Spatial single-cell multi-omics characterization of the tumor microenvironment heterogeneity by HER-2 expression status in gastric cancer.

[BACKGROUND] The success of novel antibody-drug conjugates, such as trastuzumab deruxtecan (T-DXd) and disitamab vedotin (RC48), has been pivotal in rendering “HER-2-low” gastric cancer (GC) a therapeutically targetable entity, expanding the population benefiting from anti-HER2 therapy by 2-3-fold. This advancement underscores the imperative to redefine and subclassify HER-2 status in GC. In this study, we innovatively proposed a four-tiered HER-2 classification standard for GC and, for the first time, conducted comparative analyses at the spatial single-cell multi-omics level across these four subgroups.

[METHODS] HER-2 status was recategorized into absent (immunohistochemistry (IHC) 0+, HER2_N), low (IHC 1+, HER2_L), moderate (IHC 2+/fluorescence in situ hybridization (FISH)-, HER2_M), and high (IHC 2+/FISH + or IHC 3+, HER2_H) groups. Primary tumor samples from 427 GC patients were collected and analyzed using Xenium5K in situ single-cell spatial transcriptomics ( = 153) and multiplex immunofluorescence ( = 427) detection base on tissue microassays.

[RESULTS] The proportion of samples that classified as HER2_N, HER2_L, HER2_M, and HER2_H was 43.13%, 22.88%, 11.11%, and 22.88%, respectively. Spatial HER-2 heterogeneous expression existed in 74.42% of HER-2 expressing GC and in 40.0% of HER2_H cases, which correlated with an unfavorable response to combined HER-2-targeted and immunotherapy and poor prognosis. In HER2_H group, lower infiltrates of exhausted T cells and regulatory T cells (Treg), higher cytotoxic activity of T cells, and enriched T cell-B cell niches were discovered, presenting a favorable tumor microenvironment that may benefit from immunotherapy. In HER2_M population, significantly higher CTLA4 + Treg infiltration, and their strengthened interactions with other cells via the CD80/CD86-CTLA4 axis were highlighted. HER2_L group exhibited increased infiltrates of SOX2-OT + mesenchymal cells and TGF-β-driven stromal-tumor cellular interactions. Moreover, HER2_N group presented a higher prevalence of diffuse and mixed histology, enriched endothelial cell-fibroblast-myeloid-derived suppressor cell niches, and augmented angiogenic activity.

[CONCLUSION] Differences in clinicopathologic, molecular, and immunological underpinnings were underscored across GC with differential HER-2 expression status, providing a rationale for a novel quaternary HER-2 classification system and the development of stratified therapeutic strategies.

[SUPPLEMENTARY INFORMATION] The online version contains supplementary material available at 10.1186/s12943-026-02577-x.