Evaluation of Proton-Induced DNA Damage in 3D-Engineered Glioblastoma Microenvironments

Glioblastoma (GBM) is a devastating cancer of the brain with an extremely poor prognosis. For this reason, besides clinical and preclinical studies, novel in vitro models for the assessment of cancer response to drugs and radiation are being developed. In such context, three-dimensional (3D)engineered cellular microenvironments, compared to unrealistic two-dimensional (2D) monolayer cell culture, provide a model closer to the in vivo configuration. Concerning cancer treatment, while X-ray radiotherapy and chemotherapy remain the current standard, proton beam therapy is an appealing alternative as protons can be efficiently targeted to destroy cancer cells while sparing the surrounding healthy tissue. However, despite the treatment's compelling biological and medical rationale, little is known about the effects of protons on GBM at the cellular level. In this work, we designed novel 3D-engineered scaffolds inspired by the geometry of brain blood vessels, which cover a vital role in the colonization mechanisms of GBM cells. The architectures were fabricated by two-photon polymerization (2PP), cultured with U-251 GBM cells and integrated for the first time in the context of proton radiation experiments to assess their response to treatment. We employed Gamma H2A.X as a fluorescent biomarker to identify the DNA damage induced in the cells by proton beams. The results show a higher DNA doublestrand breakage in 2D cell monolayers as compared to cells cultured in 3D. The discrepancy in terms of proton radiation response could indicate a difference in the radioresistance of the GBM cells or in the rate of repair kinetics between 2D cell monolayers and 3D cell networks. Thus, these biomimetic-engineered 3D scaffolds pave the way for the realization of a benchmark tool that can be used to routinely assess the effects of proton therapy on 3D GBM cell networks and other types of cancer cells. KEYWORDS: engineered cell microenvironments, two-photon polymerization, cancer, glioblastoma, proton therapy, DNA damage

Automated summary

A novel 3D-engineered scaffold inspired by the geometry of brain blood vessels was designed to assess the effects of proton therapy on glioblastoma (GBM) cells. The results showed a higher DNA double-strand breakage in 3D-cultured cells compared to 2D cell monolayers, indicating potential differences in radioresistance of GBM cells between different culture configurations. These biomimetic-engineered 3D scaffolds provide a benchmark tool for evaluating the effects of proton therapy on 3D GBM cell networks and other types of cancer cells.