Spatiotemporal analysis reveals tsMHC-I downregulation-induced neutrophil extracellular traps as a driver of lung adenocarcinoma neoplastic evolution

Background: Loss of tumor-specific MHC class I (tsMHC-I) is a recurrent feature of lung adenocarcinoma (LUAD) progression and is associated with immune escape and poor outcomes. However, how tsMHC-I downregulation shapes tumor evolution and remodels the immune microenvironment remains unclear. Methods: We integrated single-cell RNA sequencing, spatial transcriptomics, and multiplex imaging from human LUAD samples spanning precancerous lesions to advanced tumors, together with longitudinal genetically engineered mouse models (GEMMs). Functional assays-including neutrophil co-culture, assessment of neutrophil extracellular traps (NETs), and in vivo tumor studies-were performed to define mechanistic consequences of tsMHC-I loss. Results: Progressive tsMHC-I downregulation was observed across LUAD evolution and validated in GEMMs. Single-cell trajectory analysis showed that reduced tsMHC-I is linked to loss of alveolar type II identity, increased chromosomal instability, heightened tumor plasticity, and acquisition of epithelial-mesenchymal transition (EMT) programs. Functionally, tsMHC-I Low malignant cells displayed increased invasiveness and metastatic capacity. Spatial and immunophenotypic profiling revealed that tsMHC-I downregulation reshapes the tumor microenvironment toward a neutrophil-enriched, cytotoxic cell-excluded niche. tsMHC-I Low regions exhibited dense tumor-associated neutrophil aggregates and depletion of CD8⁺ T cells, NK cells, and other effector populations. Mechanistic studies showed that tsMHC-I Low malignant cells induce robust NET formation. In vitro assays identified Annexin A2, a tumor-secreted protein, as a key driver of NET induction. Annexin A2 expression and secretion were consistently elevated in tsMHC-I Low cells across human and mouse models, and Anxa2 knockdown markedly reduced NET formation. Targeting NETs in vivo with DNase I selectively suppressed growth and metastatic spread of tsMHC-I Low tumors, restored cytotoxic immune infiltration, and reversed the immune-excluded phenotype. In tsMHC-I heterogeneous tumors, combining NET inhibition with anti-PD-1 enhanced tumor control and survival. Conclusions: Our integrated analyses reveal that tsMHC-I downregulation drives neutrophil recruitment and NET formation through Annexin A2 secretion, creating an immunosuppressive barrier that promotes LUAD progression and limits immunotherapy response. These findings identify a previously unrecognized tsMHC-I-NET axis as a therapeutic vulnerability in LUAD.