Cell lines and cell culture

The HER2-positive human GC cell lines NCI-N87 and SNU216 were kindly provided by the Beijing Institute of Genomics, Chinese Academy of Sciences. The cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin in a humidified atmosphere at 37 °C with 5% CO2.

Plasmids and transfection

Plasmids encoding different domains of HER2 were kindly provided by Professor Yongliang Zhao from the Beijing Institute of Genomics, Chinese Academy of Sciences. Cell transfection was performed following the instructions of the DNA transfection reagent LipoMax (Sudgen, Nanjing, China).

Western blot analysis

Protein extraction from cells and tissues was performed using RIPA buffer (Solarbio, Beijing, China). Protein concentrations were determined using a BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Equal protein amounts were separated by Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis and transferred onto polyvinylidene fluoride membranes (Millipore, Massachusetts, MA, USA). The membranes were blocked for 1 h at room temperature with either 5% skimmed milk or 3% bovine serum albumin. Primary antibodies (Supplementary Table 1) were then applied, and the membranes were incubated overnight at 4 °C. After thorough washing, secondary antibodies were applied at room temperature for 1 h, and the protein bands were visualized using enhanced chemiluminescence.

Co-immunoprecipitation

NCI-N87 and SNU216 cell lines were subjected to lysis using IP lysis buffer (Beyotime, Shanghai, China), followed by overnight incubation with either anti-HER2 or IgG antibodies at 4 °C. On the next day, 20 μL of Protein A/G PLUS-agarose beads were applied to each tube, with gentle rotation at 4 °C for 8 h. The beads were then subjected to a series of washes, and the resulting samples were analyzed via Western blotting to detect bands corresponding to ubiquitin, HER2, or HSP90.

RNA extraction and qRT‑PCR

RNA was extracted from cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Subsequently, cDNA synthesis was carried out according to the manufacturer’s instructions using the PrimeScript™ RT reagent Kit (TaKaRa, Tokyo, Japan). Quantitative PCR (qPCR) was conducted with TB Green® Premix Ex Taq™ II (TaKaRa, Tokyo, Japan). The data were normalized to the expression of the GAPDH internal control gene. The qRT-PCR primer sequences were as follows: HER2: forward, 5′-TGTGACTGCCTGTCCCTACAA-3′; and reverse, 5′-CCAGACCATAGCACACTCGG-3′. GAPDH: forward, 5′-GGAGCGAGATCCCTCCAAAAT-3′; and reverse, 5′-GGCTGTTGTCATACTTCTCATGG-3′.

Hematoxylin–eosin (H&E) and Immunohistochemistry (IHC) staining

Tissue specimens were sectioned and stained with Hematoxylin and Eosin (H&E) or Immunohistochemistry (IHC). For the H&E staining, tissue sections were baked at 65 °C for 2 h, followed by automated staining. For IHC staining, paraffin-embedded tissue sections underwent dewaxing, hydration, antigen retrieval, and inhibition of endogenous peroxidase activity. Afterward, primary antibody was applied for 60 min, followed by secondary antibody for 15 min, after which 3,3′-Diaminobenzidine was used for color development. HER2 positivity was based on brownish yellow staining on the cell membrane and was quantified using ImageJ (National Institutes of Health, Bethesda, MD, USA).

Immunofluorescence staining

Cells were seeded onto glass coverslips in 24-well plates (0.5 × 104 cells per well) and cultivated for 24 h. Subsequently, the cells were fixed with 4% paraformaldehyde solution for 10 min. To enable permeabilization, 0.1% Triton X-100 solution was then applied for 10 min at room temperature. After thorough washing, the cells were incubated overnight at 4 °C with primary antibody. On the next day, the cells were rinsed in phosphate-buffered saline and then incubated with Alexa Fluor 488-labeled or Alexa Fluor 594-labeled secondary antibody at room temperature for 2 h. The cellular nuclei were stained with DAPI (Solarbio, Beijing, China) prior to mounting onto microscope slides and visualization by confocal microscopy (Nikon, Tokyo, Japan).

pHrodo-ADC assay

To assess the impact of pyrotinib on T-Dxd endocytosis, the pHrodo™ Deep Red Antibody Labeling Kit (Invitrogen, Carlsbad, CA, USA) was used according to the manufacturer’s instructions. For our experimental setup, 0.5 × 104 cells were seeded into 24-well plates with pre-placed coverslips. The cells were divided into two groups: the T-Dxd group and the T-Dxd combined with pyrotinib group. After overnight incubation, both groups received pHrodo-T-Dxd (1 μg/mL) at 4 h time points, while the combined group was supplemented with 0.1 μM pyrotinib 30 min subsequent to pHrodo-Dxd incubation. After treatment, the cells were fixed in 4% paraformaldehyde solution and visualized by laser confocal microscopy.

In vitro cell growth assay

Cells were seeded in 96-well plates (8000 cells/well for NCI-N87, 3000 cells/well for SNU216). After overnight incubation, serial dilutions of pyrotinib or lapatinib were added. Cell viability was assessed after 24 h using CCK8 (ShareBio, Shanghai, China) according to the manufacturer’s instructions. To study synergy, the cells were divided into two groups: T-Dxd and T-Dxd combined with HER2-TKI. Based on the HER2-TKI results, doses selected with minimal proliferation impact for each cell type were combined with varying T-Dxd doses. After 24 h, the cell viability was measured using CCK8.

In vivo xenograft studies

Animal experiments followed protocols approved by the Animal Research Committee of Bethune International Peace Hospital. Female BALB/c nude mice (5 weeks old) were obtained from the Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China) and housed in a controlled, pathogen-free environment. Subcutaneous injections of 5 × 106 NCI-N87 cells were administered into the right flanks, and treatments were initiated when the appropriate tumor volumes were reached. To mitigate gastrointestinal reactions linked to both HER-TKIs and T-Dxd, a lower intervention dose was selected. Xenografts were randomly assigned to treatment groups: T-Dxd (0.25 mg/kg, intravenously every 3 weeks) [12], pyrotinib (2 mg/kg, orally 5 days a week) [13], a combination of both, or vehicle control (sterilized water, orally 5 days a week). Daily observations were carried out to monitor potential toxicity. Tumor dimensions were measured biweekly using a digital caliper, and the tumor volume was calculated using the formula: tumor volume = (length × width2)/2. Subsequently, histological examinations, including H&E staining, IHC, and Western blotting assays were conducted on excised subcutaneous tumors.

Statistical analysis

IBM SPSS Statistics Version 26.0 (IBM, New York, NY, USA) was used for data analysis and GraphPad Prism 8 (GraphPad Software, San Diego, CA, USA) for graph creation. Data were expressed as mean ± standard deviation. Two-group differences were evaluated by two-sample two-tailed Student’s t-test, while multiple-group comparisons were evaluated by one-way analysis of variance (ANOVA) with Dunnett’s post hoc test. A significance level was set at α = 0.05, with significance denoted as P < 0.05.