PURPOSE: HER2-positive gastric cancer (HER2+ GC) exhibits significant intra-tumoral heterogeneity and frequent development of resistance to HER2-targeted therapies. This study aimed to characterize the spatial tumor microenvironment (TME) in HER2+ GC and identify mechanisms of resistance to HER2 blockade including trastuzumab and trastuzumab deruxtecan (T-DXd), with the goal of informing novel therapeutic strategies. PATIENTS AND METHODS: We performed spatial transcriptomics on pre- and post-treatment samples from patients with HER2+ metastatic GC who received trastuzumab-based therapy. We also established patient-derived organoids (PDOs) to investigate mechanisms of trastuzumab resistance in vitro. RESULTS: ERBB2-high tumor regions were found to be "immune cold", characterized by low PD-L1 expression and reduced lymphocyte infiltration. We identified two distinct mechanisms of acquired trastuzumab resistance: epithelial-mesenchymal transition (EMT) and upregulation of the endoplasmic reticulum-associated protein degradation (ERAD) pathway. EMT-positive tumors showed increased expression of immune checkpoints, including PD-L1, and the chemokine CCL2. Non-EMT tumors exhibited upregulation of the ERAD pathway, highlighting it as a potential therapeutic target. Importantly, we observed increased expression of the promising therapeutic target CLDN18.2, in trastuzumab-resistant tumors. Additionally, loss of HLA was identified as a potential mechanism of resistance to trastuzumab deruxtecan (T-DXd). CONCLUSION: Our spatial profiling study reveals distinct TME features and resistance mechanisms in HER2+ GC, providing a valuable resource for future research and therapeutic development. The identification of potential therapeutic targets, such as CLDN18.2, may pave the way for novel treatment strategies to overcome resistance and improve outcomes for patients with HER2+ GC.

R. Sundar reports grants from National Medical Research Council (NMRC) during the conduct of the study, as well as other support from Bristol Myers Squibb, Merck, Eisai, Bayer, Taiho, Novartis, Eli Lilly, Roche, AstraZeneca, DKSH, MSD, Paxman Coolers, Natera, Astellas, GSK, Ipsen, Pierre-Fabre, Tavotek, Sanofi, Daichii Sankyo, Beigene, CytoMed and Auristone outside the submitted work. F. Pietrantonio reported receiving institutional research grants from BMS, Incyte, Agenus, Amgen, Lilly and AstraZeneca, and personal fees from BMS, MSD, Amgen, Merck-Serono, Pierre-Fabre, Servier, Bayer, Takeda, Astellas, Johnson&Johnson, Rottapharm, Ipsen, AstraZeneca, GSK, Daiichi-Sankyo, Seagen/Pfizer, Beigene. P. Tan has stock in Tempus AI and Auristone Pte Ltd, previous funding from Kyowa Hakko Kirin and Thermo Fisher Scientific, and patents/other intellectual property through the Agency for Science and Technology Research, Singapore (all outside the submitted work). All other authors do not have any conflict of interest to declare.

R. Sundar is supported by the National Medical Research Council (NMRC/CIRG23Jul-0035 and NMRC/ MOH-000627). F. Pietrantonio is supported by AIRC (Associazione Italiana per la Ricerca sul Cancro), IG 2019 number 23624 and Italian Ministry of Health GR-2019-12371132. P. Tan's research is supported by the National Research Foundation, Singapore, and Singapore Ministry of Health's National Medical Research Council under its Open Fund-Large Collaborative Grant ("OF-LCG") (MOH-OFLCG18May-0003) and the Singapore Gastric Cancer Consortium. His work is also supported by the National Medical Research Council grant MOH-000967. All other authors do not have any funding sources to declare. The funders of the study had no role in study design, data collection, analysis, interpretation, or writing of the manuscript.

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