magnetic bead sorting  (Miltenyi Biotec)

Journal: eLife

Article Title: Citalopram exhibits immune-dependent anti-tumor effects by modulating C5aR1 + TAMs

doi: 10.7554/eLife.103016

Figure Lengend Snippet: ( A ) Western blotting showed the knockdown efficiency of GLUT1 in mouse Hepa1-6 cells. ( B ) GLUT1 KD Hepa1-6 cells were subcutaneously injected into the Rag1 −/− or immunocompetent C57BL/6 mice, and mice were treated with 5 mg/kg citalopram when bore visible tumors; 3 weeks later, tumor burden was examined ( n = 6–7 per group). ( C ) The growth kinetics of GLUT1 KD Hepa1-6 tumors in C5ar1 +/− and C5ar1 −/− C57BL/6 host ( n = 7). ( D ) Immunofluorescence analysis of C5a deposition in GLUT1 KD Hepa1-6 tumors from C5ar1 +/− and C5ar1 −/− C57BL/6 host. Scale bar, 50 μm. ( E ) Experimental design of bone marrow transfer experiments. ( F, G, I ) GLUT1 KD Hepa1-6 cells were subcutaneously implanted into syngeneic recipient (r) mice that had been reconstituted with bone marrow cells from either C5ar1 +/− or C5ar1 −/− donor mice. The therapeutic effect of citalopram ( F ), C5a deposition ( G ), and macrophage phagocytosis ( I ) in this model was analyzed. Scale bar, 50 μm. ( H ) The phagocytic capacity of macrophages isolated from GLUT1 KD Hepa1-6 tumors in C5ar1 +/− and C5ar1 −/− C57BL/6 host. Flow cytometry showed the infiltration of CD45 + CD11b + F4/80 + macrophages ( J ), CD206 + TAMs and CD11b + TAMs ( K ), tumor-infiltrating lymphocytes ( L ) in tumor tissues from orthotopic xenograft model, which was generated in immunocompetent C57BL/6 mice with Hepa1-6 cells ( n = 5 per group). ( M, N ) Measurement of CD8 + T cell function in tumor tissues from the groups mentioned in C and F . ( O ) The growth kinetics of GLUT1 KD Hepa1-6 tumors in C5ar1 +/− and C5ar1 −/− C57BL/6 host upon CD8 + T cell depletion ( n = 7). ( P ) Correlation analysis of C5aR1 expression and immune checkpoint molecules, gene signatures of TAMs, exhausted T cells, and effector Tregs in the TCGA cohort ( n = 371). In all panels, *p < 0.05, **p < 0.01, ***p < 0.001; ns, non-significant. Values are presented as mean ± SD and compared by two-way analysis of variance (ANOVA) with Dunnett’s multiple comparisons ( B, C, F, O ), Student’s t test ( H–M ), one-way ANOVA multiple comparisons with Tukey’s method ( B, N ), and the Spearman’s rank correlation methods ( P ). Figure 3—source data 1. Original western blots for , indicating the relevant bands. Figure 3—source data 2. Original files for western blot analysis displayed in .

Article Snippet: CD8 + T cells were isolated from the resulting cell suspensions by CD8 + magnetic bead selection (Miltenyi Biotec, Cat #130-117-044).

Techniques: Western Blot, Knockdown, Injection, Immunofluorescence, Isolation, Flow Cytometry, Generated, Cell Function Assay, Expressing

