Poster S5 - 3D neuropathological analysis of the clarified brain tumor tissues without sectioning

Introduction: The evaluation of tumor response to therapeutic agents, including nanoparticles and chemotherapy drugs, through microscopic analysis is currently limited by traditional tissue sectioning methods. These methods, involving tissue slicing followed by immunostaining and optical microscopy, are naturally destructive. Moreover, the microscopic data obtained from such techniques, typically derived from a limited number of thin tissue slices (ranging from 5 to 50 µm in thickness), may not accurately represent the treatment efficacy across the entire tumor mass [1]. This limitation is especially significant in the context of brain tumors, such as glioblastoma multiforme (GBM), known for their considerable heterogeneity. Microscopic results obtained from a single or a limited number of tissue slices may lead to misleading conclusions. Hence, there is a critical need to develop alternative methods for microscopic analysis that do not involve tissue sectioning; methods that provide comprehensive feedback on treatment efficacy across various regions of the tumor mass. Implementation of non-sectioning microscopic analysis techniques has the potential to significantly enhance treatment outcomes and reduce the likelihood of tumor recurrence. By enabling treatments to target the entire tumor more effectively, this techniques can contribute to optimizing therapeutic interventions.

Materials and

Methods: We employed a polymerization technique to convert the entire mouse brain into optically transparent acrylamide-based hydrogels, preserving the microscopic and nanoscale structure of tumors at both extracellular and intercellular levels. Orthotopic U87 tumors (human-derived GBM cells expressing GFP) were induced in the brains of mice (n=3). Therapeutic nanoparticles (gold nanoparticles labeled with Cy5 fluorescent molecules) were stereotactically injected into the tumors under magnetic resonance imaging (MRI) guidance [2]. Mice were euthanized 24 hours post-injection, and their brains were excised and immersed in a monomer solution. Subsequently, a low-temperature polymerization and tissue-clearing technique known as CLARITY were employed [3]. Finally, samples were prepared following immersion in a refractive index-matching solution for microscopic analysis using light sheet and confocal microscopy.

Results, Conclusions, and Discussions: Three-dimensional maps were constructed to assess tumor growth patterns and their interactions with nanoparticles, utilizing their GFP and Cy5 signals, respectively. Additionally, the distribution of nanoparticles within tumors and other brain regions, such as the ventricles, was investigated. The optical transparency provided by tissue clearing enabled in-depht analysis of the entire brain volume in a single microscopy session, eliminating the need for destructive tissue sectioning. Distinct growth patterns, including diffusive and bulk-shape growth, were observed among different GBM cell types, indicative of varying invasiveness levels. The uniformity of nanoparticle distribution within tumors appeared to correlate with tumor type and the precision of intracranial injection. Additionally, the ability to reverse immunostaining enables the examination of treatment biomarkers, such as cell death, necrosis, and immune response separately. Expanding upon this methodology has the potential to enhance pre-clinical and clinical investigations, facilitating the evaluation of therapeutic agent efficacy. In conclusion, our study demonstrates the efficacy of novel microscopic techniques in revealing tumor growth patterns and nanoparticle interactions within the brain. The optical transparency achieved through tissue clearing enables comprehensive analysis without the need for destructive sectioning, providing valuable insights into tumor biology and therapeutic efficacy. Through these advancements, researchers can enhance pre-clinical and clinical investigations, ultimately improving the development of effective therapeutic strategies for brain tumors and beyond.

References:
[1] L. Tseng et al. Can Vet J. 64 (2023) 389.
[2] H. Arami et al. Nat Nano 17 (2022) 1015.
[3] K. Chung et al. Nat Methods 10 (2013) 508.

Acknowledgements (Optional): This work was supported by the following NIH grants: R00EB031178 from National Institute of Biomedical Imaging and Bioengineering and R00CA234208 from National Cancer Institute.