Integrated bulk and single-cell transcriptomics to develop an efferocytosis-related prognostic model for lung adenocarcinoma and validate the key gene LDHA.

BACKGROUND: Efferocytosis, the phagocytic clearance of apoptotic cells, exerts dual anti-inflammatory and pro-tumorigenic effects, while existing studies have not well-elucidated the underlying mechanisms in Lung adenocarcinoma (LUAD). This study aims at identifying prognostic genes related to efferocytosis and potential therapeutic targets in LUAD. METHODS: LUAD-related gene expression data sets were obtained from UCSC Xena and GEO databases, while efferocytosis-related genes (ERGs) were sourced from the GeneCards database. Unsupervised consensus clustering analysis assisted in classifying LUAD patients into two distinct subgroups, after which differential gene expression was analyzed for identifying differentially expressed genes (DEGs). Subsequently, a four-gene prognostic model was built by virtue of LASSO-Cox regression and further validated by Kaplan-Meier (KM) analysis and receiver operating characteristic (ROC) curve assessment. Analysis on single-cell RNA sequencing (scRNA-seq) data engaged in investigating cell subpopulations associated with efferocytosis and to pinpoint key genes involved. In vitro experiments were conducted to ascertain the functional importance of the selected key gene in LUAD progression. RESULTS: A prognostic signature incorporating four genes was successfully constructed for LUAD, and risk scores were calculated for categorizing patients into low- and high-risk groups. Notably, the two risk groups presented different survival outcomes and immune cell infiltration (ICI). Analysis of scRNA-seq data revealed macrophages as the primary efferocytosis-related cell type in LUAD and pinpointed LDHA as a key regulatory gene. Pseudotime trajectory analysis demonstrated a progressive increase in LDHA expression and efferocytosis-related activity as macrophages differentiated into tumor-associated macrophages (TAMs). Moreover, in vitro experimental data showed that LDHA silencing in macrophages facilitated polarization toward an M1 phenotype and simultaneously inhibited M2 polarization. In addition, co-culturing LUAD cells with LDHA-silenced M2 macrophages substantially reduced cancer cell proliferation, migration, and invasion. Cell communication analyses suggested that LDHA promotes M2-type TAMs by modulating the SPP1 pathway between macrophages and cancer-associated fibroblasts (CAFs), activating ECM receptors and downstream effectors. CONCLUSIONS: We constructed an efferocytosis-related prognostic gene model for LUAD. Furthermore, LDHA may target M2 macrophages, highlighting its potential as a therapeutic target in LUAD.