Investigation of chemotherapy resistance mechanisms and therapeutic vulnerabilities in rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is a rare and aggressive malignant tumor of mesenchymal origin, representing 7–8% of all pediatric cancers and the most common soft tissue sarcoma in children and adolescents. The current standard treatment consists of chemotherapy with ifosfamide, vincristine, and actinomycin D (IVA), combined with surgery and radiotherapy. Despite improvements in survival rates for low- and intermediate-risk patients, high-risk, recurrent, and metastatic RMS still have a poor prognosis, with survival rates below 30%. The biggest challenge in treating RMS is the development of chemoresistance, leading to tumor recurrence and treatment failure. A recent 2024 study identified the presence of quiescent progenitor cells and cells with neurodevelopmental gene expression in RMS, which increased after chemotherapy, suggesting unknown resistance mechanisms associated with these cell subpopulations. This project aims to investigate the biological mechanisms underlying chemoresistance in various RMS subtypes using patient-derived organoids (PDOs) and organoids obtained from patient-derived xenografts (PDXOs). The study will focus on: (a) establishing and performing the histological and genetic characterization of PDOs and PDXOs; (b) establishing a chemoresistance protocol to IVA in RMS organoids, characterizing resistance by determining IC50 and expression of resistance markers; (c) evaluating gene expression and characterizing resistant subpopulations through single-cell RNA sequencing (scRNA-seq) to identify biomarkers associated with resistance and vulnerabilities in RMS; (d) characterizing mutations acquired after a chemoresistance protocol; and (e) conducting a screening of molecules from natural products of Brazilian biodiversity capable of reversing chemoresistance. This project will contribute to the understanding of the resistance mechanisms of rhabdomyosarcoma, a rare cancer with few effective therapeutic options. Understanding tumor biology and resistance mechanisms in RMS, as well as developing a preclinical model, may aid in the development of innovative and more personalized therapeutic strategies, opening new perspectives for reversing chemoresistance and improving patient prognosis. Keywords: Chemoresistance, Tumor heterogeneity, Molecular screening, Rhabdomyosarcoma