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

Trastuzumab-deruxtecan (T-DXd) is a clinically effective anti body−drug conjugate (ADC)withactivity acrossHER2-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 lysosomalprocessinginADCpharmacology,we investigatedthespatiotemporal intracellular response toT-DXd in humanHER2-positive breast cancer cell models.Byintegratingbiochemical analyseswithlightandelectronmicroscopy basednanoscaleimaging, andemployingorthogonalnanoscaleprobes, including BSA−goldnanoparticles andnanogold-based labeling, togetherwithproteomic profiling,wereconstructedthetemporalsequenceofT-DXdactionover72h.An early phase (2−24 h) was characterized by rapid HER2 phosphorylation, sustained ERK signaling, metabolic activation, and TFEB-driven lysosomal engagement, consistentwithactivedrugprocessing.Nanoscaleprobingof theendocyticpathwayusingBSA−goldnanoparticles demonstratedamarkedexpansionofthelysosomalcompartment,withincreasedlysosomenumberandsize,supportingtheconcept thatT-DXdactivelyremodelslysosomalarchitectureratherthenpassivelyexploitingitasadeliverysite.Atransitionalphaseat48h revealedpronounced lysosomal accumulationofT-DXdandextensiveorganelle remodeling.By72h, cellsentereda latephase markedbymitochondrial dysfunction, nuclear envelope stress, andDNAdamage, accompaniedby theemergenceof nanoscale contactsamonglysosomes,mitochondria,andthenucleus, suggestiveofcoordinatedorganellefailure.Proteomicanalysisindicated activationofinflammatoryandstress-associatedpathways, includingTNFα/NF-κBsignaling,supportedbyincreasedreleaseofIL-6, IL-8, andTNF-α.Collectively, thisspatiotemporal frameworkidentifiescompartment-specificnanoscalevulnerabilitiesengagedby T-DXdandhighlightsERK-dependent signalingandlysosomal functionaspotential targets for rational combinationstrategies in HER2-positivebreastcancer.