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Publications

Journal Article Epublish

PubMed: 42729999 DOI: 10.1021/acsnanomed.6c00097

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

ACS Nano Med 1(8):2071-2090

Authors: Erica Tagliatti, Maria Cristina Gagliani, Grazia Bellese, Shahnaz Salamat, Martina Crippa, Manuela Sollazzo, Andrea Abbona, Matteo Paccagnella, Pietro Arnaldi, Lucilla Rossi, Andrea Petretto, Paola Rusmini, Valeria Crippa, Marco Carlo Merlano, Ornella Garrone, Anna Maria Porcelli, Michela Matteoli, Paola Falletta, Patrizio Castagnola, Katia Cortese

Affiliations

  • Laboratory of Pharmacology and Brain Pathology, IRCCS Humanitas Research Hospital, Via Alessandro Manzoni 56, 20089 Rozzano, Milan, Italy.
  • DIMES, Department of Experimental Medicine, MorphoLAB, University of Genoa, Via Antonio De Toni 14, 16132 Genoa, Italy.
  • Experimental Imaging Center, IRCCS Ospedale San Raffaele, Via Olgettina 60, 20132 Milan, Italy.
  • FABIT, Department of Pharmacy and Biotechnology, University of Bologna, Via Zamboni, 33, 40126 Bologna, Italy.
  • Fondazione Arco, Via Antonio Carle, 25, 12100 Cuneo, Italy.
  • Azienda Ospedaliera Santa Croce e Carle, Via Michele Coppino 26, 12100 Cuneo, Italy.
  • CNR Institute of Neuroscience, Via Raoul Follereau 3, 20854 Milano, Italy.
  • Core Facility for Omics Sciences, IRCCS Istituto Giannina Gaslini, Via Gerolamo Gaslini 5, 16147 Genoa, Italy.
  • Dipartimento di Scienze Farmacologiche e Biomolecolari "Rodolfo Paoletti", Dipartimento di Eccellenza 2018-2027, Università degli Studi di Milano, Via Giuseppe Balzaretti 9, 20133 Milan, Italy.
  • Scientific Direction, Candiolo Cancer Institute, FPO-IRCCS, Viale della Ricerca 7, 10060 Candiolo, Italy.
  • Medical Oncology, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Via Francesco Sforza 28, 20122 Milano, Italy.
  • FABIT, Department of Pharmacy and Biotechnology and CIRI, Interdepartmental Centre for Industrial Research 'Scienze Della Vita e Tecnologie per La Salute', University of Bologna, Via Zamboni 33, 40126 Bologna, Italy.
  • IRCCS Azienda Ospedaliero-Universitaria di Bologna, Via Giuseppe Massarenti 9, 40138 Bologna, Italy.
  • Vita-Salute San Raffaele University, Via Olgettina 60, 20132 Milan, Italy.
  • IRCCS Azienda Ospedaliera Metropolitana, Largo Rosanna Benzi 10, 16132 Genova, Italy.

Abstract

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.

MeSH

Organelles, Cells, Breast Neoplasms, Nanoparticles, Pharmaceutical Preparations, Stress, Physiological, Neoplasms, Mitochondria, Microscopy, Electron, Breast, Lysosomes, Trastuzumab