Myxofibrosarcoma characterization to generate novel preclinical models and test tailored therapies

Introduction Myxofibrosarcoma (MFS) is a rare and biologically heterogeneous soft tissue sarcoma characterized by a high rate of local recurrence, marked genomic instability, and limited availability of representative preclinical models. Although alterations of the TP53 pathway are frequently reported, their functional significance and relationship with the tumor microenvironment (TME) remain incompletely understood. The aim of this study was to generate patient-derived preclinical models of MFS, characterize their molecular and immunological features, and evaluate their response to therapeutic agents. Methods Tumor specimens and plasma samples were collected from seven MFS patients treated at IRCCS Ospedale Policlinico San Martino between 2023 and 2025. Primary cell lines were established from surgical samples and characterized through morphological, immunophenotypic, molecular, and functional analyses. The TME was investigated using multiparametric flow cytometry, while soluble immune mediators were assessed in plasma by Ella multiplex assays. TP53 status was evaluated through Western blotting, Functional Analysis of Separated Alleles in Yeast (FASAY), and Sanger sequencing. Drug sensitivity to doxorubicin and palifosfamide was assessed in 2D cultures, and three-dimensional collagen-based scaffolds were used to reproduce tumor architecture in vitro. Results Three stable MFS primary cell lines were successfully generated and maintained in long-term culture, preserving fibroblast-like morphology and heterogeneous proliferative behavior. Immunophenotypic analyses demonstrated high expression of HLA class I molecules and stromal markers, whereas PD-L1 expression was variable. TME characterization revealed a markedly immunosuppressive milieu enriched in exhausted PD-1⁺ T cells, regulatory T cells, M2-like macrophages, and myeloid-derived suppressor cells. Plasma analyses showed increased IFN-γ levels and detectable soluble immune checkpoint molecules, supporting the presence of chronic immune activation and T-cell exhaustion. Functional characterization of TP53 demonstrated alterations in the majority of analyzed MFS models, including heterozygous and homozygous pathogenic mutations. Irradiation experiments highlighted differential activation of the p53/p21 pathway among cell lines, suggesting functional heterogeneity of TP53 alterations. Drug testing demonstrated dose-dependent sensitivity to doxorubicin and palifosfamide, with greater efficacy observed for doxorubicin. Finally, 3D collagen-based cultures reproduced morphological features more closely resembling the original tumors, supporting their value as biologically relevant preclinical platforms. Conclusion Overall, this work provides an integrated molecular and immunological characterization of MFS and establishes novel patient-derived preclinical models for future translational studies. These findings support the development of biology-driven therapeutic strategies and combinatorial approaches targeting both tumor cells and the immunosuppressive microenvironment in myxofibrosarcoma.