Immunomodulatory effects of trastuzumab deruxtecan through the cGAS-STING pathway in gastric cancer cells

Abstract

Although the efficacy of trastuzumab deruxtecan (T-DXd) against HER2-positive gastric cancers (GCs) has driven its clinical application, the precise mechanisms governing its immunomodulatory role remain unclear. In this study, we examined the immune-related mechanisms of action of T-DXd in GC cells. T-DXd exhibited potent antitumor effects in GC cells across diverse HER2 expression levels by inducing DNA damage and apoptosis. Activation of the DNA damage response by T-DXd led to increased PD-L1 expression. RNA-Seq analysis revealed that T-DXd modulated immune-related pathways, resulting in the upregulation of genes associated with inflammation and IFN signaling. Importantly, T-DXd activated the cGAS-STING pathway, inducing an IFN-I response in HER2-positive GC cells. Furthermore, T-DXd activated dendritic cells via the cancer cell-intrinsic cGAS-STING-IFN axis and enhanced PBMC-mediated tumor cell killing by activating CD8⁺ T cells. These findings provide valuable insights into the role of the cytosolic DNA sensing pathway in the action of T-DXd and offer a compelling rationale for combining T-DXd with immune checkpoint blockade therapies in GC treatment.

Keywords: DNA damage; ErbB-2; Trastuzumab deruxtecan; Type-1 IFN; cGAS-STING signaling.

Fig. 1

T-DXd induces DNA damage response and potent cytotoxic effects against GC cell lines with various HER2 expression levels. A GC cells were treated with varying concentrations of T-DXd for six days, and cell viability was measured using MTT assays. The results of four independent experiments are presented as the mean ± SEM of the percentage of viable cells. B Whole lysates were prepared from GC cells and subjected to immunoblotting to determine the basal expression level of HER2. C The nonparametric Spearman’s correlation between the growth inhibition rate of GC cells in response to T-DXd (Fig. 1A) and the mRNA expression level of HER2, obtained from the RNA-Seq-based CCLE expression dataset (DepMap Public 22Q2). D GC cells were treated with 1 µg/ml of trastuzumab or T-DXd for 10 days and subjected to clonogenic assays. Relative colony numbers are presented as mean ± SEM *, p < 0.05; **, p < 0.005 of three biological replicates. E The cell cycle distribution was evaluated by flow cytometry after five days of treatment with 1 µg/ml T-DXd in indicated GC cells, and data from at least three independent experiments are shown as mean ± SEM *, p < 0.05, **, p < 0.005. F GC cells treated with 1 µg/ml of trastuzumab or T-DXd for five days were prepared into whole lysates for immunoblotting. G Comet analysis was conducted in GC cells treated with 1 µg/ml of T-DXd for 72 h. Left panel: representative images of the three independent experiments are shown. Right panel: each scattered dot represents the tail intensity of single cells and the sum of three biological replicates. Data are shown as mean ± SEM **, p < 0.005, ***, p < 0.001. H Immunofluorescence analysis was performed to detect Rad51 foci. Left panel: GC cells treated with 1 µg/ml of T-DXd for 72 h were permeabilized with 0.5% Triton X-100 and stained for Rad51 (green), and counterstained with DAPI (blue). Representative images from three independent experiments are shown. Right panel: the relative number of cells positive for Rad51 foci was determined by counting over 100 cells in each dataset and shown as mean ± SD *, p < 0.05; ***, p < 0.001

Fig. 2

T-DXd-induced DDR activation regulates PD-L1 transcription through IRF1 in GC cells. A PD-L1 expression in GC cells treated with different concentrations of T-DXd (0, 0.04, 0.2, 1, and 5 µg/ml) for 120 h was assessed by immunoblotting. B Quantitative PCR analysis of PD-L1 expression in GC cells treated with 1 µg/ml of T-DXd for 72 h. β-Actin was used as a normalization control. Data are presented as mean ± SD ***, p < 0.001. C Flow cytometric analysis of surface PD-L1 expression in GC cells treated with 1 µg/ml of T-DXd for 72 h. Data from at least three independent biological replicates are shown as mean ± SD. ***, p < 0.001. D PD-L1 mRNA levels in NCI-N87 cells treated with T-DXd (1 µg/ml) and/or AZD0156 (ATMi, 0.1 µM), and/or AZD6738 (ATRi, 0.1 µM) for 72 h, were determined by RT-qPCR. Data from three independent experiments are represented as mean ± SEM *, p < 0.05; ***, p < 0.001. E Immunoblot analysis of PD-L1 and IRF1 expression levels in cell lysates from NCI-N87 cells treated with T-DXd (1 µg/ml) and/or inhibitors of ATM and ATR. F Immunoblot analysis of PD-L1 and IRF1-related gene expression levels in GC cells transfected with siRNAs targeting IRF1, with or without T-DXd treatment (1 µg/ml)

