Abstract 6129: Cancer cell attenuate tumor-associated macrophage TLR9 pathway through lipid-TFEB-PLD3 axis to induce immune suppression environment in ESCC

Abstract Introduction: Esophageal squamous cell carcinoma (ESCC) is one of the most threaten malignancies in China. Immunotherapy targeting PD-1/PD-L1 and CTLA-4 has achieved progress, while there remains about 30% patients that did not response. Tumor-associated macrophages (TAMs) are an important component of tumor microenvironment (TME), which may contribute to immunosuppression, tumor progression, and metastasis. Thus, targeting to TAMs in ESCC is with potential translational value. Phospholipase D3 (PLD3) is an exonuclease to degrade single strand DNA/RNA in lysosome to attenuate toll-like receptor 9 signaling (TLR9) in myeloid cells. Mice with PLD3 homozygous deletion sufferd from severe autoimmune disease. However, the role of PLD3 in TME has not been reported. Considering the innate immune inhibition effect of PLD3, we decide to explore its function in TAMs. Material: We collected normal tissue, tumor, and metastatic lymph node samples from ESCC patients for single-cell RNA sequencing. After quality control, we got a total of 85263 cells and clustered cells by Seurat package. Then we integrated our cohort with 3 other ESCC single-cell sequencing cohorts and focused on macrophages. clusterProfiler package were used to do pathway enrichment analysis. JASPR were used for transcription factor prediction. THP-1 cells were cultured in complete RPMI1640 medium. Mouse bone marrow-derived macrophages (BMDM) were isolated and cultured as mentioned in references. KYSE-30, 180, 410, and 520 cell lines were cultured in complete RPMI1640 medium. mEC25 and mEC35 cell lines were cultured in complete DMEM medium. Result: After integration and clustering, we found PLD3 was specifically expressed in macrophages among all cell types. After sub-clustering, only a small part of macrophages showed high expression of PLD3, which was defined as PLD3-high macrophages. PLD3-high macrophages presented an enrichment in tumors and metastatic lymph nodes, and pathway analysis revealed an inhibition of innate immune response and upregulation of cholesterol metabolism and lysosome pathway in these macrophages. Immunofluorescence staining verified these macrophages in ESCC patient samples. In vitro results showed that oxidized low-density lipoprotein and tumor cell conditioned medium could increase PLD3 expression both in THP-1 macrophages and BMDM, and attenuated TLR9 downstream cytokines expression. Cholesterol assay found ESCC tumor cells can secret considerable amount of cholesterol to extracellular environment. Since TFEB is the key transcription factor in macrophage lysosome biogenesis, we found TFEB may bind to the promoter region of PLD3 to start its transcription. Conclusion: Tumor cells can secret cholesterol to trigger TFEB/PLD3 signaling and suppress TLR9 activation in TAMs to achieve an innate immune inhibition effect in TME. Citation Format: Licheng Tan, Beilei Liu, Hongyu Zhou, Shuang Zhang, Bowen Yao, Jiayi Huang, Xinyuan Guan. Cancer cell attenuate tumor-associated macrophage TLR9 pathway through lipid-TFEB-PLD3 axis to induce immune suppression environment in ESCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6129.