Effect of hyaluronic acid on drug resistance in 3D in vitro models of brain tumors

BACKGROUND: On-treatment drug resistance is a major concern in brain tumor therapy, significantly reducing the efficacy of conventional chemotherapy. Three-dimensional (3D) cell models are a powerful tool in experimental oncology for assessing drug resistance mechanisms in tumor cells, including the role of extracellular matrix-activated signaling pathways.

AIM: The work aimed to assess the effect of hyaluronic acid on doxorubicin resistance in 3D in vitro models of brain tumors.

METHODS: 2D monolayer cultures were obtained from continuous cell lines of brain tumors in laboratory animals. The cell lines were derived from the unique collection of the A.P. Avtsyn Research Institute of Human Morphology of the B.V. Petrovsky Russian Research Center of Surgery. 3D spheroids were formed using ultra-low attachment plates with or without hyaluronic acid. The cytotoxicity of doxorubicin was assessed using the ССК-8 kit. Fluorimetry was used to measure doxorubicin efflux efficiency and CD44 production, whereas flow cytometry was used to assess the fraction of CD44+ cells. The expression of genes responsible for drug resistance was assessed using real-time polymerase chain reaction.

RESULTS: The IC50 of doxorubicin in 2D models was as follows: 180 nM for oligodendroglioma 51/7, 280 nM for glioblastoma 14-60-4, 500 nM for astrocytoma 10-17-2, and 2750 nM for neurinoma RGGN2. The IC50 of doxorubicin determined for 2D models had no significant effect on the viability of cells in spheroids. However, a fourfold increase in its concentration induced a cytotoxic effect, the severity of which was determined by spheroid compaction. Hyaluronic acid increased doxorubicin resistance in 3D models of brain tumors in a dose-dependent manner. This effect was associated with CD44 receptor activation and enhanced drug efflux from cells, which were mediated by changes in the expression of ABC transporter genes (Abcb1, Abcg2, Abcc1) and DNA repair genes (Mgmt, Top2a).

CONCLUSION: The findings clarify the role of hyaluronic acid and the CD44 receptor in drug resistance in brain tumors, facilitating the development of more relevant in vitro models. Furthermore, the findings have practical significance for developing drug resistance-overcoming strategies, such as targeted inhibition of ABC transporters or suppressed interactions between hyaluronic acid and its CD44 receptor.