Project Summary Despite multimodal treatment, cancer-related mortality in pediatric brain cancers remains high and survivors often suffer from serious, life-long, therapy-related side effects and secondary malignancies. There is a clear need for more effective therapies, including for the most common malignant pediatric brain cancer medullo- blastoma, a tumor that originates in the cerebellum. Mechanosensitive signaling pathways have emerged as powerful targets in cancer drug discovery, including for the treatment of medulloblastoma. Yet, when targeting signaling pathways that serve as sensors for a tumor cell's microenvironment, traditional monolayer cultures that are most commonly used in cell-based high-throughput drug discovery, do not accurately recapitulate critical environmental cues such as tissue stiffness or extracellular matrix composition. Drug discovery aimed at key mediators of mechanosensitive signaling require cell-based screening assays in a cell culture environment that more closely resembles in vivo tissue. Three-dimensional (3D) cell cultures have moved to the forefront in the effort to create more in vivo-like experimental environments that can mimic intricate cell-cell and cell-extracellular matrix interactions found in tissue. Our previous collaborative work demonstrated the suitability of the self- assembling and hydrogelating MAX8 β-hairpin peptide as a 3D cell culture scaffold for automated high- throughput drug discovery. We demonstrated that MAX8 combines biocompatibility and tunability in function and stiffness with unique mechanical properties (e.g., shear-thinning, injectable solid with immediate rehealing) that allow automatic handling with standard high-throughput screening (HTS) liquid handling equipment commonly found in a drug discovery laboratory. The primary objective of this proposal is to use the versatile and tunable MAX8 peptide to develop a 3D cell culture scaffold that mimics key features of brain extracellular matrix while also retaining material properties critical for use with automated liquid handling equipment, all for a high- throughput drug discovery approach targeting mechanosignaling. Aim 1 will establish a targeted assay for a well- characterized mechanosensitive signaling pathway that is compatible with MAX8 peptide hydrogel scaffold- based 3D cell cultures in a high throughput-compatible setup. Aim 2 will examine how tuning hydrogel stiffness and peptide functionalization with brain extracellular matrix components affects assay performance and phenotype of cerebellar neurons and pediatric brain cancer cells. Aim 3 will validate the newly developed assay platform by performing a pilot drug screen and in vivo efficacy testing of candidate compounds. The outcome of these studies will be a 3D cell culture platform that will provide fundamental understanding of how extracellular matrix composition and tissue stiffness regulate mechanosignaling in both normal neurons and pediatric brain cancer cel...