A novel sarcoma tumoroid platform preserving the tumor microenvironment to guide personalized immunotherapy

Sarcomas are rare, aggressive mesenchymal cancers with over 80 subtypes. Although they represent a small fraction of adult cancers, sarcomas comprise over 20% of pediatric solid tumors and carry poor outcomes for patients with advanced disease. Preclinical modeling remains challenging: traditional 2D cultures fail to preserve the tumor microenvironment (TME), while conventional 3D organoid platforms rely on enzymatic dissociation, which disrupts cellular interactions essential for TME integrity. Patient-derived xenografts, though more representative, are costly and low-throughput. To address these limitations, this study aims to develop reproducible 3D sarcoma tumoroids as a translational platform for functional studies and therapeutic testing that preserves the TME. Freshly resected sarcoma specimens were mechanically cut into small fragments without enzymatic dissociation, preserving the TME in each fragment, and cultured in suspension or matrix-embedded conditions. Media formulations and growth factor combinations were evaluated across sarcoma subtypes to optimize culture performance. Tumoroid formation and growth were monitored for 14 days using brightfield microscopy, quantitative area analysis, and alamarBlue viability assay. Structural integrity and TME preservation were assessed by hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC). Cytotoxic response assays were performed using standard chemotherapeutic agents, including doxorubicin and methotrexate, to evaluate drug sensitivity in 3D culture over a 5-day treatment period. Established tumoroids maintained consistent 3D morphology and viability over 14 days, with Mammocult medium plus growth factors supporting the strongest proliferation. Quantitative growth analysis showed a progressive increase in tumoroid area and metabolic activity, indicating stable expansion under optimized conditions. H&E staining confirmed retention of tumor cells, stroma, and histologic architecture across osteosarcoma, dedifferentiated liposarcoma, and chondrosarcoma for over 10 days. IHC staining for CD45 confirmed the presence of immune and stromal cell populations within the cultures, indicating preservation of the native TME. Chemotherapeutic testing using methotrexate and doxorubicin revealed cytotoxic effects through H&E and Live/Dead staining, validating the model's responsiveness and functional relevance. These findings demonstrate that mechanically dissociated sarcoma tissues can generate viable, subtype-representative tumoroids while maintaining key TME features. Future efforts will focus on expanding therapeutic testing, integrating immune co-culture systems to better recapitulate the sarcoma TME, and applying spatial transcriptomics and single-cell RNA sequencing to uncover cellular heterogeneity and guide precision oncology for sarcoma patients.