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

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  1. Time-dependent modulation of HER2 signaling, lysosomal response, and apoptosis upon T-DXd treatment in HER2+ breast cancer cells. (A) Immunoblot analysis of HER2 phosphorylation at Y1248, total HER2 levels, and downstream signaling pathways (pAKT S473 and pERK1/2) in SKBR3 cells treated with 10 μg/mL T-DXd for 2 and 72 h, compared to IgG control. Actin was used as a loading control. (B) Quantification of Western blot in A. Phosphorylated proteins were first normalized to their corresponding total protein levels (pHER2 Y1248/total HER2, pAKT S473/total AKT, and pERK1/2/total ERK1/2) and then expressed relative to the corresponding IgG-treated control. All other protein levels were normalized to actin and expressed as relative optical density compared with IgG-treated controls. Data are presented as mean ± SEM from three independent experiments (N = 3). Statistical significance was determined using one-way ANOVA. (C) Immunoblot analysis of TFEB, LAMP1, LC3I/II, and p62 in response to T-DXd treatment. Actin was used as a loading control. (D) Quantification of Western blot in C. Each bar graph represents the mean relative optical density quantification relative to IgG-treated…

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  2. Time-dependent metabolic adaptations induced by T-DXd treatment in SKBR3 cells. (A,B) Left, Average oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) kinetics showing the response of SKBR3 cells to 10 μg/mL T-DXd and Tz for 2 h compared to IgG-treated cells. Mean ± SEM. Right, Quantification of basal, maximal, ATP-coupled respiration, and nonmitochondrial oxygen consumption (OCR) and ECAR acidification rate (ECAR). 2 h post-treatment, OCR measurements show a significant increase in maximal respiration and nonmitochondrial respiration, accompanied by an elevation in ECAR. N = 3 independent experiments, 30 replicates/condition. (C,D) Left, Average OCR and ECAR kinetics showing the response of SKBR3 cells to 10 μg/mL T-DXd and Tz for 72 h. Mean ± SEM. Right, Quantification of basal, maximal, ATP-coupled respiration, and nonmitochondrial oxygen consumption (OCR) and ECAR acidification rate (ECAR). 72 h post-treatment, oxygen consumption rate (OCR) measurements show a significant decrease in basal, ATP-linked, maximal respiration and nonmitochondrial respiration, accompanied by a reduction in extracellular acidification rate (ECAR). N = 3 independent…

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  3. Mitochondrial network dynamics upon T-DXd treatment in SKBR3 cells. (A) Confocal microscopy images of SKBR3 cells treated with IgG or T-DXd for 72 h, stained with MitoTracker Green and LAMP1 antibody. T-DXd-treated cells exhibit mitochondrial disorganization and an extended, fused mitochondrial network. Scale bars: 10 μm. (B) Quantification of MitoTracker morphology in IgG- and T-DXd-treated cells at 72 h. Morphometric analysis of mitochondrial area (B), branches (C), and branch junctions (D) was performed using the Mitochondria Analyzer ImageJ plugin. Data are presented as mean ± SEM from three independent experiments, with 50 cells analyzed per condition (unpaired t test). (E) Representative electron microscopy (EM) images of SKBR3 cells treated with IgG or T-DXd for 72 h. In T-DXd-treated cells, mitochondria appear fused and display cristae alterations and swelling. N: nucleus; mit: mitochondria. The asterisk indicates a mitochondrial region with poorly preserved or no longer discernible cristae (F). Quantification of mitochondrial long axis length by EM morphometry. Mitochondria were measured in 30 whole cells per experimental condition (see material and methods) and plotted…

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  4. T-DXd internalization and subcellular localization. (A) Real-time live-cell imaging of SKBR3 cells treated with T-DXd over 72 h using the IncucyteSX5 system. Scale bars: 200 μm. (B) Cell proliferation is shown as % of cell confluence. (C) HER2 internalization was monitored with an anti-HER2 antibody directly conjugated to CoraLite488 and quantified as HER2-associated fluorescent puncta per image. (D) Confluence-normalized quantification of HER2-associated fluorescent puncta. HER2 CoraLite Count per image was normalized to the corresponding phase-contrast-derived cell confluence area at each time point and then expressed relative to the initial time point. N = 3 experiments, 3 replicates each condition, 4 images/well. Statistical significance was determined by two-way ANOVA followed by Sidak’s multiple comparisons test comparing IgG and T-DXd at each time point. P < 0.05 (*), P < 0.0001 (****). (E, F) Immunofluorescence microscopy images of T-DXd-treated SKBR3 cells at 2, 24, 48, and 72 h post-treatment. Cells were stained with an anti-LAMP1 antibody (red) to assess lysosomal localization and antihuman cy2 (green) to track T-DXd. Co-localization of T-DXd with LAMP1 (yellow arrows)…

