Hydrogel-based engineering of glioblastoma for three-dimensional visualization of tumor invasion and micro/nano-environment

Abstract

Glioblastoma (GBM) is a refractory malignant brain tumor with high invasiveness and recurrence. To elucidate the pathogenesis of invasive GBM and evaluate treatment strategies, it is necessary to observe the three-dimensional invasion process of the tumor and its interaction with surrounding tissues in three dimensions at the cellular level or below in animal models. Tissue clearing and tissue expansion methods, which have been rapidly developed in the past decade, have made it possible to investigate three-dimensional large-scale morphologies and analyze micro-/nano-structures in existing animal models of brain diseases, respectively. However, despite researchers’ demands for these techniques to investigate tumor tissues, it has been difficult to apply them to tumor tissue due to the mechanochemical properties that differ from normal tissue. In this study, I attempted to develop tissue clearing and tissue expansion techniques suitable for application to an invasive mouse GBM model using hydrogel and chemical engineering approaches that changed the mechanochemical properties of tumor tissue. I revealed that the hybridization method controlling the chemical interaction between the hydrogel and tissue, which I termed controlled hybridization (cHyb), exhibited superior fluorescence preservation compared to the conventional hydrogel–tissue hybridization method with uncontrolled chemical interaction. By combining this hybridization method with the existing non-ionic surfactant-based tissue clearing technique, a new tissue clearing platform that satisfied all of fluorescence preservation, discoloration, and structural preservation was developed. I demonstrated a three-dimensional investigation of tumor invasion on a large scale using the new platform. I found that three-dimensional tumor micro/nano-environment analysis, such as glutamatergic interaction between the synapse and the invading GBM cell, was possible through the combination of N-hydroxysuccinimide chemical staining and tissue expansion using physical hybridization. Notably, a new buffer designed for deep tissue chemical staining resolved the issue with the original protocol, preventing tissue expansion. Finally, the developed tissue clearing and tissue expansion methods were combined and applied to a GBM model, thereby establishing a three-dimensional, multiscale tumor microenvironment analysis platform. I expect that various information obtained through the three-dimensional multi-scale tumor microenvironment analysis platform of the invasive GBM model will facilitate elucidating the pathological mechanisms of GBM invasion and establishing a treatment strategy.