Journal: Cell Reports Medicine
Article Title: CAR-T triggers TAM reeducation and adaptive anti-tumor response via TREM2 deficiency or CD40 agonist
doi: 10.1016/j.xcrm.2025.102539
Figure Lengend Snippet: CD40 agonism triggers a metabolic reprogram of TAMs similar to that of Trem2 -KO (A) Heatmap of differential gene set analysis in TAM subpopulations. Genes were enriched in Mac_Cxcl9 cluster. (B) The network diagram illustrated the highly expressed genes in Mac_Cxcl9 cluster and their interactions with shared transcription factors. (C) Correlation analysis between the ratio of Cd40 + TAMs and Cxcl9 + TAMs . Each dot represented a single-cell sequencing sample of tumor and spleen. (D) BMDMs isolated from Trem2 -KO or WT mice were educated for 24 h in a transwell system with cancer cells, with or without IFN-γ intervention. Relative protein expression levels of CD40 were assessed by WB and normalized to β-actin. (E) Schematic representation of in vitro tumor-educated macrophage model. BMDMs isolated from WT mice were educated for 24 h in a transwell system with cancer cells, with or without IFN-γ (40 ng/mL) and CD40 agonism (crosslinked FGK45, 20 ng/mL) intervention. (F) Relative mRNA expression levels of Cxcl9 and Spp1 in BMDMs were quantified by RT-qPCR, and the ratio of Cxcl9/Spp1 was calculated. n = 3. (G and H) The real-time changes of OCR of BMDMs were stimulated with or without IFN-γ and CD40 agonism in the basal state and following the additions of oligomycin (Oligo), FCCP, etomoxir (Eto), and rotenone + antimycin A (Rot/AA). The average of basal OCR, maximal OCR, and Eto-sensitive OCR were revealed. n = 3. (I and J) The real-time measurement of ECAR of BMDMs were stimulated with or without IFN-γ and CD40 agonism in the basal state and following the additions of glucose (Gluc), oligomycin (Oligo), and 2-deoxy-D-glucose (2-DG). The average of basal ECAR, maximal ECAR, and glycolytic reserve were revealed. n = 3. (K, L, O, and P) BMDMs from WT mice were co-cultured with tumor cells in transwells for 24 h with or without IFN-γ (40 ng/mL) and CD40 agonism (crosslinked FGK45, 20 ng/mL) (K and O). For inhibitor studies, BMDMs were pre-treated with AMPK inhibitor (20 μM) (L) or STAT1 inhibitor (100 μM) (P) for 2 h prior to co-culture. Protein levels were analyzed by western blot and normalized to β-actin. (M and N) AMP and ATP concentrations in BMDMs were measured using dedicated assay kits, and the AMP/ATP ratio was calculated from these values. n = 3. (Q) Mechanism schema diagram. Data were represented by mean ± SEM. ns, no significance, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.
Article Snippet: STAT1 α Rabbit mAb , CST , Cat. #9172.
Techniques: Sequencing, Isolation, Expressing, In Vitro, Quantitative RT-PCR, Cell Culture, Co-Culture Assay, Western Blot
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