Multiomic Integration for Atypical Teratoid Rhabdoid Tumours to Identify Plasma Membrane Proteins for Therapeutic Targets

Abstract AIMS AT/RT’s - (Atypical Teratoid Rhabdoid Tumours) are rare, malignant hindbrain tumours, primarily diagnosed in children under three. Survival rates are low, radiation is generally avoided due to long-term health complica- tions in the developing brain of children, and chemotherapeutics have not demonstrated clinical benefits. AT/RT residual disease is a severe clinical complication, with post-surgery tumour replication a common occurrence. Plasma membrane proteins were hypothesised as revealing easily druggable therapeutic targets. METHODS AT/RT cells were co-cultured with human cerebellum astrocytes and decelullarised human cerebellum ECM to recapitulate a residual post-surgery environment and separated via FACS following seven-day co-cultures. 3D AT/RT and astrocyte membrane proteins and metabolites were analysed through liquid chromatography-mass spectrometry (LC-MS) for differential proteomic and metabolomic profiling between healthy and malignant states. AP2A1, AP2S1, CAV1 and CAV2 membrane proteins ubiquitously expressed across all AT/RT cell lines were subjected to drug assays using endocytosis inhibitors. RESULTS Cell separation following FACS was visualised via fluorescent tags – (eGFP and Cell Trace Violet for tumour and astrocyte respectively) following co-culture. AT/RT Metabolomic PCA Plots demonstrated distinct differential metabolite clustering between AT/RT cells and human cerebellum astrocytes. Volcano Plots illustrate differen- tially abundant metabolites ubiquitously expressed across all AT/RT cell lines when normalised against astro- cytes quantified from Log2FoldChanges. Enriched metabolites ubiquitous across all AT/RT cell lines included Vitamin B6, Tyrosine, Propanoate, Pyrimidine and beta-alanine. Chlorpromazine and Genistein endocytosis in- hibitors conferred reduced metabolic activity across all AT/RT lines. CONCLUSION AT/RT cells retrieved following astrocyte and cerebellum ECM co-culture require protein changes to be identified via LC-MS. This intends to specifically identify plasma membrane proteins influenced by astrocytes and ECM, and furthermore to elucidate specific proteins responsible for influencing molecular crosstalk between healthy and malignant brain cells. Metabolomics data will be further integrated with membrane proteomic data to further our understanding of AT/RT membrane biology.