HER2 Breast Cancer Drug T-DXd Triggers Senescence as a Hidden Resistance Mechanism

Breast cancer is the leading cause of cancer death in women worldwide, and HER2-positive tumors — roughly 15% of all cases — carry elevated relapse risk. Trastuzumab-deruxtecan (T-DXd, Enhertu) has rapidly become the preferred treatment across multiple clinical settings, outperforming older ADCs in progression-free survival in the pivotal DESTINY-Breast03 phase 3 trial. Yet primary and acquired resistance remain poorly understood, especially at the mechanistic level. This study set out to define exactly how T-DXd acts on HER2-positive breast cancer cells and how those cells escape its effects.

The team used two HER2-positive cell lines — BT474 (HER2/ER co-positive) and SKBr3 (HER2-positive) — plus trastuzumab-resistant derivatives (BT474-R and SKBr3-R) generated by 10-month continuous trastuzumab exposure. They compared the effects of T-DXd, trastuzumab alone, T-DM1, free DXd payload, and datopotamab-DXd (Dato-DXd, which targets TROP2) across 2D monolayer cultures, 3D mammospheres, and a novel 3D bioprinted scaffold system incorporating tumor cells alongside fibroblasts and endothelial cells to model the tumor microenvironment. Drug-to-antibody ratio of 8 was used to calculate molar-equivalent DXd doses.

T-DXd treatment induced robust senescence in all HER2-positive cell lines, including trastuzumab-resistant models. Key hallmarks were confirmed: beta-galactosidase positivity (a classical senescence marker), enlarged and flattened cell morphology, p53 stabilization, p21 upregulation, and secretion of canonical SASP factors including IL-6, IL-8, and MMP-9. Reactive oxygen species were significantly elevated in treated cells, and metabolic profiling revealed a shift away from oxidative phosphorylation consistent with the Warburg-like metabolic rewiring seen in senescent cells. Lysosomal and mitochondrial morphology were also markedly altered, with enlarged lysosomes and fragmented mitochondrial networks observed by confocal and electron microscopy.

To dissect whether senescence arose from the antibody or payload component, the investigators tested trastuzumab alone versus free DXd at molar-equivalent concentrations. Trastuzumab alone did not induce senescence markers, whereas DXd alone recapitulated all major features: p53/p21 activation, beta-galactosidase positivity, ROS elevation, and SASP secretion. This was confirmed in both adherent and mammosphere cultures. The finding was further validated by testing Dato-DXd (which carries an identical DXd payload but targets TROP2 instead of HER2): TROP2-positive BT474 and triple-negative MDA-MB-231 cells treated with Dato-DXd showed the same senescence program, Topoisomerase I downregulation, lysosomal enlargement, and metabolic changes, strongly implicating DXd as the shared driver across both ADCs.

A parallel resistance mechanism was identified: Topoisomerase I protein levels — the direct intracellular target of DXd — were substantially reduced in both T-DXd- and Dato-DXd-treated cells, even in HER2-low and HER2-negative lines exposed via bystander effect in the 3D co-culture scaffolds. The 3D bioprinted heterotypic model revealed that the bystander effect of released DXd reached fibroblasts and endothelial cells in adjacent compartments, inducing low-level senescence markers in stromal cells as well, raising questions about off-target effects in the tumor microenvironment. The authors conclude that DXd-based ADCs share a previously unrecognized mechanism of resistance — therapy-induced senescence — and propose that adding senolytic agents (which eliminate senescent cells) or senomorphic drugs (which suppress SASP) to ADC regimens could restore tumor cell killing and prevent pro-tumorigenic SASP-driven microenvironment remodeling.