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Published on: February 12, 2022
CD19 CAR-macrophage suppressed the progression of DLBCL via modulating SYK/SHP2/TBK1/TFEB signaling
Xikui Zhang1, Tao Wen2, Pengfei Chen1
1Department of Ophthalmology, The Fourth Hospital of Hebei Medical University, 051000, China.
Objective:
To explore the specific molecular mechanisms of CD19 CAR-Macrophage (CD19 CAR-M) in treating Diffuse Large B-cell Lymphoma (DLBCL).
Methods:
Differential expression and pathway enrichment analyses of GEO dataset GSE66770 were performed to construct a SYK/SHP2/TBK1/TFEB interaction network. CD19 CAR-M were generated by lentiviral transduction of THP-1-derived macrophage-like cells and co-cultured with U2932 cells. CCK-8, colony formation and Transwell assays were used to evaluate U2932 proliferation, invasion and migration; IL-6 and IFN-γ levels were measured by ELISA; ROS was detected by DHE staining. RT-qPCR quantified macrophage markers CD14 (M0), CD163 and CD206 (M2), and M1 marker IL-12. Western blot analyzed p-SYK, p-SHP2, NOX2, p-TBK1, TFEB, LC3B and phosphorylated/total protein ratios; immunofluorescence assessed endosomal markers EEA1 and LAMP1. Phagocytosis was evaluated by fluorescent microsphere assay; U2932 apoptosis was determined by flow cytometry after FcγR blockade. Flow cytometry was additionally performed to validate the maintenance of macrophage phenotype, CAR transduction efficiency, and the purity of cell separation after co-culture. NOD-scid mouse DLBCL xenografts were subjected to in vivo imaging for therapeutic validation.
Results:
Bioinformatic analysis identified TBK1 as a key immune-evasion node positively correlated with SYK and negatively with SHP2. Flow cytometric analysis confirmed successful construction of the CD19 CAR-M cell model, with stable macrophage phenotype and specific CAR expression. CD19 CAR-M significantly inhibited U2932 proliferation, invasion and migration, elevated IL-6, IFN-γ and ROS, and induced M1 polarization. Phosphorylation of SYK/SHP2/TBK1/TFEB axis proteins and LC3B expression were markedly increased; EEA1/LAMP1 fluorescence intensities were enhanced. Fluovrescent microsphere uptake was elevated and reversible by FcγR inhibitor elsubrutinib, accompanied by increased U2932 late-apoptosis rate. In vivo experiments confirmed that CD19 CAR-M reduced tumor burden via activation of the SYK/SHP2/TBK1/TFEB axis.
Conclusion:
CD19 CAR-M kills DLBCL through macrophage phagocytosis mediated by the SYK/SHP2/TBK1/TFEB axis.
Insights
CD19 CAR-Macrophage therapy effectively treats Diffuse Large B-cell Lymphoma by activating the SYK/SHP2/TBK1/TFEB pathway, enhancing macrophage phagocytosis to eliminate cancer cells.
Area of Science:
- Immunology
- Oncology
- Cell Biology
Background:
- Diffuse Large B-cell Lymphoma (DLBCL) is an aggressive non-Hodgkin lymphoma.
- Chimeric Antigen Receptor (CAR) engineered macrophages represent a novel therapeutic strategy.
- Understanding the molecular mechanisms of CAR-Macrophage therapy is crucial for optimizing treatment.
Purpose of the Study:
- To elucidate the specific molecular mechanisms by which CD19-targeted CAR-Macrophage (CD19 CAR-M) therapy exerts its effects against DLBCL.
- To identify key signaling pathways involved in CD19 CAR-M mediated anti-lymphoma activity.
Main Methods:
- Bioinformatic analysis of gene expression data (GEO dataset GSE66770) to identify key signaling nodes.
- Generation and characterization of CD19 CAR-M using lentiviral transduction.
- In vitro assays to assess DLBCL cell proliferation, invasion, migration, cytokine production, and apoptosis.
- Western blot and immunofluorescence to analyze protein expression and cellular localization.
- In vivo studies using DLBCL xenograft mouse models to evaluate therapeutic efficacy.
Main Results:
- Bioinformatics identified a SYK/SHP2/TBK1/TFEB interaction network, with TBK1 as a crucial immune-evasion node.
- CD19 CAR-M successfully inhibited DLBCL cell growth, migration, and invasion, while promoting M1 polarization and cytokine release (IL-6, IFN-γ).
- Activation of the SYK/SHP2/TBK1/TFEB pathway and increased LC3B expression were observed, alongside enhanced phagocytosis and DLBCL apoptosis.
- In vivo studies demonstrated tumor burden reduction mediated by CD19 CAR-M via SYK/SHP2/TBK1/TFEB axis activation.
Conclusions:
- CD19 CAR-M therapy effectively eliminates DLBCL cells.
- The mechanism involves macrophage-mediated phagocytosis, driven by the activation of the SYK/SHP2/TBK1/TFEB signaling axis.
- This study provides critical insights into the molecular underpinnings of CAR-Macrophage therapy for DLBCL.
