Ethanol-Fixed, Paraffin-Embedded Tissue Imaging: Implications for Alzheimer's Disease Research.

Mass spectrometry imaging (MSI) is rapidly becoming a crucial tool in disease research. Fresh-frozen tissue is ideal for MSI because the protein and lipid structures are undisturbed by chemical fixatives; however, that means long-term preservation is limited. Formalin-fixed paraffin-embedded tissue has a virtually infinite shelf life, but whole proteins are difficult or impossible to image directly. To bridge this gap, we examine the use of ethanol-fixed, paraffin-embedded (EFPE) tissue for the localization of intact proteins and lipids and comment on implications in Alzheimer's disease (AD) research. The new sample preparation methods for EFPE tissues have allowed us to greatly broaden the information we can extract from MSI experiments. Our methods involve a xylene-free deparaffination for lipid analysis and an intact protein method for visualizing amyloid-beta plaques from human AD brain tissue. This unique combination streamlines the MSI sample preparation process while allowing for the most biologically and pathologically relevant information to be extracted from a single tissue source.

Systems Biology Approach Identifies Prognostic Signatures of Poor Overall Survival and Guides the Prioritization of Novel BET-CHK1 Combination Therapy for Osteosarcoma.

Osteosarcoma (OS) patients exhibit poor overall survival, partly due to copy number variations (CNVs) resulting in dysregulated gene expression and therapeutic resistance. To identify actionable prognostic signatures of poor overall survival, we employed a systems biology approach using public databases to integrate CNVs, gene expression, and survival outcomes in pediatric, adolescent, and young adult OS patients. Chromosome 8 was a hotspot for poor prognostic signatures. The MYC-RAD21 copy number gain (8q24) correlated with increased gene expression and poor overall survival in 90% of the patients (n = 85). MYC and RAD21 play a role in replication-stress, which is a therapeutically actionable network. We prioritized replication-stress regulators, bromodomain and extra-terminal proteins (BETs), and CHK1, in order to test the hypothesis that the inhibition of BET + CHK1 in MYC-RAD21+ pediatric OS models would be efficacious and safe. We demonstrate that MYC-RAD21+ pediatric OS cell lines were sensitive to the inhibition of BET (BETi) and CHK1 (CHK1i) at clinically achievable concentrations. While the potentiation of CHK1i-mediated effects by BETi was BET-BRD4-dependent, MYC expression was BET-BRD4-independent. In MYC-RAD21+ pediatric OS xenografts, BETi + CHK1i significantly decreased tumor growth, increased survival, and was well tolerated. Therefore, targeting replication stress is a promising strategy to pursue as a therapeutic option for this devastating disease.