TRACKING HER2 TRAFFICKING AND ORGANELLE CROSSTALK IN TRASTUZUMAB-DERUXTECAN–TREATED BREAST CANCER CELLS USING MULTIMODAL IMAGING

E. Tagliatti¹, M. C. Gagliani², G. Bellese², M. Crippa³, A. Abbona⁴, M. Paccagnella⁴, P. Arnaldi⁵, M. C. Merlano⁶, O. Garrone⁷, A.M. Porcelli⁸, M. Matteoli¹, P. Falletta⁹, P. Castagnola¹⁰, K. Cortese² | ¹IRCCS Humanitas Research Hospital, Laboratory of Pharmacology and Brain Pathology, Rozzano, Milano, Italy; ²DIMES, Department of Experimental Medicine, Cellular Electron Microscopy Lab, University of Genoa, Genoa, Italy; ³Experimental Imaging Center, IRCCS Ospedale San Raffaele, Milan, Italy; ⁴Health Department S. Croce e Carle Teaching Hospital, Cuneo, Italy; ⁵IRCCS Ospedale Policlinico San Martino, Genova, Italy; ⁶Scientific Direction, Candiolo Cancer Institute, FPO-IRCCS, Candiolo, Italy; ⁷Medical Oncology, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milano, Italy; ⁸FABIT, Department of Pharmacy and Biotechnology and CIRI, Interdepartmental Centre for Industrial Research ‘Scienze Della Vita e Tecnologie per La Salute’, University of Bologna, Bologna, Italy; ⁹Vita-Salute San Raffaele University, Milan, Italy; ¹⁰IRCCS Ospedale Policlinico San Martino, Genova, Italy

Trastuzumab-deruxtecan (T-DXd) is a clinically effective antibody–drug conjugate (ADC) for HER2+ and HER2-low breast cancers, yet its intracellular mechanisms remain elusive. We used a multimodal imaging approach including confocal, immuno-EM, and biochemical assays to investigate T-DXd dynamics in HER2+ breast cancer cells. Cells were treated with T-DXd (10 µg/mL, 2– 72 h) and compared to IgG controls. Early-phase responses (2–24 h) included HER2 phosphorylation, sustained ERK signaling despite total ERK reduction, and AKT downregulation. Imaging revealed active lysosomal remodeling, increased TFEB, and reduced LAMP1 and LC3, indicating modulation of autophagic flux. In late stages (48–72 h), T-DXd–HER2 complexes accumulated in lysosomes, forming previously undescribed lysosome–mitochondria–nucleus contact sites. These triads correlated with mitochondrial damage, γH2AX activation, nuclear envelope stress, and LaminB1 increase. Secreted cytokines (IL-6, IL-8, TNF-α) induced M2-like macrophage polarization. This study highlights the value of integrated imaging to dissect the morphofunctional responses induced by ADCs and proposes new cellular targets, such as lysosomal hubs or ERK signaling, for future therapeutic combinations.