Secretomic profiling of triple-negative breast cancer media using Mag-Net™ HP

Introduction: Secretomics provides a real-time view of tumor biology by profiling proteins that cancer cells release into their microenvironment and circulation. In breast and other cancers, secreted factors can serve as predictive and prognostic markers of disease state and treatment response. However, conventional mass-spectrometry workflows often miss low-abundance cytokines, growth factors, and signalling mediators masked by highly abundant plasma proteins. Ex vivo secretome analyses in controlled culture conditions allow reproducible characterization of secreted proteomic signatures, which can then guide targeted validation in larger clinical cohorts. Mag-Net™ enrichment, as demonstrated by Wu et al., offers cost-effective capture of extracellular vesicle-linked, low-abundance proteins from plasma and other biofluids, enhancing downstream MS sensitivity. Here, we present Mag-Net™ HP enabled secretome profiling to sensitively track dose responses to doxorubicin in an in vitro model of triple-negative breast cancer (TNBC). Methods: BT-20 cells (1 × 10 5 cells/well) were cultured in 24-well plates and treated in triplicate with doxorubicin (0.1, 0.3, 0.7 or 1.5 µM), or DMSO. After 72 h, conditioned media were collected and incubated with Mag-Net™ HP beads for secretome enrichment. Vesicle capture, clean up and digestion was performed in a semi-automated manner on a KingFisher™ Flex using the Mag-Net™ HP kit. Peptides were loaded onto Evotips and analysed using an Evosep One coupled to a Bruker timsTOF HT system. Results: Mag-Net™ HP yielded substantial improvements in secretomic proteome coverage compared with conventional methods i.e. ~4-fold improvement compared to protein aggregation capture. Approximately 300 proteins were significantly (FDR<0.05) changed proportional to dose-mostly decreasing, negative correlations and others increasing, positive correlations. Gene set enrichment analyses of proteins positively correlated with dose showed, consistent with doxorubicin's mechanism of action, induction of pathways related to DNA damage, p53-related and checkpoint networks (via ATM/ATR, RB1, and MECP2 pathways) potentially indicative of cell-cycle arrest and apoptosis initiation. Together, these results show that Mag-Net™ HP -enhanced secretome profiling enables coherent, reproducible, and biologically interpretable proteomic readouts of drug response that may warrant further investigation in clinical applications. Conclusion: Mag-Net™ HP enrichment markedly improves detection of low-abundance secretome proteins in doxorubicin-treated TNBC cells in which doxorubicin drives a clear, dose-graded shift in the secretome. This streamlined approach enhances MS-based secretome workflows and supports discovery of clinically relevant biomarkers. Ongoing studies extend this pipeline to additional cell lines and patient-derived organoids.