Fusion-Negative Rhabdomyosarcoma xenografts in murine tongue provide a model for the study of local invasion, intravasation and metastasis at cellular and subcellular levels in live animals

Purpose PAX3/7 Fusion-negative rhabdomyosarcoma (FN-RMS) is a malignancy of the mesodermal lineage that predominantly occurs in children and adolescents in the head and neck and genitourinary system. Currently implemented therapeutic approaches improved the response and overall survival of patients with early-stage FN-RMS; however, treatment of metastatic or relapsed FN-RMS remains a challenge. Advances in our understanding of molecular mechanisms of invasion and metastasis might identify new therapeutic avenues for metastatic FN-RMS. As a step towards achieving a more complete understanding, we developed an orthotopic tongue xenograft model of FN-RMS for the examination of molecular basis and events of FN-RMS invasion and metastasis. Experimental design The behavior of human-derived FN-RMS cell lines injected orthotopically into the tongue of athymic nude mice was examined with a combination of in vivo bioluminescence imaging, non-invasive two-photon intravital microscopy (IVM), and histopathology. Tumor progression in this orthotopic xenograft model was compared to the two prevailing hindlimb intramuscular and subcutaneous xenograft models. Results FN-RMS cells were retained in the tongue and progressed to invade into muscle mysial spaces and neighboring tissues including mandibular bone and musculature and salivary glands, while evidence of local invasion was absent in tumors engrafted in the hindlimb or subcutaneously. Vascular invasion and hematogenous dissemination to the lungs occurred in tongue and intramuscular xenografts. Lymphatic dissemination and metastasis to the lymph nodes was only observed in tongue xenografts. IVM of tongue xenografts shows dynamic growth and shifts in cell morphology longitudinally. Transcriptome analysis revealed enrichment for significantly upregulated genes implicated in myogenic development and differentiation in tongue tumors but not submandibular metastases. IVM also shows dynamic interaction between FN-RMS cells and surrounding elements of the microenvironment including homing to blood and lymphatic vessels, lymphatic intravasation, and dynamic membrane protrusion formation. Conclusion Based on these findings, the tongue orthotopic xenograft of FN-RMS is a valuable model for short-term and longitudinal studies of tumor progression at the tissue, cellular and subcellular levels providing insight into kinetics and molecular bases of tumor invasion and metastasis and, hence, new therapeutic avenues for advanced FN-RMS. ### Competing Interest Statement The authors have declared no competing interest.