Abstract Background: Enhertu (trastuzumab-deruxtecan) is a HER2-targeting antibody-drug conjugate (ADC) that has demonstrated remarkable efficacy in HER2-positive cancers. Its design features a GGFG tetrapeptide linker that is selectively cleaved by lysosomal enzymes such as cathepsins to release the potent cytotoxic payload, deruxtecan (DXd). However, the precise intracellular trafficking, timing of linker cleavage, and kinetics of payload release remain poorly understood. This study aimed to elucidate these mechanisms using advanced 3D imaging platforms. Methods: HER2-positive NCI-N87 and HER2-negative AGS gastric cancer cell lines were used for comparative analysis. Enhertu was labeled with Alexa 647 dye using a commercial labeling kit. Cells were stained with LysoTracker and treated with fluorescence labeled Enhertu, followed by 3D correlative imaging with holotomography (HT) and multichannel fluorescence imaging machine (HT-X1 Plus; Tomocube). The target specificity of Enhertu was confirmed by pretreating cells with an excess of trastuzumab before Enhertu treatment. HER2-positive gastric cancer organoids (SC128) were cultured in both 2D and 3D conditions. After Enhertu treatment, 3D HT and fluorescence imaging were taken, followed by HER2 immunofluorescence staining after fixation. Results: Enhertu selectively bound to the plasma membrane of NCI-N87 cells, followed by internalization and subsequent merging with lysosomes, as visualized by LysoTracker staining. Blocking experiments validated the HER2 specific binding of Enhertu. Enhertu demonstrated higher cytotoxicity in HER2-positivie NCI-N87 cells compared to HER2-negative AGS cells and induced a bystander effect in co-culture, highlighting its potential to overcome tumor heterogeneity. In SC128 organoids, Enhertu bound to apical regions with strong HER2 expression and gradually internalized into the organoid interior. Prolonged exposure disrupted the organoid membrane and led to cell death, consistent with DXd-mediated cytotoxic effects. Conclusions: This study provides a comprehensive analysis of Enhertu’s intracellular trafficking and functional mechanisms. Using 3D holotomography and multichannel fluorescence imaging, we visualized the processes of HER2-specific binding, cleavage by lysosome, and DXd release. These findings will enhance our understanding of ADC mechanisms and offer valuable insights for optimizing future ADC designs to improve therapeutic efficacy. Citation Format: Seock-Jin Chung, Eric Sha, Minji Kim, Youngwon Cho, Seunghyi Kook, Soonyoung Lee, Jongseong Jang, Eunyoung Choi, Tae Hyun Hwang. AI-driven live 3D/4D spatial multimodal evaluation of a HER2-targeting ADC: delivery, lysosomal cleavage, payload action, and bystander effects resolved by holotomography and fluorescence abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 716.
Chung et al. (Fri,) studied this question.