CXCL9/SPP1-polarized tumor-associated macrophages exert dual roles in regulating anti-tumor immunity in lung adenocarcinoma

BACKGROUND: CS polarity, the mutually exclusive expression of CXCL9 and SPP1, is a key feature of tumor-associated macrophages (TAMs) in the lung adenocarcinoma (LUAD) microenvironment. The mechanisms and impact of CS polarity on tumor progression remain incompletely understood. The study aims to define the molecular basis of CS polarity and its role in LUAD progression to inform new immunotherapies. METHODS: Single-cell RNA sequencing was used to characterize TAM subsets within the LUAD microenvironment, with survival analysis assessing their prognostic values and CellChat mapping cell-cell communication. In the functional validation section, a CS polarization regulation model of THP-1 cells was established. The Transwell co-culture system was utilized to evaluate its effects on the malignant behaviors of LUAD cells. CCK-8 assay, colony formation assay and cell migration assay were performed to detect cell proliferation and metastatic capacities. Flow cytometry was applied to determine the polarized phenotypes of TAMs (CD86⁺/CD163⁺). Dual-luciferase reporter assay was conducted to verify the direct transcriptional regulation of HPGDS promoter by MEF2C. Western blot, immunocytochemistry and qRT-PCR were adopted to analyze the activity of key signaling pathways. Furthermore, the above findings were further validated in vivo using a subcutaneous transplantation tumor model in nude mice. RESULTS: TAMs participated in immune suppression and tumor-promoting processes, correlating with poor outcome. In vitro, increasing the CXCL9/SPP1 ratio synergistically promoted macrophages toward an M1 phenotype and suppressed LUAD cell proliferation, migration and invasion. In addition, CS polarity reversal further promoted MEF2C nuclear translocation and HPGDS transcription by activating the p38/MAPK signaling axis. Dual-luciferase reporter assays directly confirmed that MEF2C activated HPGDS transcription via specifically binding to its promoter, which served as a critical molecular event that induced phenotypic switching of TAMs and triggered their antitumor functions. In vivo experiments using the subcutaneous transplantation tumor model in nude mice further verified that combined treatment with KO-SPP1 and OE-CXCL9 markedly inhibited tumor growth and facilitated the polarization of intratumoral macrophages toward an antitumor phenotype, which was highly consistent with the in vitro findings. CONCLUSION: CS polarity modulates TAM polarization and their anti-tumor function through the p38/MAPK-MEF2C-HPGDS axis, offering theoretical foundation and potential targets for macrophage reprogramming-based LUAD immunotherapy.