Modeling osteosarcoma invasion with fibroblast and tumor spheroids in functionalized hydrogels beads

: Osteosarcoma (OS) is a highly aggressive bone tumor with a strong propensity for metastasis. To develop a physiologically relevant in vitro platform for tumor invasion studies and drug testing, we engineered a three-dimensional OS model that mimics the tumor microenvironment using hydrogel-based multicellular spheroid beads. Tumor spheroids (∼125 μm) were generated and co-cultured with WS1 fibroblasts in VitroGel® RGD, a hydrogel matrix that promotes cell adhesion via integrin binding. The hydrogel beads, approximately 2-3 mm in size, supported robust growth of both MG63 and highly metastatic 143B cells. Notably, 143B spheroids exhibited pronounced invasion into the matrix, recapitulating their aggressive phenotype. To evaluate therapeutic potential, the antitumoral compound Licochalcone A (Lic-A) was applied to beads at varying concentrations. Lic-A induced a dose-dependent reduction in metabolic activity and membrane integrity, with minimal cytotoxic effects on stromal fibroblasts. Gene and protein expression analyses revealed that Lic-A significantly downregulated Ki67 and MMP-9, while increasing BMP2 and VEGF-C expression. Interestingly, vimentin mRNA was upregulated, yet its filamentous structure was disrupted, indicating stress- or apoptosis-related cytoskeletal remodeling. Live-cell imaging and SEM confirmed extensive 143B cell invasion, along with cytoskeletal changes and ECM degradation. Altogether, this study establishes a scalable 3D OS model that captures key features of the tumor-stroma interface and supports dynamic invasion processes. The model demonstrates the multifaceted anticancer effects of Lic-A and provides a valuable platform for studying OS biology and testing anti-metastatic therapies in a biomimetic setting.