Single-cell and spatial profiling of cysteine cathepsins identifies tumor states relevant to antibody-drug conjugates in breast cancer

Breast cancer is a highly heterogeneous disease shaped by dynamic interactions between malignant cells, immune infiltrates, stromal compartments, and the extracellular matrix. Among the molecular regulators of these interactions, cysteine cathepsins and legumain have emerged as important proteases involved in tissue remodeling, immune regulation, and tumor progression, yet their distribution and functional status across human breast cancer ecosystems remain insufficiently defined. Here, we performed an integrated protease-centric analysis of breast cancer specimens from 66 patients using high-dimensional single-cell mass cytometry of matched peripheral blood and tumor samples, imaging mass cytometry of intact tissues, and activity-based TOF probes for in situ detection of active proteases. Systemic immune profiling identified two patient clusters associated primarily with neoadjuvant therapy and tumor grade, accompanied by distinct cytokine and circulating protease patterns. In tumors, single-cell analysis revealed pronounced interpatient heterogeneity in tissue architecture and immune infiltration, while protease profiling uncovered reproducible cell type-associated modules, including cathepsin B/L-cystatin C and legumain-cystatin E/M axes. Cathepsins B and L were prominent in tumor-infiltrating immune cells and variably expressed in epithelial cells, whereas cathepsin D showed broader tumor distribution and cathepsin S remained more restricted. In epithelial cells, HER2 expression did not consistently coincide with high cathepsin B or L abundance, enabling identification of a limited subgroup of patients with combined HER2-high/protease-high states relevant to protease-cleavable antibody-drug conjugates. Spatial imaging further localized cathepsins B and D to tumor-stroma interfaces and macrophage-rich niches, and activity-based IMC confirmed the presence of catalytically active cathepsin B in human breast tumor tissue. Together, these findings define cysteine cathepsins as spatially and cellularly organized components of breast tumor ecosystems and provide a framework for protease-informed patient stratification and biomarker-protease pairing in targeted therapy. This study provides a single-cell and spatial framework for understanding how cysteine cathepsins are organized across breast cancer ecosystems and how their activity may influence protease-dependent therapeutic strategies. By showing that HER2 expression does not necessarily coincide with high cathepsin abundance or activity, our findings suggest that ADC patient selection should incorporate both target expression and the proteolytic state of tumor cells. More broadly, this work establishes protease-informed stratification as a rational approach for improving the design and clinical use of protease-cleavable ADCs.