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1-Methylnicotinamide is an immune regulatory metabolite in human ovarian cancer

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Science Advances  20 Jan 2021:
Vol. 7, no. 4, eabe1174
DOI: 10.1126/sciadv.abe1174
  • Fig. 1 Tumor cells have greater glucose uptake but similar mitochondrial activity to T cells.

    (A and B) Representative plot (left) and tabulated data (right) for median fluorescence intensity (MFI) of glucose uptake (2-NBDG) (A) and mitochondrial activity (MitoTracker Deep Red) (B) of CD4+ T cells, CD8+ T cells, and EpCAM+CD45 tumor cells from ascites and tumor. (C) Proportion of CD4+ and CD8+ cells (of CD3+ T cells) within ascites and tumor. (D) Proportion of EpCAM+ (of CD45) tumor cells within ascites and tumor. (E and F) Representative plot (left) and tabulated data (right) for glucose uptake (2-NBDG) (E) and mitochondrial activity (MitoTracker Deep Red) (F) of EpCAM+CD45 tumor and EpCAMCD45 stromal cells from ascites and tumor. (G) Representative plots for CD25, CD137, and PD1 expression by flow cytometry. (H and I) CD25, CD137, and PD1 expression on CD4+ T cells (H) and CD8+ T cells (I). (J and K) Naïve, central memory (Tcm), effector (Teff), and effector memory (Tem) phenotype based on CCR7 and CD45RO expression. Representative plot (left) and tabulated data (right) for CD4+ T cells (J) and CD8+ T cells (K) from ascites and tumor. P values determined by paired t test (*P < 0.05, **P < 0.01, and ***P < 0.001). Lines indicate matched patients (n = 6). FMO, fluorescence minus one; MFI, median fluorescence intensity.

  • Fig. 2 Metabolite profiling of matched ascites and tumor reveals key differences between tumor cells and T cells.

    (A) Schematic of magnetic bead enrichment. Cells underwent three consecutive rounds of magnetic bead enrichment or remained on ice before analysis by LC-MS/MS. (B) Impact of enrichment type on metabolite abundance. Means of triplicate measurements for each enrichment type ±SE shown. Gray line represents 1:1 relationship. Intraclass correlation (ICC) for replicate measurements shown in axis labels. NAD, nicotinamide adenine dinucleotide. (C) Schematic of patient metabolite profiling workflow. Ascites or tumor was collected from patients and cryopreserved. A fraction of each sample was analyzed by flow cytometry, while the remaining sample underwent three rounds of enrichment for CD4+, CD8+, and CD45 cells. These cell fractions were analyzed using LC-MS/MS. (D) Heatmap of normalized metabolite abundance, with dendrograms representing Ward’s clustering of Euclidean distances among samples. (E) Principal components analysis (PCA) of sample metabolite profiles, showing triplicate replicates of each sample, with samples from the same patients joined by lines. (F) PCA of sample metabolite profiles conditioned on patient (i.e., using partial redundancy); sample types are circumscribed by convex hulls. PC1, principal component 1; PC2, principal component 2.

  • Fig. 3 MNA is more abundant in T cells from the tumor compared with ascites.

    (A) Normalized abundance of MNA in CD4+, CD8+, and CD45 cells from ascites and tumor. Boxplots show medians (lines), interquartile range (box hinges), and range of data up to 1.5× interquartile range (box whiskers). P values are determined using limma with patient as a random effect, as described in Materials and Methods (*P < 0.05 and **P < 0.01). (B) Schematic of MNA metabolism (60). Metabolites: S-adenosyl-l-methionine; SAH, S-adenosyl-l-homocysteine; NA, nicotinamide; MNA, 1-methylnicotinamide; 2-PY, 1-methyl-2-pyridone-5-carboxamide; 4-PY, 1-methyl-4-pyridone-5-carboxamide; NR, nicotinamide riboside; NMN, nicotinamide mononucleotide. Enzymes (green): NNMT, nicotinamide N-methyl transferase; SIRT, sirtuins; NAMPT, nicotinamide phosphoribosyltransferase; AOX1, aldehyde oxidase 1; NRK, nicotinamide riboside kinase; NMNAT, nicotinamide mononucleotide adenylyltransferase; Pnp1, purine nucleoside phosphorylase. (C) t-SNE of scRNA-seq of ascites (gray) and tumor (red; n = 3 patients). (D) NNMT expression in different cellular populations identified using scRNA-seq. (E) Expression of NNMT and AOX1 in SK-OV-3, human embryonic kidney (HEK) 293T, T cells, and T cells treated with MNA. Fold expression is shown relative to SK-OV-3. Means of expression (n = 6 healthy donors) with SEM shown. A Ct values greater than 35 is considered undetectable (U.D.). (F) Expression of SLC22A1 and SLC22A2 in SK-OV-3, HEK293T, T cells, and T cells treated with 8 mM MNA. Fold expression is shown relative to SK-OV-3. Means of expression (n = 6 healthy donors) with SEM shown. A Ct value greater than 35 is considered undetectable (U.D.). (G) Cellular MNA content in activated healthy donor T cells after 72-hour incubation with MNA. Means of expression (n = 4 healthy donors) with SEM shown.

