Targeting Ornithine Decarboxylase by α-Difluoromethylornithine Inhibits Tumor Growth by Impairing Myeloid-Derived Suppressor Cells

Abstract

α-Difluoromethylornithine (DFMO) is currently used in chemopreventive regimens primarily for its conventional direct anticarcinogenesic activity. However, little is known about the effect of ornithine decarboxylase (ODC) inhibition by DFMO on antitumor immune responses. We showed in this study that pharmacologic blockade of ODC by DFMO inhibited tumor growth in intact immunocompetent mice, but abrogated in the immunodeficient Rag1(-/-) mice, suggesting that antitumor effect of DFMO is dependent on the induction of adaptive antitumor T cell immune responses. Depletion of CD8(+) T cells impeded the tumor-inhibiting advantage of DFMO. Moreover, DFMO treatment enhanced antitumor CD8(+) T cell infiltration and IFN-γ production and augmented the efficacy of adoptive T cell therapy. Importantly, DFMO impaired Gr1(+)CD11b(+) myeloid-derived suppressor cells (MDSCs) suppressive activity through at least two mechanisms, including reducing arginase expression and activity and inhibiting the CD39/CD73-mediated pathway. MDSCs were one primary cellular target of DFMO as indicated by both adoptive transfer and MDSC-depletion analyses. Our findings establish a new role of ODC inhibition by DFMO as a viable and effective immunological adjunct in effective cancer treatment, thereby adding to the growing list of chemoimmunotherapeutic applications of these agents.

Figure 1. Antitumor effect of DFMO is dependent on CD8⁺ T cells

B16F10 (A, B) or B16-SIY (C) cells were injected s.c. into WT (A, C) or Rag^−/− C57BL/6 mice (B) (5 mice per group). DFMO was administered as a 1% solution in drinking dH₂O to mice starting 1 day after tumor injection. The mean DFMO consumption of mice was approximately 1.5 g/kg/day. Mice fed with dH₂O without DFMO were used as controls (5 mice per group). (C) Depletion of CD8⁺ T cells was achieved by twice-weekly i.p. injection of depleting mAb clone 53.6.7 (anti-CD8α, 200 μg), starting 1 day prior to tumor challenge. Tumor volumes were measured every 2 or 3 days (5 mice per group). Data (mean ± SEM) are representative of at least 3 independent experiments. *, p< 0.05; **, p<0.01.

Figure 2. DFMO adminstration enhances tumor-infiltrating T cell immunity

(A) Representative flow cytometric analysis of tumor-infiltrating CD8⁺TCRVβ⁺ T cells (5 mice per group). (B) Percent CD8⁺TCRVβ⁺ cells and (C) absolute number of gp100-specific tetramer⁺CD8⁺TCRVβ⁺ cells per 10⁶ cells in tumor infiltrates (5 mice per group). (D) Percent IFN-γ secreting CD3⁺CD8⁺ T cells in tumor infiltrates. Cells were collected from B16F10-bearing DFMO treated or control mice 14 days after tumor inoculation (5 mice per group). Data (mean ± SEM) are representative of 3 independent experiments. *, p< 0.05.

Figure 3. DFMO treatment augments IFN-γ production of CD8⁺ T cells in response to tumor antigen

(A) Representative ELISPOT images. Negative control wells contain splenic CD8⁺ T cells from B16F10-bearing DFMO treated or control mice collected 14 days after tumor inoculation (3 mice per group). Other triplicated wells contain CD8⁺ T cells and dendritic cells at the ratio of 2 to 1 in the presence of tumor antigen gp100 peptides (1 μg/ml). Each spot represents an IFN-γ secreting cell. The digital image analysis showed the total number (B), diameter of spots (C) and mean spot size (D) were significantly increased following DFMO treatment. Data (mean ± SEM) are representative of 2 independent experiments. **p< 0.01, ***p< 0.001.

Figure 4. Charaterization of phenotypic tumor-associated MDSCs following DFMO treatment

(A) Percent splenic Gr1⁺CD11b⁺ MDSCs were determined by flow cytometry from B16F10-bearing mice. Percent CD11b⁺Ly6G⁺Ly6C^low (granulocytic) and CD11b⁺Ly6G⁻Ly6C^high (monocytic) MDSCs were indicated within plots (5 mice per group). (B) Percent Gr1⁺CD11b⁺ MDSCs, CD11b⁺Ly6G⁺Ly6C^low (granulocytic) and CD11b⁺Ly6G⁻Ly6C^high (monocytic) MDSCs in spleen and tumor tissues from B16F10-bearing mice were summarized (5 mice per group). (C) Measurement of ODC activity in Gr1⁺CD11b⁺ cells from naïve and B16F10 tumor-bearing (TB) mice treated by DFMO or dH₂O (5 mice per group). (D) Expression levels of CD39, CD73, CD115, MHC-II, B7H1, DCFDA (ROS indicator) and arginase-I among both tumor-infiltrating CD11b⁺Ly6G⁺Ly6C^low (granulocytic) and CD11b⁺Ly6G⁻Ly6C^high (monocytic) MDSCs were determined by flow cytometry. Cells were collected from B16F10-bearing DFMO treated or control mice 14 days after tumor inoculation (5 mice per group). Data (mean ± SEM) are representative of 2 independent experiments. *, p< 0.05.

