Identifying Ovarian Cancer-Associated EV mRNA Expression Profiles Using Unsupervised Machine Learning and Non-Negative Matrix Factorization.

Extracellular vesicle (EV) transcriptomic data provides a high-dimensional representation of cellular states but remains challenging to interpret due to noise, redundancy, and limited sample sizes. Most existing approaches rely on supervised differential expression analyses, which can be biased and may fail to capture latent structure in small datasets. In this study, we propose an unsupervised machine learning framework based on non-negative matrix factorization (NMF) to identify latent expression programs from EV mRNA profiles. A structured preprocessing pipeline combining expression filtering, variance selection, ANOVA-based feature selection, and correlation pruning was used to reduce dimensionality and improve signal quality prior to matrix factorization. NMF was applied to decompose the data into interpretable gene modules and sample-specific activation patterns. Model selection was performed using reconstruction error and component stability across multiple initializations. Candidate features were prioritized using a composite ranking score integrating module loadings, group-level expression differences, and model stability. The approach identified a stable low-rank representation capturing dominant patterns in the data and a compact set of informative features. These results demonstrate that unsupervised matrix factorization can effectively extract structured, interpretable signals from small-scale transcriptomic datasets and provide a robust framework for feature prioritization and representation learning in high-dimensional biological data.