Intracellular Nanopharmacology of Trastuzumab Deruxtecan Reveals Lysosome-Centered Organelle Vulnerabilities in HER2-Positive Breast Cancer Cells

Trastuzumab-deruxtecan (T-DXd) is a clinically effective antibody–drug conjugate (ADC) with activity across HER2-amplified, HER2-low, and ultralow breast cancers. Despite its clinical success, how its nanoscale intracellular trafficking and processing shape therapeutic outcomes remains incompletely defined. Given the central role of HER2 internalization and lysosomal processing in ADC pharmacology, we investigated the spatiotemporal intracellular response to T-DXd in human HER2-positive breast cancer cell models. By integrating biochemical analyses with light and electron microscopy-based nanoscale imaging, and employing orthogonal nanoscale probes, including BSA–gold nanoparticles and nanogold-based labeling, together with proteomic profiling, we reconstructed the temporal sequence of T-DXd action over 72 h. An early phase (2–24 h) was characterized by rapid HER2 phosphorylation, sustained ERK signaling, metabolic activation, and TFEB-driven lysosomal engagement, consistent with active drug processing. Nanoscale probing of the endocytic pathway using BSA–gold nanoparticles demonstrated a marked expansion of the lysosomal compartment, with increased lysosome number and size, supporting the concept that T-DXd actively remodels lysosomal architecture rather then passively exploiting it as a delivery site. A transitional phase at 48 h revealed pronounced lysosomal accumulation of T-DXd and extensive organelle remodeling. By 72 h, cells entered a late phase marked by mitochondrial dysfunction, nuclear envelope stress, and DNA damage, accompanied by the emergence of nanoscale contacts among lysosomes, mitochondria, and the nucleus, suggestive of coordinated organelle failure. Proteomic analysis indicated activation of inflammatory and stress-associated pathways, including TNFα/NF-κB signaling, supported by increased release of IL-6, IL-8, and TNF-α. Collectively, this spatiotemporal framework identifies compartment-specific nanoscale vulnerabilities engaged by T-DXd and highlights ERK-dependent signaling and lysosomal function as potential targets for rational combination strategies in HER2-positive breast cancer.