Background: Rhabdomyosarcoma (RMS) is the most prevalent soft tissue sarcoma in children and adolescents. It is divided in two major histological subtypes, being the Alveolar (ARMS) the one associated to a poorer prognosis1. The cure rate for these tumors is up to 70% for children with localized disease, but this numbers decrease notably to a 20% 5-year survival rate for patients that show metastatic spreading. However, mechanisms leading to metastatic disease remain poorly understood. Hence, there is an urgent need to better understand this process, which will hopefully lead to novel targeted therapies and improved survival rates for these patients.
Methods: ARMS cells (RH4) were injected in the gastrocnemius muscle of Athymic Nude-Foxn1nu mice2. Primary tumors (800mm3) and peritoneal metastases (visible by IVIS®) were collected, and RNA extracted. Xenograft-derived metastatic cell lines were obtained directly as a primary culture from mice metastases. Characterization of paired tumors and metastases was performed using Clariom™ D microarray (Applied Biosystems). After data normalization and comparative analyses (R; RMA and limma), a list of differentially expressed transcripts between sample groups was obtained. These are being validated by qPCR and Western Blot.
Results: Transcriptomic profiling of tumors (n=3), paired metastases (n=3), xenograft-derived metastatic cell lines (n=3) and RH4 cell line (n=3) revealed 3420 differentially expressed transcripts (2747 increased in metastases, 673 in tumors). Several transcripts of interest are overexpressed in metastases samples compared to primary tumors as showed by microarray analysis and validated by qPCR. These targets are related in literature to metastasis in other cancers, as well as lymph nodes metastasis, which occurs to happen in our model and in ARMS clinics3. Additionally, some transcripts are overexpressed in tumor samples, which are linked to inhibition of proliferation and motility.
Conclusions/Future Work: In vitro phenotypic characterization of the obtained targets will involve proliferation (MTT), migration (Transwell®) and clonogenic assays, including loss-of-function (siRNA and commercial inhibitors). Using stable knock-out and knock-in models of the genes of interest, their role in ARMS metastasis will be further studied in vitro and in vivo. The clinical relevance of these targets will be explored in several cohorts of patients by immunohistochemistry on Tissue Microarrays. Studying the profile of paired tumors and metastases provides a unique tool to decipher the mechanisms of tumor progression. Fully understanding the metastatic process will ultimately lead to the development of better targeted therapies and thus to improved outcomes for ARMS patients. Also, these same steps are being followed in our laboratory for Ewing Sarcoma, another developmental tumor with discouraging survival rates for metastatic patients (<30%)4.
Citation Format: Sara Sanchez-Serra, Mariona Chicon-Bosch, Paola Monaco, Laura Santana-Viera, Susana Maqueda-Marcos, Manuel Gris-Lorente, Roser Lopez-Alemany, Oscar M. Tirado. Transcriptomic analysis to unveil alveolar rhabdomyosarcoma metastatic process [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 1299.
