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

Abstract

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.

Two-Photon Polymerization of 2.5D and 3D Microstructures Fostering a Ramified Resting Phenotype in Primary Microglia

“…The most significant limitation of 2PP is the relatively long printing time hindering the upscaling of the technology ( Moroni et al, 2018 ). Multiple polymeric, hydrogel or composite materials were employed in combination with 2PP to fabricate micro- and nano-patterns as well as 3D structures for in vitro cellular studies involving neuroblastoma, glioblastoma, prostate cancer, murine cerebellar granule, chondrocytes, macrophages, neuronal and stem cells ( Marino et al, 2013 ; Accardo et al, 2017 , 2018 ; Turunen et al, 2017 ; Maciulaitis et al, 2019 ; Fendler et al, 2019 ; Babi et al, 2021 ; Bertels et al, 2021 ; Maciulaitis et al, 2021 ; Nouri-Goushki et al, 2021 ; Akolawala et al, 2022 ; Costa et al, 2022 ).…”

3D Polymer Architectures for the Identification of Optimal Dimensions for Cellular Growth of 3D Cellular Models

“…To create such scaffolds, TPP have shown to be an ideal method of fabrication, where several size orders of magnitude can be spanned needed for the incorporation of 3D morphological hierarchy, thereby replicating the structural influence of the ECM on cells. While 3D TPP has been used to mimic ECM structures for example in the context of cell differentiation [ 30 , 31 ], and stem cell therapy [ 32 , 33 ], or to generate 3D microenvironments suited to conduct in vitro tests of medical therapies [ 34 ], here we propose the application of TPP to generate 3D metrology structures for the analysis of cell packing and cell motility. The systematically changed dimensions of wall separations and niches allow us to use the 3D polymer structures to “act as rulers“, that are able to operate on the typical dimensions of cellular sizes.…”

3D‐Engineered Scaffolds to Study Microtubes and Localization of Epidermal Growth Factor Receptor in Patient‐Derived Glioma Cells

“…[20,24,25] Importantly, the recent development of a biocompatible and low-autofluorescent methacrylate photosensitive polymer (IP-Visio) for 2PP has facilitated the use of this approach for life science applications. [23,26,27] Here, we have studied patient-derived glioma cells cultured on biomimetic 3D-engineered IP-Visio microscaffolds, fabricated by the 2PP technique, and compared them to standard 2D control models. Our results show that a standard glioma cell line (U-87) as well as four patient-derived glioma cultures can efficiently adhere and colonize the structures.…”