Polymer-Based Transparent Brains for 3D Evaluation of Brain Tumors without Tissue Sectioning

Conventional tissue sectioning, staining, and optical microscopy are used as standard clinical<br/>methods for diagnosis of the brain tumors (i.e., glioblastoma multiforme or GBM), studying their<br/>anatomical phenotypes and evaluating their response to therapies. Sectioning of the tissues is<br/>destructive and stained and analyzed tissues cannot be re-used for evaluation of additional<br/>biomarker. Also, histological microscopy data obtained from each tissue section are limited to<br/>that area of the tumor and are not ideal representatives of the whole tumor mass, especially for<br/>highly heterogeneous tumors such as GBM. Here, we will discuss a method for transforming the<br/>whole brain tumor tissues to optically transparent hydrogel masses without losing any<br/>anatomical, extracellular or sub-cellular features within the tissues. Different types of orthotopic<br/>brain tumors (e.g., human-derived U87 GBM with GFP signal) were generated in mouse brains<br/>(n=3-5) and analyzed using magnetic resonance and bioluminescent imaging (MRI and BLI).<br/>Brains were excised and immersed in an acrylamide monomer solution, followed by the<br/>polymerization of these monomer molecules within the entire brain tissue at 37°C. Brains were<br/>clarified by removing the lipid molecules from the tissue microenvironment. Transparent brains<br/>were analyzed using confocal and light-sheet microscopy techniques after being immersed in<br/>Focus Clear solution to adjust their refractive index for optimized microscopy. Three-<br/>dimensional macroscopic and microscopic maps were generated from the whole brains for<br/>analyses of the tumor growth pattern and their response to therapies, without losing the<br/>intactness of the proteins and tissue structure. Using various monomer derivatives enabled the<br/>tuning of the cross-linking rate and optical transparency of the hydrogels for improved analysis<br/>of heterogeneous tumor masses. This method enabled series of reversible staining de-staining,<br/>and re-staining of the tissues for clinical analysis of different brain tumors and a variety of<br/>neuropathological biomarkers.