  • Fig. 4 Exogenous MNA enhances TNFα expression and inhibits IFN-γ production in T cells.

    (A) Total live cell count and population doubling (PD) directly from culture on day 7. Bar graphs represent means + SEM of six healthy donors. Data representative of at least n = 3 independent experiments. (B to D) T cells were activated using CD3/CD28 with IL-2 in respective concentrations of MNA for 7 days. Cells were stimulated with PMA/ionomycin with GolgiStop for 4 hours before analysis. TNFα (B) expression in T cells. Example plot of TNFα expression in live cells (left) and tabulated data (right). IFN-γ (C) and IL-2 (D) expression in T cells. Cytokine expression was measured by flow cytometry. Bar graphs represent means (n = 6 healthy donors) + SEM. P value determined using a one-way ANOVA with repeated measures (*P < 0.05 and **P < 0.01). Data representative of at least n = 3 independent experiments. (E to G) T cells were activated using CD3/CD28 with IL-2 in respective concentrations of MNA for 7 days. Media was collected before (Ctrl) and after PMA/ionomycin stimulation for 4 hours. Concentration of TNFα (E), IFN-γ (F), and IL-2 (G) was measured by ELISA. Bar graphs represent means (n = 5 healthy donors) + SEM. P value determined using a one-way ANOVA with repeated measures (*P < 0.05). Dotted line indicates the limit of detection for the assay. (H) Cytolytic assay. FRα-CAR-T or GFP-CAR-T cells were conditioned with adenosine (250 μM) or MNA (10 mM) for 24 hours, or left untreated (Ctrl). Percentage of killing was measured against SK-OV-3 cells. P value determined by Welch’s t test (*P < 0.5 and **P < 0.01).

  • Fig. 5 MNA increases binding of Sp1 to the promoter of TNFα, increasing TNFα transcription and cytokine production.

    (A) Fold change in expression of TNFα in T cells treated with MNA over T cells cultured without MNA. Means of expression (n = 5 healthy donors) with SEM shown. Data representative of at least n = 3 independent experiments. (B) NFAT and Sp1 binding to the TNFα promoter of T cells treated with or without 8 mM MNA before (Ctrl) and after 4-hour stimulation by PMA/ionomycin. Immunoglobulin G (IgG) and H3 were used as negative and positive controls, respectively, for the immunoprecipitation. Quantification of ChIP shows the fold increase in Sp1 and NFAT binding to the TNFα promoter in MNA-treated cells compared with control. Data representative of at least n = 3 independent experiments. P value determined by multiple t tests (***P < 0.01). (C) T cells (noncytotoxic) show increased expression of TNF in the tumor relative to the ascites of HGSC. Colors represent different patients. Displayed cells have been randomly subsampled to 300 and jittered to limit overplotting (**Padj = 0.0076). (D) Proposed model of MNA in ovarian cancer. MNA is produced in tumor cells and fibroblasts in the TME and taken up by the T cells. MNA increases binding of Sp1 to the promoter of TNFα, leading to increased transcription of TNFα and cytokine production of TNFα. MNA also leads to a decrease in IFN-γ. The resulting inhibition of T cell function leads to decreased killing capacity and increased tumor growth.

Supplementary Materials

  • Supplementary Materials

    1-Methylnicotinamide is an immune regulatory metabolite in human ovarian cancer

    Marisa K. Kilgour, Sarah MacPherson, Lauren G. Zacharias, Abigail E. Ellis, Ryan D. Sheldon, Elaine Y. Liu, Sarah Keyes, Brenna Pauly, Gillian Carleton, Bertrand Allard, Julian Smazynski, Kelsey S. Williams, Peter H. Watson, John Stagg, Brad H. Nelson, Ralph J. DeBerardinis, Russell G. Jones, Phineas T. Hamilton, Julian J. Lum

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