Drug repurposing via high throughput 3D spheroids screening in rare cancer treatment

Rare cancers comprise over 200 distinct types and account for 20–25% of U.S. cancer diagnoses yet receive limited therapeutic investment due to small patient populations and resource constraints. Drug repurposing offers a viable strategy, and large-animal models such as minipigs provide physiologically realistic platforms for preclinical evaluation. However, scalable systems for systematically screening large drug libraries in rare cancer models remain unavailable. We developed an integrated pipeline combining a minipig spinal cord glioma (SCG) model with miniaturized high-throughput screening (HTS). Primary SCG cells were cultured in 3D platforms for rapid screening of bioactive compounds. Lead candidates underwent validation through 3D spheroid assays, in vivo xenografting, RNA sequencing, immunohistochemistry, and histological analysis. Human SCG tissue samples and population-level datasets assessed translational relevance. HTS screening identified Clofoctol as a lead compound with potent glioma growth inhibition in both primary SCG cells and xenograft models. Studies revealed pleiotrophin (PTN) as a key target, with expression patterns in human SCG samples, establishing association and potential translational validity. Integration of large-animal cancer models with drug-repurposing pipelines and HTS platforms is feasible and effective for rare cancer drug discovery. This approach successfully identified Clofoctol, an FDA-approved compound with therapeutic potential for spinal cord gliomas and establishes a scalable model applicable to other orphan cancers.