Fig. 3

T-DXd provokes a gene expression signature related to enhanced tumor immunogenicity in GC cell lines with high and low HER2 expression. RNA-Seq was performed using NCI-N87 cells after treatment with the vehicle, trastuzumab (1 µg/ml), and T-DXd (1 µg/ml) for 72 h. A Hierarchically clustered heatmap of 1,310 DEGs (| fold change |≥ 2 and raw p < 0.05, n = 3). B The top 20 enriched Hallmark gene sets in response to T-DXd ranked by normalized enrichment score from GSEA. C The result of the GO enrichment analysis showing the top 28 enriched gene sets following T-DXd treatment. D Representative images of GSEA showing the enrichment of gene signatures associated with antitumor immune response by T-DXd treatment. E Heatmap illustrating Reactome Interferon alpha/beta signaling geneset from RNA-Seq data

Fig. 4

T-DXd activates the cytosolic DNA recognition pathway in GC cells. A Heatmap of cytosolic pattern recognition receptor pathway genes showing differential expression after T-DXd treatment. B NCI-N87 cells treated with 1 µg/ml of T-DXd or 10 µM of camptothecin for indicated times were subjected to slot blot assays to measure TOP1cc levels. Representative images from biological duplicates are shown. C Immunofluorescence analysis was conducted in SNU-216 cells treated with 1 µg/ml of T-DXd for 72 h to detect the formation of DNA damage foci and MN. Permeabilized cells were stained for γ-H2AX (red) and DNA compartment (blue). Left panel: representative images from four independent experiments are shown, indicating the γ-H2AX-positive MN and γ-H2AX-negative MN. Right panel: the percentage of cells with MN was measured by counting more than 100 cells of each experiment and shown as mean ± SEM **, p < 0.005. D Immunofluoresence analysis using SNU-216 cells treated with 1 µg/ml of T-DXd for identifying the TOP1cc (green) formation and its colocalization with MN. E Immunofluorescence analysis of cytoplasmic dsDNA (green) in SNU-216 cells treated with 1 µg/ml of T-DXd, 0.1 µM AZD0156 (ATR inhibitor), 0.1 µM AZD6738 (ATM inhibitor), or their combination with T-DXd for 48 h. Nuclei were counterstained with DAPI (blue), and Lamin B1 (red) was used as a nuclear envelope marker. Left panel: representative images from four biological replicates are shown. Right panel: the percentage of cells positive for cytoplasmic dsDNA is shown as mean ± SEM. ns: not significant; **, p < 0.005; ***, p < 0.001. F Immunoblotting of genes associated with the canonical cGAS-STING pathway in NCI-N87 cells treated with varying concentrations of T-DXd (0, 0.04, 0.2, 1, and 5 µg/ml). G Immunofluorescence analysis of SNU-216 cells treated with 1 µg/ml of T-DXd showing the upregulation of cGAS (Red) expression. The nuclei were counterstained by DAPI (blue) and representative images from three biological replicates are shown. H The synthesis (left panel) and secretion (right panel) levels of cGAMP were determined by ELISA in NCI-N87 cells treated with 1 µg/ml of trastuzumab or T-DXd for 72 h. Every symbol represents one biological replicate from five experiments. Statistical significance is shown as *, p < 0.05; **, p < 0.005. I Quantitative PCR analysis determined the gene expression level of ISGs in NCI-N87 cells in response to 1 µg/ml of T-DXd treatment. Each dot represents one biological replicate and data are shown as mean ± SEM, ns: not significant, *, p < 0.05; **, p < 0.005. J Secretion levels of CCL5 (left panel) and CXCL10 (right panel) in NCI-N87 cells treated with 1 µg/ml trastuzumab or T-DXd, measured by ELISA