Journal: eLife

Article Title: Citalopram exhibits immune-dependent anti-tumor effects by modulating C5aR1 + TAMs

doi: 10.7554/eLife.103016

Figure Lengend Snippet: ( A ) Schematic depicting macrophage blockade with clodronate liposomes. Twelve days before tumor inoculation, C57BL/6 mice were pretreated with clodronate liposomes or phosphate-buffered saline (PBS) liposomes. Subsequently, GLUT1 KD Hepa1-6 cells were subcutaneously injected into the C57BL/6 mice. The effect of citalopram (5 mg/kg) on the tumor burden was evaluated after 18 days of drug treatment. ( B ) Immunofluorescence analysis of F4/80 + macrophages in the liver and tumor tissues of indicated groups. ( C ) In C57BL/6 mice, the effect of citalopram on the GLUT1 KD Hepa1-6 xenograft tumors was measured in the presence of macrophage depletion ( n = 7 per group). ( D ) Immunohistochemical analysis of cleaved caspase-3 (CCS3) and Ki67 in GLUT1 KD Hepa1-6-bearing subcutaneous xenograft tumors, treated with DMSO or 5 mg/kg citalopram ( n = 7 per group). Scale bar, 50 μm. ( E ) In the context of macrophage depletion, measurement of CD8 + T cell function in tumor tissues upon DMSO or citalopram treatment. Values are presented as mean ± SD and compared by the Student’s t test ( C–E ).

Article Snippet: CD8 + T cells were isolated from the resulting cell suspensions by CD8 + magnetic bead selection (Miltenyi Biotec, Cat #130-117-044).

Techniques: Liposomes, Saline, Injection, Immunofluorescence, Immunohistochemical staining, Cell Function Assay

Journal: eLife

Article Title: Citalopram exhibits immune-dependent anti-tumor effects by modulating C5aR1 + TAMs

doi: 10.7554/eLife.103016

Figure Lengend Snippet: ( A ) Real-time qPCR revealing the mRNA expression of M1-oriented ( Il6 , Ifnb1 , and Nos2 ) and M2-oriented ( Mrc1 , Il10 , and Arg1 ) markers in isolated macrophages from orthotopic Hepa1-6 tumors ( n = 3 per group). ( B, C ) Gating strategies used for flow cytometry analysis of tumor and splenic lymphocytes. Panel A : Identification of CD4 + T cells, CD8 + T cells, and dendritic cells (DC). Panel B : Identification of B220 + B cells, tumor-associated macrophages (TAMs), and natural killer (NK) cells. ( D ) Flow cytometry showed the infiltration of CD45 + CD11b + F4/80 + macrophages, CD4 + T cells, CD8 + T cells, B220 + B cells, CD11c + DC cells, and NK1.1 + NK cells in spleen tissues from orthotopic xenograft model, which generated in immunocompetent C57BL/6 mice with Hepa1-6 cells ( n = 5 per group). ( E ) Real-time qPCR analysis of Glut1 and Glut3 expression in intratumoral CD8 + T cells ( n = 3 per group). In all panels, *p < 0.05, **p < 0.01. Values as mean ± SD and compared by the Student’s t test.

Article Snippet: CD8 + T cells were isolated from the resulting cell suspensions by CD8 + magnetic bead selection (Miltenyi Biotec, Cat #130-117-044).

Techniques: Expressing, Isolation, Flow Cytometry, Generated

Journal: eLife

Article Title: Citalopram exhibits immune-dependent anti-tumor effects by modulating C5aR1 + TAMs

