Macrophage remodeling during lung adenocarcinoma progression revealed by single-cell RNA sequencing and multiplex immunohistochemistry

Lung adenocarcinoma (LUAD) represents the most prevalent subtype of lung cancer, characterized by a high metastatic potential and a poor prognosis. Tumor-associated macrophages are recognized as influential regulators of the tumor microenvironment (TME); however, the stage-specific evolution of their heterogeneity and transcriptional reprogramming during the progression from localized disease to distant metastasis remains inadequately understood. Multiplex immunohistochemistry (mIHC) was performed on 118 LUAD specimens, complemented by single-cell RNA sequencing (scRNA-seq) analysis of 28 primary tumor biopsies obtained from 26 LUAD patients across independent cohorts. Patients were stratified into three clinically defined disease-extent groups: Group 1, representing localized early-stage disease; Group 2, encompassing locoregional and proximal spread, such as intrapulmonary metastasis or regional lymph node involvement; and Group 3, reflecting distant metastatic disease. Analyses focused on macrophage spatial distribution, transcriptional profiles, subtype dynamics, and intercellular communication networks. mIHC revealed predominant localization of macrophages within the tumor stroma, with an increased accumulation of M2-like macrophages observed in more advanced disease-extent groups. Larger tumor size and lymph node metastasis were significantly correlated with higher M2-like macrophage infiltration, whereas M1-like macrophage abundance remained relatively consistent across different stages. The complementary independent scRNA-seq cohort analysis of primary tumors identified eight transcriptionally distinct macrophage clusters, highlighting disease extent-associated remodeling of macrophages toward M2-like polarization. This remodeling was marked by upregulated M2-associated regulatory genes and lysosomal–phagocytic programs, accompanied by diminished antigen presentation and reduced MHC class II gene expression in distant metastatic disease. Macrophage subsets within localized tumors retained features characterized by inflammatory activity and support for T cells, including an M1-like population that became less abundant in distant metastatic tumors. In contrast, distant metastatic tumors exhibited enrichment of M2-like macrophage subsets typified by immunosuppressive and metabolically quiescent transcriptional programs. Cell–cell communication analysis predicted reduced interactions between pro-inflammatory M1-like macrophages and T cells, alongside increased autocrine signaling centered on macrophages, with alterations in macrophage–T cell ligand–receptor interactions inferred in metastatic disease. Progression of LUAD was associated with a marked increase in M2-like macrophage abundance and the transcriptional remodeling of macrophage programs. The transition from an immune-supportive to an immunosuppressive TME corresponded with reduced macrophage-mediated antigen-presentation characteristics and notable alterations in cellular communication dynamics. These findings, derived from parallel cohort analyses, delineate stage-associated macrophage remodeling during LUAD progression and provide critical groundwork for future investigations into the potential functional role of macrophage plasticity in metastatic dissemination.