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: Trem2 -KO combined with IFN-γ promotes metabolic reprogramming in TAMs (A) Heatmap of KEGG pathways and differential gene set analysis of glycolysis, TCA, and fatty acid metabolism. Results were applied separately to Cxcl9 + and Spp1 + subtypes. (B) Heatmap of differential gene set analysis of glycolysis, TCA, and fatty acid metabolism. Results were applied separately to each group of CAR-T-treated WT or Trem2 -KO mice. (C) Schematic representation of in vitro tumor-educated macrophage model. 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 (40 ng/mL) intervention. (D) Relative mRNA expression levels of Cxcl9 and Spp1 in BMDMs were quantified by RT-qPCR, and the ratio of Cxcl9 / Spp1 was calculated. (E) AMP and ATP concentrations in BMDMs were measured using dedicated assay kits, and the AMP/ATP ratio was calculated from these values. Relative mRNA expression levels of Ldha, Idh2, and Cpt1 in BMDMs were quantified by RT-qPCR. (F) Relative protein expression levels of indicated molecules were assessed by western blot (WB) and normalized to β-actin. (G, H, and O) BMDMs were pretreated for 2 h with the AMPK inhibitor compound C (20 μM; G), mTOR inhibitor rapamycin (1 μM; H), or SYK inhibitor R406 (1 μM; O), followed by 24 h co-culture with tumor cells in the presence or absence of IFN-γ (40 ng/mL). Protein expression was analyzed by WB and normalized to β-actin. (I and J) The real-time changes of OCR of BMDMs were stimulated with or without IFN-γ in the basal state and following the additions of oligomycin (Oligo), fluorocarbonyl cyanide phenylhydrazone (FCCP), etomoxir (Eto), and rotenone + antimycin A (Rot/AA). The average of basal OCR, maximal OCR, and Eto-sensitive OCR were revealed. (K and L) The real-time measurement of ECAR of BMDMs was stimulated with or without IFN-γ 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. (M and N) The relative protein expression levels of indicated molecular were assessed by WB and normalized to β-actin. (P) The iBMDMs were transiently transfected to overexpress SOCS1 for 48 h, followed by 24-h stimulation with or without IFN-γ (40 ng/mL). Indicated molecular protein expression levels were tested by WB and normalized to β-actin. Data were represented by mean ± SEM. ns, no significance, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: SYK inhibitor R406 , Selleck , Cat. #S1533.

Techniques: In Vitro, Isolation, Expressing, Quantitative RT-PCR, Western Blot, Co-Culture Assay, Transfection