Engineered 3D osteosarcoma microenvironment model: Bridging in vitro - in vivo gap in cancer research and anticancer drug screening

Abstract

Introduction Current treatments for osteosarcoma typically include surgical excision followed by neoadjuvant and adjuvant chemotherapy. Research attempts to streamline and improve these treatments, but progress is slow mostly due to limited translation of in vitro to in vivo studies. The aim of this work was to develop and validate an engineered three-dimensional (3D) osteosarcoma model based on macroporous composite scaffolds, as cell carriers, and biomimetic perfusion bioreactor for osteosarcoma research and anticancer drug screening. Material and method The scaffolds (4 mm thick discs, 9 mm in diameter) were produced by controlled gelation of hydroxyapatite (HAP) suspension in Na-alginate solution (2 wt.% alginate and 2 wt.% HAP) followed by freeze-drying and rehydration in the culture medium. Murine osteosarcoma K7M2-wt cells were seeded onto the scaffolds (15x106 cells cm-3 scaffold volume) and cultivated for 7 days in "3D Perfuse" bioreactors under continuous medium superficial velocity of 40 μm s-1, while static cultures served as a control. To evaluate this model for anticancer drug screening, bioreactor cultures were treated with doxorubicin (1 μg cm-3), on day 1 (first study) or on day 7 (second study) and lasted for 1 day, while untreated bioreactor culture served as a control. The scaffolds were assessed regarding the cell metabolic activity by MTT, morphology and distribution by histological and scanning electron microscopy analyses. Masson-trichrome and reticulin staining were used for extracellular matrix (ECM) analysis, while quantitative real-time PCR (qRTPCR) assessed osteosarcoma marker expression.Result and discussion After short-term cultures, biological assessment showed that the cells stayed viable and metabolically active, produced ECM, expressed osteosarcoma markers and spontaneously formed aggregates under both culture conditions. However, cells in the bioreactor culture exhibited higher metabolic activity, while the cell aggregates were slightly larger (~1.2-fold), more compact with higher amounts of reticular fibers, more numerous and more uniformly distributed throughout the scaffold compared to the static culture. These results could be explained by positive effects of flow on cells due to enhanced mass transport and adequate hydrodynamic shear stresses. Evaluation of the model for anticancer drug screening has shown a negligible effect of doxorubicin on individual cells as well as cell aggregates implying that the developed model more closely mimics in vivo drug responses than 2D cultures. Conclusion This study has shown potentials of engineered 3D osteosarcoma microenvironment model based on macroporous composite scaffolds, and perfusion bioreactor for relevant and reliable osteosarcoma research and anticancer drug screening.EACR 2025 Congress Abstract