2ⁿᵈ Edition of the Cancer R&D World Conference 2026

Abstract

Trastuzumab–deruxtecan (T-DXd) demonstrated strong clinical efficacy in HER2⁺ and HER2-low breast cancers, however, its intracellular mechanisms underlying its action remain poorly understood. To investigate T-DXd dynamics in HER2⁺ breast cancer cells, we implemented multimodal imaging techniques combining live-cell imaging, confocal microscopy, immuno-EM, and biochemical assays. HER2 over-expressing SK-BR-3 cells were exposed to T-DXd (10 µg/mL for 2–72 h) and compared to IgG controls. During the adaptation phase (2–24 h), we observed HER2 phosphorylation, sustained ERK activation despite a decrease in total ERK, and down-regulation of AKT signaling.

Simultaneously, TFEB and LAMP1 expression increased, suggesting early lysosomal biogenesis. At 24 h, we observed the unexpected release of T-DXd–positive sphereosomes, a specialized form of extracellular vesicles revealed by under electron microscopy. At later stages (48–72 h), live-cell imaging showed maximal intracellular accumulation of T-DXd HER2, while confocal and immuno-EM imaging revealed that these assemblies concentrated within enlarged, metabolically active lysosomes that formed tripartite contact sites with both mitochondria and the nucleus. The formation of these structures coincided with γH2AX activation, Lamin B1 upregulation, and marked nuclear envelope stress. Collectively, these findings illustrate how imaging uncovers ADC induced cellular remodeling and pinpoint lysosomal hubs, and ERK signaling as possible targets for combination therapy, highlighting the value of advanced multimodal workflows in defining ADC mechanisms of action.