The immune-dependent and SERT-independent anti-tumor effects of citalopram in HCC.

(A, B) Mouse HCC cells, Hepa1-6 or Hep53.4, were subcutaneously injected into Rag1-/-C57BL/6 or immunocompetent C57BL/6 mice (n = 5-7 per group). When bore visible tumors, 5 mg/kg citalopram was treated daily for 15 or 25 days. Tumors were excised after mice were sacrificed, and the tumor weight was measured. (C, D) Western blotting showed the knockdown efficiency of SERT in Hepa1-6 and Hep53.4 cells. (E, F) shControl and shSlc6a4 Hepa1-6 and Hep53.4 cells were subcutaneously injected into Rag1-/- C57BL/6 or immunocompetent C57BL/6 mice (n = 5-6 per group). When bore visible tumors, 5 mg/kg citalopram was treated daily for 15 or 25 days. Tumors were excised after mice were sacrificed, and the tumor weight was measured. In all panels, *p < 0.05, **p < 0.01, ***p < 0.001. Values as mean ± SD and compared by one-way ANOVA multiple comparisons with Tukey’s method (for bar chart comparison) and two-way ANOVA with Dunnett’s multiple comparisons (for survival curve comparison).

C5aR1 is a direct target of citalopram.

(A) Gene Set Enrichment Analysis (GSEA) identified GLUT1 and C5aR1 as two top hits related to SSRI-induced gene changes. (B) GSEA of HCC RNAseq data (TCGA cohort) with the SSRI-related gene signature. Sample grouping was made based on the median expression of C5aR1. (C) Representative immunohistochemical images showed the expression pattern and cellular distribution of C5aR1 in human HCC tissues. Scale bar, 50 μm. (D) Single cell RNA sequencing analysis showed the expression pattern of C5aR1 with the immune microenvironment of HCC. (E) Co-immunofluorescence of C5aR1 (green) with CD163 (red) in HCC samples. Scale bar, 10 μm. (F) The DARTS assay and immunoblot analysis showed C5aR1 protein stability against 5 μg/mL pronase in the presence and absence of 100 μM citalopram treatment. (G) The DARTS assay and immunoblot analysis showed C5aR1 protein stability against 5 μg/mL pronase in the presence of different concentration of citalopram treatment. (H) The overall conformation of citalopram binding to C5aR1. (I) Representative models of citalopram in pose-1 (left), pose-2 (middle) and allosteric site (right). Several polar interactions were indicated by black dashed lines. (J) HEK293T cells were transfected with either WT or mutant C5aR1 expression plasmids for 48 h, followed by DARTS assay with immunoblotting analysis of C5aR1 protein levels. In all panels, *p < 0.05, **p < 0.01. Values as mean ± SD and compared by the Student’s t test (F) or one-way ANOVA multiple comparisons with Tukey’s method among groups (G, J).

Citalopram targets C5aR1+ TAMs

(A) Western blotting showed the knockdown efficiency of GLUT1 in mouse Hepa1-6 cells. (B) GLUT1KD 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; three weeks later, tumor burden was examined (n = 6-7 per group). (C) The growth kinetics of GLUT1KD Hepa1-6 tumors in C5ar1+/- and C5ar1-/- C57BL/6 host (n = 7). (D) Immunofluorescence analysis of C5a deposition in GLUT1KD 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) GLUT1KD 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 were analyzed. Scale bar, 50 μm. (H) The phagocytic capacity of macrophages isolated from GLUT1KD Hepa1-6 tumors in C5ar1+/- and C5ar1-/- C57BL/6 host. (J-L) 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 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 GLUT1KD 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 as mean ± SD and compared by two-way 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).

Citalopram activates CD8+ T cells.

(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 MASH mice (n = 5 per group); Basal 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-HT levels in GLUT1KD Hepa1-6 tumor-bearing mice fed with chow diet or CDAHFD, with the presence or absence of citalopram treatment (n = 5 per group). (F) Serum TNF-α, IL-1β, and IL-6 levels in GLUT1KD 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 GLUT1KD 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 as mean ± SD and compared by the Student’s t test (A-G), one-way ANOVA multiple comparisons with Tukey’s method (I), and two-way ANOVA with Dunnett’s multiple comparisons (H, J).

Mechanism model.

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 (Dong et al., 2024) 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.