SPP1-positive myeloid cell subpopulations associated with resistance to PD-1/L1 immunotherapy in lung adenocarcinoma.

PD-1/PD-L1 immune checkpoint inhibitors have significantly improved outcomes in Lung adenocarcinoma (LUAD), yet many patients remain unresponsive due to resistance. This study aimed to identify key cellular subpopulations associated with immunotherapy resistance in LUAD. Clinical data from 197 LUAD patients treated with PD-1/PD-L1 inhibitors were retrospectively analyzed, including 79 with response and 118 with resistance. Fresh tumor tissues from three patients per group underwent single-cell RNA sequencing (scRNA-seq), and one sample per group was analyzed using spatial transcriptomics. Immunohistochemistry (IHC) and immunofluorescence (IF) were used to validate protein expression and overlapping spatial signals. scRNA-seq revealed a distinct SPP1⁺ myeloid subpopulation enriched in resistant tumors. Pseudotime analysis suggested these cells are in a differentiation state linked to tumor progression. SCENIC analysis indicated activation of transcriptional networks potentially contributes to immune suppression. Spatial transcriptomics confirmed SPP1⁺ myeloid cells were localized near tumor nests in resistant tissues, suggesting an association with immunosuppressive microenvironment. IHC and IF further validated increased SPP1 expression and its overlapping spatial signals with macrophage markers CD68 and CD163, confirming their tumor-associated macrophage identity. This study identifies SPP1⁺ myeloid cells as a subpopulation closely associated with LUAD immunotherapy resistance. They may serve as a potential candidate biomarker for predicting PD-1/PD-L1 treatment outcomes and represent targets for further functional exploration.