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  5. Ultrastructural analysis of lysosomal remodeling and organelle interactions induced by trastuzumab and T-DXd in HER2+ breast cancer cells. (A) Representative TEM micrographs of SKBR3 cells treated with HRP-conjugated trastuzumab (Tz) for 24, 48 and 72 h, followed by DAB development, showing HRP-positive lysosomal structures (lys) and their interactions with mitochondria (white boxes). (B) Representative TEM micrographs of SKBR3 cells treated with HRP-conjugated T-DXd for 24, 48, and 72 h. T-DXd-positive lysosomes appeared enlarged, frequently associated with mitochondria and the nuclear envelope, and contained electron-dense intraluminal material at later time points. (C) Quantification of lysosome number per image in IgG-, Tz-, and T-DXd-treated cells at 24 and 48 h. Data are presented as box plots with individual data points. Trastuzumab induced a modest transient increase in lysosome number, whereas T-DXd treatment resulted in a more pronounced increase at 24 and 48 h. (D) Quantification of lysosomal size in IgG-, Tz-, and T-DXd-treated cells. Tz induced a significant increase in lysosomal size at 48 h (****p < 0.0001), whereas T-DXd treatment induced a pronounced enlargement…

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  6. Assessment of nuclear damage and nuclear envelope alterations induced by T-DXd treatment. (A) Western blot analysis of γH2AX protein levels in SKBR3 cells treated with T-DXd at 2 and 72 h. Actin was used as a loading control. (B). Quantification of Western blot in A. Each bar graph represents the mean relative optical density quantification of γH2AX relative to IgG-treated control. Error bar + SEM. Significance was determined using one-way ANOVA from N = 3 independent experiments. (C) Confocal analysis of γH2AX foci formation in SKBR3 cells treated with IgG and T-DXd for 72 h. γH2AX foci, indicative of DNA double-strand breaks (DSBs), were detected using anti- γH2AX antibody (red). DAPI (blue) was used to detect nuclei. (D) Histogram showing quantification of γH2AX foci per nucleus. N = 3 independent experiments. Scale bars: 20 μm. (E) Immunoblot analysis of Lamin1B showing increased expression upon 72 h od T-DXd treatment (F) Quantification of LaminB1 expression from E. Each bar graph represents the mean relative optical density quantification of LaminB1 normalized to IgG-treated controls. Significance was determined using one-way ANOVA from N = 3 independent experiments. (G)…

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  7. Gene set enrichment analysis resulting from proteomic analysis of lysosome enriched fraction of SKBR-3 cells treated for 48 h with T-DXd or normal human IgGs used as controls. Positive Normalized Enrichment Scores (NES) are represented by blue bars and indicate upregulation induced by T-DXd while negative NES scores (orange bars) indicate downregulation. (A) Enriched transcriptional pathway modulated by T-DXd. (B) Enriched Biological processes modulated by T-DXd. (C) Enriched Molecular Function modulated by T-DXd. (D) Enriched Cellular Component modulated by T-DXd. A color saturation scale is provided to indicate the False Discovery Ratio (FDR) associated with the modulation.

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  8. Analysis of cytokine content in the conditioned medium (CM) of T-DXd–treated breast cancer cell lines. CM was collected from HER2+ (SKBR-3, BT474, MDA-MB-361) cell lines treated with 10 μg/mL of T-DXd or control IgG for 72 h and 10 days. Cytokine concentrations (IL-2, IL-6, IL-8, IL-10, TNF-α, IFN-γ) were measured using the Ella automated ELISA platform. Concentration values were converted to a log10 scale, and the ratio between T-DXd and IgG treatments was calculated as log10(T-DXd)/log10(IgG). These ratios are shown on the y-axis, with the red line indicating the IgG reference value. Cell lines are plotted on the x-axis. Data are presented as median with range. Statistical significance refers to comparisons between T-DXd and IgG conditions for each cytokine and time point. IL-6 levels were significantly increased in SKBR-3 cells treated with T-DXd at both 72 h and 10 days (p < 0.0001). IL-8 concentrations were consistently elevated in T-DXd-treated SKBR-3 (72 h and 10 d), BT474 (72 h and 10 d), MDA-MB-361 (72 h and 10 d) (all p < 0.0001). TNF-α levels were also significantly higher in SKBR-3 cells at both time points (p < 0.0001). No significant differences were detected for…