Journal: Journal of Translational Medicine

Article Title: Glutamine-driven metabolic reprogramming promotes CAR-T cell function through mTOR-SREBP2 mediated HMGCS1 upregulation in ovarian cancer

doi: 10.1186/s12967-025-06853-0

Figure Lengend Snippet: Glutamine enhances metabolic activity and functional phenotypes of CAR-T cells. A Schematic of experimental: Analysis of metabolic and functional phenotypes in CAR-T cells. CAR-T cells were pretreated with glutamine (2 or 5 mM) for 2 days. B CAR expression levels in CAR-T cells were detected by flow cytometry after co-incubation with 2 or 5 mM glutamine for 2 days. C Intracellular ATP levels in CAR-T cells after co-incubation with 2 or 5 mM glutamine for 2 days. D Flow cytometry analysis of CAR-T cell subsets after glutamine pre-treatment. The gating strategy for memory phenotype identification was as follows: (1) live cell populations were gated, with a negative gate established using CAR-T cells without antibody staining; (2) single staining for CD45RA and CCR7 was used to define positive gates; (3) a double staining for CD45RA and CCR7 was applied to identify the subsets: TSCM (CD45RA + CCR7 +), TCM (CD45RA – CCR7 +), TEM (CD45RA – CCR7 –), and TEFF (CD45RA + CCR7 –). E Co-expression level of exhaustion-related inhibitory receptors (PD – 1and TIM – 3) in CAR-T cells after co-incubation with 2 or 5 mM glutamine for 2 days. The co-expression of PD-1 and TIM-3 was quantified as the percentage of double-positive cells (PD – 1 + TIM – 3 +) within the live CAR-T cell population. The gating strategy involved defining the live cell population and identifying PD-1 and TIM-3 positive cells. F ROS levels in CAR-T cells by flow cytometry. G Apoptosis analysis of CAR T cells by flow cytometry. H On Day 4, CAR-T cells were labeled with CFSE, and after two days of treatment with 2 mM or 5 mM glutamine, cell proliferation was assessed by flow cytometry. I Flow-cytometric analysis of activation (CD25 and CD69) in CAR-T cells after treatment with glutamine (2/5 mM). J , K Mitochondrial function and glycolysis: ( J ) Measurement of oxygen consumption rates (OCRs); ( K ) Seahorse glycolytic stress test showing glycolytic capacity of CAR-T and CAR-T + Gln cells. L α-KG levels in CAR-T cells. Glutamine pretreatment (5 mM, 48 h) significantly increased intracellular α-KG concentration compared to untreated controls. Measured by Amplex Red-based enzymatic assay. M NAD +/NADH ratio in CAR-T cells. Glutamine-treated cells showed elevated NAD +/NADH ratio, indicating improved redox state. Quantified by WST-8 enzymatic cycling assay. Data represent mean ± SEM (n = 3); * p < 0.05, ** p < 0.01, *** p < 0.001 (t test)

Article Snippet: Briefly, cells were collected by centrifugation at 400 × g for 10 min at room temperature, and the supernatant was transferred to new tubes for cytokine release assays using the BD Human Th1/Th2 Cytokine Cytometric bead array (CBA) kit (Becton–Dickinson).

Techniques: Activity Assay, Functional Assay, Expressing, Flow Cytometry, Incubation, Staining, Double Staining, Labeling, Activation Assay, Concentration Assay, Enzymatic Assay

Journal: Journal of Translational Medicine

Article Title: Glutamine-driven metabolic reprogramming promotes CAR-T cell function through mTOR-SREBP2 mediated HMGCS1 upregulation in ovarian cancer

doi: 10.1186/s12967-025-06853-0

Figure Lengend Snippet: Glutamine enhanced antitumor efficacy of CAR-T cells in vivo. A Schematic illustration of experimental protocol in vivo. NKG mice were intravenously (i.v.) injected with OVCAR3 cells and infused with CAR-T or CAR-T + Gln cells 16 days later. Tumor burden was assessed every 2 days. The peripheral blood were collected for analysis on day 4, 8, 12, 16, 20. B Tumor growth curves measured every two days (n = 6). The length (L) and width (W) of the tumors were measured every two days by caliper, and tumor volume was calculated using the standard formula: Tumor Volume (mm3) = L × W 2 /2. C Weight of mice measured every two days (n = 6). D Survival of NKG mice i.v. infused with CAR-T or CAR-T + Gln cells (n = 6 mice/group). E Frequency of central memory CAR-T cells remained in the peripheral blood from different groups (n = 3). F Co-expression level of inhibitory receptors (PD-1 and TIM3) in CAR-T cells in the peripheral blood from different groups (n = 3). G Numbers of CAR-T cells remained in the peripheral blood from different groups (n = 3). H MSLN + CAR copies in peripheral blood from different groups (n = 3). I – L Cytokine levels (IFN-γ, TNF-α, IL-6, IL-10) in tumor tissues measured using Cytometric Bead Array (CBA) (n = 3). M Representative immunohistochemical staining for CD45 (brown) and corresponding hematoxylin and eosin (H&E) staining on day 8 (n = 3). Scale bars: 50 µm. N Quantitative analysis of CD45 + cell relative Integrated Optical Density (IOD) (n = 3). O Representative H&E staining of tissues at Day 28 endpoint in mouse model. At the Day 28 endpoint, heart, liver, spleen, lung, kidney, and brain tissues were collected from the mouse model and subjected to H&E staining. The results show no significant tissue damage, indicating the safety of the treatment. The log-rank test was used for the survival analysis ( D ). * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: Briefly, cells were collected by centrifugation at 400 × g for 10 min at room temperature, and the supernatant was transferred to new tubes for cytokine release assays using the BD Human Th1/Th2 Cytokine Cytometric bead array (CBA) kit (Becton–Dickinson).

Techniques: In Vivo, Injection, Expressing, Immunohistochemical staining, Staining