Fig. 5

cGAS is essential for the T-DXd-mediated promotion of IFN-I response in GC cells. A The impact of cGAS knockdown on cell proliferation (left panel) and sensitivity to T-DXd (right panel) was assessed by MTT assays using NCI-N87 cells transfected with the indicated siRNAs. Data represent the mean ± SD from three independent experiments, ns: not significant. B The immunoblot assay determined the effect of cGAS deficiency on T-DXd-induced STAT1 pathway activation in NCI-N87 cells. C Quantitative PCR analysis evaluated the mRNA expression levels of ISGs in NCI-N87 cells transfected with specific siRNAs. Data represent the mean ± SD from three independent experiments, ns: not significant, *, p < 0.05; **, p < 0.005; ***, p < 0.001. D ELISA measured the cytokine release from cancer cells transfected with specific siRNAs in response to T-DXd treatment. The relative secretion levels of CCL5 and CXCL10 are depicted as mean ± SD, ns: not significant, **, p < 0.005; ***, p < 0.001. E Immunoblotting was employed to evaluate the restoration of T-DXd-induced IRF1 induction, which was attenuated by cGAS depletion upon cGAMP transfection in NCI-N87 cells

Fig. 6

T-DXd induces DC activation and enhances PBMC-mediated tumor cell killing. A THP-1 cells were differentiated into TDDCs by adding a cocktail of 20 ng/ml of PMA and recombinant human IL-4. The morphologies of THP-1 cells and TDDCs were observed under a light microscope. B Flow cytometry analysis was performed to determine the relative proportion of CD11c⁺ cells in TDDCs at day 6 post-differentiation. C Flow cytometry analysis was performed to assess the expression level of HLA-DR in CD11c⁺/CD209⁺ TDDCs treated with the vehicle, T-DXd, LPS, or transfected with 2 µg/ml of cGAMP. D NCI-N87 cells and TDDCs were cocultured at a ratio of 3:1 for 72 h with 1 µg/ml of trastuzumab or T-DXd. Flow cytometry analysis was performed to determine the expression level of HLA-DR in CD11c⁺/CD209⁺ TDDCs. E NCI-N87 cells transfected with specific siRNAs were pretreated with T-DXd in the presence of STINGi (H-151, 1 µg/ml), human type I IFN neutralizing antibody mixture, or cGAMP transfection for 6 h. Subsequently, cells were harvested and cocultured with TDDCs for an additional 72 h. Flow cytometry analysis was performed to determine the expression level of HLA-DR in CD11c⁺/CD209⁺ TDDCs. F TDDCs were generated from THP-1 cells in the lower chambers of Transwell plates. NCI-N87 cells were seeded in the upper chambers of Transwell plates and treated with trastuzumab or T-DXd (1 µg/ml) for 72 h. Flow cytometry analysis determined the expression level of CD86 (left panel) and HLA-DR (right panel) in CD11c⁺/CD209⁺ TDDCs. Data from three independent experiments are presented as mean ± SEM, ns: not significant. G Agarose gel electrophoresis of exosomal dsDNA isolated from the culture medium of NCI-N87 cells treated with 1 µg/ml of T-DXd. Exosomes were isolated from the conditioned medium, followed by dsDNA enrichment. Representative images from two independent experiments are shown. H Bar graph showing the expression levels of CD86 and HLA-DR in CD11c⁺/CD209⁺ TDDCs after 48 h incubation with exosomes isolated from the culture medium of NCI-N87 cells treated with T-DXd. Flow cytometry was performed to assess the activation markers. Data represent the mean ± SEM from three independent experiments. *, p < 0.05. I An immunoblot assay was performed on lysates from TDDCs after 48 h of indirect coculture with NCI-N87 cells in the presence of 1 µg/ml of trastuzumab or T-DXd. J RT-qPCR was used to measure the expression levels of IL-12 in TDDCs that were indirectly cocultured with NCI-N87 cells for 48 h in the presence of 1 µg/ml of trastuzumab or T-DXd. Data from three independent experiments were shown as mean ± SD, *, p < 0.05; ***, p < 0.001. K PBMC-mediated killing assay of NCI-N87 cells pretreated with T-DXd or the vehicle. Following a 72-h coculture, flow cytometry was used to determine the percentage of live HER2⁺ cells. L Flow cytometry analysis of CD69 expression, as well as intracellular staining of Granzyme B and IFN-γ in CD8⁺ T cells from PBMCs and NCI-N87 cocultures, with or without T-DXd pre-treatment. Left panel; representative images from at least 11 experiments. Right panel; frequencies of CD8⁺ T cells expressing CD69, Granzyme B, and IFN-γ. Statistical significance was determined using a paired Wilcoxon test. M Scatterplots illustrating the correlation between cGAS expression (log2TPM) and the tumor infiltration of immune cells in TCGA-STAD, including subsets of CD8⁺ T cells, DCs, and macrophages, analyzed using the TIMER2.0 tumor immune estimation resource