Targeting MDA-MB-231 Cancer Stem Cells With Temsirolimus in 3D Collagen/PGA/Na2SiO3-Based Bone Model

Abstract

This study evaluated the efficacy of temsirolimus in a 3D model mimicking breast cancer stem cell (CSC) metastasis to bone. A composite material (collagen/PGA/Na₂SiO₃) is used to create a scaffold with bone marrow mesenchymal stem cells (BM-MSCs) and human umbilical vein endothelial cells (HUVECs). MSCs maintained over 80% viability for 21 days on the scaffold. Calcium analysis shows increased calcium release in both control (3-fold, 2.7-fold) and osteogenic (2.3-fold, 2.5-fold) mediums on days 14 and 21. Gene expression analysis reveals higher levels of Osteopontin (OPN) (6-fold), Osteocalcin (OCN) (12-fold), and RUNX Family transcription factor 2 (RUNX2) (1.8-fold) in the osteogenic medium (p < 0.05). The impact of 5 µM temsirolimus treatment for 6 hours is assessed under control and dynamic culture conditions, reducing mammalian target of rapamycin (mTOR)-related protein levels (phospho (p)-mTOR and p-AKT) in CSCs. The composite material is characterized through viscosity and compression testing, confirming its suitability for supporting osteogenic differentiation and cell viability (WST-1, Alizarin Red Staining, Calcium Assay, RT-qPCR). Gene expression of CSCs, selected based on CD133 expression, shows elevated stemness-associated genes (OCT4, NANOG), EMT (MMP2, CXCR4, CDH1, CDH2), and drug resistance (ABCG1, ABCG2) in the CD133⁺ group in dynamic condition. Temsirolimus treatment reduces the expression of these genes by 21-fold to 7.52-fold (p < 0.05). These findings suggest temsirolimus as a promising therapeutic for CSC metastasis to bone. [Correction added on April 8, 2025, after first online publication: the word CHD is updated to CDH in this version.].