3D Engineered Glioblastoma Microenvironments for Immunofluorescence and Genetic Expression Analysis in the Context of Proton Radiobiology

Abstract

Glioblastoma (GBM) is the most aggressive and lethal primary brain tumor, presenting significant challenges in treatment due to its heterogeneity, invasiveness, and resistance to conventional therapies. Proton radiotherapy offers a promising avenue for precise tumor targeting while sparing surrounding healthy tissues. However, its effectiveness study is limited by the complex interactions between the radiation and the tumor microenvironment (TME). Understanding these interactions is critical to improving treatment outcomes, mainly through studying radiation-induced DNA damage and repair pathways.

In vitro models play a vital role in elucidating cellular behavior and disease mechanisms within controlled, ethical, and cost-effective environments. They enable detailed drug screening and mechanistic investigations before in vivo studies. Among these, both 2D and 3D models have distinct roles; while 2D models are easier to use and cost-efficient, they often alter cell morphology and function, potentially skewing biological responses to treatments. In contrast, 3D models more accurately recapitulate in vivo-like architecture, cell-cell interactions, and tissue-specific behavior. Therefore, minimizing contamination from 2D-grown cells is crucial in assays such as gene expression analysis, where cell context directly influences biological outcomes.
This report focuses on designing three-dimensional (3D) engineered scaffolds that replicate the native glioblastoma microenvironment for use in advanced analytical studies. These scaffolds are optimized for compatibility with immunofluorescence and quantitative polymerase chain reaction (qPCR) techniques, essential for assessing DNA damage and repair mechanisms following proton radiotherapy.

This study demonstrates that the integration of a non-cell-adhesive Lipidure® coating with two-photon polymerized 3D scaffolds effectively minimizes 2D cell growth, allowing for more reliable biological characterization of glioblastoma (GBM) cells in a 3D environment. The scaffold design proved compatible with both immunofluorescence imaging and qPCR analysis, enabling precise detection of DNA damage markers and sufficient RNA yield for gene expression profiling.