Optimising osteosarcoma differentiation

Abstract

Osteosarcoma (OS) is a primary bone malignancy characterised by disrupted terminal differentiation. Despite advances in conventional treatments, the prognosis remains poor, especially for patients with metastases. This highlights the urgent need for novel therapeutic approaches. Therapeutic strategies aimed at restoring osteogenic differentiation offer a promising targeted approach to improve treatment efficacy and enhance patient survival. This study aims to optimise differentiation in SaOS-2 cells using ecdysteroids and bone remodelling agents, while optimising experimental parameters to ensure robust and reproducible data that supports their therapeutic use. Experimental conditions, including cell seeding density, alkaline phosphatase (ALP) lysis buffer choice, and flow cytometry plate formatting were optimised. A seeding density of 4000 cells per well ensured consistent spectrophotometric data and morphological analysis, 1% Triton X-100 buffer provided the most reliable ALP activity measurements, and 48-well plates ensured efficient cell yields for resource-efficient flow cytometry analysis. Following optimisation, cells were treated with ecdysteroids and bone remodelling compounds, and their effects on cell viability and differentiation were assessed through 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) and ALP assays. The most promising results were assessed morphologically by Alizarin Red S staining and cell cycle analysis. Ajugasterone C, Rubrosterone, 20-hydroxyecdysone, and Polypodine B were found to enhance early osteogenic markers and promote calcium deposition, to different extents, indicating osteogenic differentiation potential, whilst Alendronate and Dexamethasone predominantly exerted anti-proliferative effects. These findings suggest that some ecdysteroids have differentiation-inducing potential in OS cells, whilst remodelling agents primarily exert cytostatic effects. Therefore, it would be worthwhile for future studies to explore late-stage differentiation markers and combinatorial regimens, to validate and advance the therapeutic potential of differentiation-based strategies in OS.