doi: 10.7554/eLife.103016

Figure Lengend Snippet: ( A, B ) Measurement of CD8 + T cell function and glycolysis in orthotopic tumor tissues from WT C57BL/6 mice ( n = 5 per group). ( C, D ) Measurement of CD8 + T cell function and glycolysis in orthotopic tumor tissues from metabolic dysfunction-associated steatohepatitis (MASH) mice ( n = 5 per group); basal extracellular acidification rate (ECAR) indicates glycolysis after the addition of glucose, and ΔECAR represents the difference between oligomycin-induced ECAR and 2-DG-induced ECAR. ( E ) Serum 5-hydroxytryptamine (5-HT) levels in GLUT1 KD Hepa1-6 tumor-bearing mice fed with chow diet or choline-deficient, amino acid-defined high-fat diet (CDAHFD), with the presence or absence of citalopram treatment ( n = 5 per group). ( F ) Serum TNF-α, IL-1β, and IL-6 levels in GLUT1 KD Hepa1-6 tumor-bearing mice fed with chow diet or CDAHFD, with the presence or absence of citalopram treatment ( n = 5 per group). ( G ) Serum 5-HT levels in WT C57BL/6 and Tph1 −/− mice, with the presence or absence of citalopram treatment ( n = 5 per group). ( H ) Tumor growth of WT and Tph1 −/− mice after subcutaneous injection of Hepa1-6 cells and treatment with citalopram. ( I ) Measurement of CD8 + T cell function in tumor tissues from the groups mentioned in H . ( J ) The therapeutic effect of citalopram on GLUT1 KD Hepa1-6 tumor was tested in the presence or absence of CD4 + T or CD8 + T cell depletion. In all panels, *p < 0.05, **p < 0.01, ***p < 0.001; ns, non-significant. Values are presented as mean ± SD and compared by the Student’s t test ( A–G ), one-way analysis of variance (ANOVA) multiple comparisons with Tukey’s method ( I ), and two-way ANOVA with Dunnett’s multiple comparisons ( H, J ).

Article Snippet: CD8 + T cells were isolated from the resulting cell suspensions by CD8 + magnetic bead selection (Miltenyi Biotec, Cat #130-117-044).

Techniques: Cell Function Assay, Injection

Journal: eLife

Article Title: Citalopram exhibits immune-dependent anti-tumor effects by modulating C5aR1 + TAMs

doi: 10.7554/eLife.103016

Figure Lengend Snippet: ( A ) Splenic CD8 + T cells isolated from C57BL/6 mice were stimulated with plate-bound α-CD3 plus α-CD28 for 72 hr, and T-cell proliferation (carboxy fluorescein succinimidyl ester [CFSE] staining) upon citalopram treatment was determined by flow cytometry ( n = 3 per group); ACT: activated T cells. ( B ) Splenic CD8 + T cells from C57BL/6 mice were stimulated with plate-bound α-CD3 and α-CD28 for 72 hr, and CD44 and CD62L levels in activated CD8 + T cells upon citalopram treatment were determined by flow cytometry ( n = 3 per group). ( C–E ) Intracellular GZMB, IFN-γ, and TNF-α were detected in activated CD8 + T cells upon citalopram treatment ( n = 3 per group). In all panels, **p < 0.01, ***p < 0.001. Values are presented as mean ± SD and compared by the Student’s t test.

Article Snippet: CD8 + T cells were isolated from the resulting cell suspensions by CD8 + magnetic bead selection (Miltenyi Biotec, Cat #130-117-044).

Techniques: Isolation, Staining, Flow Cytometry

Journal: eLife

Article Title: Citalopram exhibits immune-dependent anti-tumor effects by modulating C5aR1 + TAMs

doi: 10.7554/eLife.103016

Figure Lengend Snippet: Model depicting the molecular mechanism by which citalopram inhibits the Warburg effect and promotes an anti-tumor response in hepatocellular carcinoma (HCC). In the primary HCC microenvironment (left panel), C5aR1-expressing tumor-associated macrophages (TAMs) exhibit reduced phagocytic capacity and an anti-inflammatory state, which correlates with diminished CD8 + T cell anti-tumor immunity and HCC progression. Upon treatment with citalopram (right panel), the drug not only inhibits the glycolytic metabolism of cancer cells by targeting GLUT1 but also acts on C5aR1 expressed by TAMs, thereby enhancing macrophage-driven anti-tumor immunity. Additionally, citalopram induces a systemic immunostimulatory effect on CD8 + T cell functions through yet-to-be-identified serotonergic mechanisms. The dotted line indicates a causal relationship that has not been fully established through direct evidence.

Article Snippet: CD8 + T cells were isolated from the resulting cell suspensions by CD8 + magnetic bead selection (Miltenyi Biotec, Cat #130-117-044).

Techniques: Expressing