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

Abstract

Abstract Background Breast cancer contains heterogeneous malignant, immune, and stromal compartments that influence biomarker performance and response to targeted therapy. Cysteine cathepsins and legumain regulate tissue remodeling, immune function, and intracellular processing of protease-cleavable antibody-drug conjugates, but their cellular distribution and functional activity in human breast tumors remain incompletely defined. We aimed to map protease abundance and activity across systemic and intratumoral breast cancer ecosystems and to evaluate their relevance for biomarker-informed patient stratification. Methods Peripheral blood and breast tumor specimens from 66 patients were analyzed. Whole-blood leukocytes and dissociated tumors were profiled by high-dimensional mass cytometry, plasma soluble mediators by multiplex bead assay, intact tissue sections by imaging mass cytometry, and active cathepsin B by a mass cytometry-compatible activity-based probe (TOF-probes). Breast cancer cell lines were used to test human HER2-directed antibody-drug conjugates. Unsupervised analyses included principal component analysis, k-means clustering, viSNE, FlowSOM, and hierarchical clustering. Associations and group differences were assessed using Spearman rank correlation and two-sided Welch tests. Results Peripheral immune profiling identified two patient clusters associated mainly with neoadjuvant therapy and nodal status, with distinct cytokine and circulating protease patterns. Tumor single-cell profiling revealed marked interpatient heterogeneity in epithelial, stromal, endothelial, and immune compartments. Protease profiling identified reproducible, cell type-associated modules, including cathepsin B/L-cystatin C and legumain-cystatin E/M axes. Cathepsins B and L were enriched in tumor-infiltrating immune cells and variably expressed in epithelial cells, whereas cathepsin D was broadly distributed and cathepsin S was more restricted to antigen-presenting cell-like populations. In epithelial tumor cells, high human HER2 expression did not consistently coincide with high cathepsin B or L abundance, defining only limited subgroups with combined target-high/protease-high states. Spatial imaging localized cathepsins to tumor-stroma interfaces and macrophage-rich niches, and activity-based imaging confirmed catalytically active cathepsin B in human breast tumor tissue. Conclusions Cysteine cathepsins are spatially and cellularly organized components of breast cancer ecosystems. Integrating target antigen expression with protease abundance, protease activity, or lysosomal state may improve biomarker-guided selection and design of protease-cleavable antibody-drug conjugates.

The authors' abstract, as published at the source. Breast Cancer Research, 2026 · DOI ↗