle of immunocompromised mice, produces highly encapsulated, non-invasive, non-metastatic tumors. While this model has shown a reasonable recapitulation of tumor proliferation response, investigators using this model are unable to assess the effect of their investigational treatment on local invasion or distant metastasis. Moreover, transgenic mouse models of RMS, while commonly metastatic, diverge significantly from the transcriptomic signature of human tumors. With these considerations, we hypothesized that an alternative orthotopic site, the tongue, may provide a more representative xenograft model of RMS. In a proof of principal experiment, the fusion negative RMS (FN-RMS) cell line RD was injected into the skeletal muscle of the tongue, a lingual intramuscular (LIM) injection. Tongue tumors establish in approximately 60-80% of mice injected within approximately 40 days. By histopathology, the primary tumors show evidence of local invasion. Interestingly, approximately 60% of these tumors also spread to local cervical lymph nodes, a process not observed in hindlimb orthotopic models or subcutaneous models. We expanded our investigation to include additional FN-RMS cell lines: RMS-YM, SMS-CTR, and a MEK inhibitor resistant cell line CTR-557. Mice injected with RMS-YM cells established tumors in at least 60% of mice by 75 days post injection. Additionally, enlarged local and distant lymph nodes are observed in 50% of these mice. Intriguingly, mice injected with SMS-CTR or its MEK inhibitor resistant derivative, CTR-557, frequently have tumors in both local and distant lymph nodes without evidence of a primary tongue tumor. One mouse in each group also had pulmonary metastases. Taken together, this model not only demonstrates the first orthotopic xenograft model of spontaneous metastasis for FN-RMS, it also mimics the spread of disease that is observed clinically in RMS originating in the head and neck. Ongoing and future studies include 1) generating highly metastatic human FN-RMS cell lines; 2) using spatial transcriptomics to compare the tumor microenvironment of non-metastatic hindlimb tumors and invasive and/or metastatic lingual tumors; and 3) identifying drivers of FN-RMS dissemination through transcriptomic, genome accessibility, and phospho-proteomic methods.
Citation Format: Katie E. Hebron, Kristine Isanogle, Amy James, Simone Difilippiantonio, Marielle E. Yohe. A novel orthotopic xenograft model of spontaneous metastasis for rhabdomyosarcoma using lingual intramuscular injection. [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 3537.