Figure 5. DFMO impairs MDSC function by reducing arginase expression and activity

(A) Real-time quantitative RT-PCR analysis of arginase-I expression in MDSCs from DFMO-treated and control mice (5 mice per group). Arginase-I activity of DFMO-treated MDSCs was compared with that of control MDSCs (3 mice per group). (B) Suppressive activity of DFMO-treated MDSCs versus control MDSCs. MDSCs were added at different ratios to eFluor450-labeled CD4⁺ T responder cells (Tres) stimulated with anti-CD3 plus antigen-presenting cells (APC) for 3 days and T cell proliferation was measured by flow cytometric eFluor450 dye dilution (3 mice per group). Arginase-I inhibitor nor-NOHA was able to blunt the suppressive activity of control MDSC but not DFMO-treated MDSCs. *, p<0.05; **, p<0.01. Data are representative of 3 independent experiments.

Figure 6. DFMO impairs MDSC function by reducing CD39/CD73-mediated adensinergic effect

(A) Representatvie flow dot plots show the gating strategy for CD11b⁺Ly6G⁺Ly6C^low (granulocytic) and CD11b⁺Ly6G⁻Ly6C^high (monocytic) BM-cultured MDSCs, and the percent CD73, CD39 or CD115 in each MDSC subset treated by DFMO or dH₂O as controls. (B) Flow quantification of CD73, CD39 or CD115 expression in DFMO-treated MDSCs versus control MDSCs (5 mice per group). *, p<0.05; **, p<0.01. (C) Suppressive activity of MDSCs as shown by quantification of eFluor450-labeled CD4⁺ T responder cells (Tres) cocultured with the indicated Gr1⁺CD11b⁺ MDSCs treated with or without CD73 inhibitors APCP or CD39 inhibitors ARL67156. The ratio of T cell/MDSC was 2:1. (D) Suppressive activity of MDSCs as shown by quantification of eFluor450-labeled Tres cocultured with the indicated Gr1⁺CD11b⁺ MDSCs treated with or without DFMO. The ratio of T cell/MDSC was 2:1. **, p<0.01; ***, p<0.001 (3 mice per group). Data (mean ± SEM) are representative of 2 independent experiments.

Figure 7. DFMO targets MDSCs to inhibit tumor growth

Mice were injected s.c. with 10⁶ B16F10 tumor cells. Depletion of MDSC was achieved by either twice-weekly i.p. injection of (A) 5-Fluorouracil (5-FU) or (B) anti-Gr1 antibodies starting 2 days after tumor challenge (5 mice per group). (C) Splenic Gr1⁺CD11b⁺ MDSCs from B16F10-bearing mice treated with DFMO or dH₂O were injected i.v. into B16-bearing mice at d7 and d14. Mice receiving PBS without MDSCs were controls. Tumor volume was measured and plotted at indicated times (5 mice per group). (D) Flow cytometry analysis of expression of ki67 and Annexin V in gp100-specific tetramer⁺CD8⁺TCRVβ⁺ cells, and absolute number of these tetramer⁺CD8⁺TCRVβ⁺ cells per 10⁶ cells in tumor infiltrates 3d after the initial MDSC transfer (5 mice per group). *, p<0.05; **, p<0.01. Data (mean ± SEM) are representative of 2 independent experiments.

Figure 8. DFMO augments the efficacy of adoptive T cell therapy

(A) Mice were s.c. injected with 10⁶ B16-SIY cells (5 mice per group). DFMO was administered as a 1% solution in drinking water starting 1 day after tumor injection. Mice fed with dH₂O without DFMO were used as controls. 7 days after tumor inoculation, activated SIY-specific 2C CD8⁺ T cells were i.v. injected into tumor-bearing mice. (B) Mice were s.c. injected with 10⁶ B16F10 cells (5 mice per group). DFMO was administered as a 1% solution in drinking water starting 7 day after tumor injection. Mice fed with dH₂O without DFMO were used as controls. Activated gp100-specific Pmel CD8⁺ T cells were i.v. injected into tumor-bearing mice on the same day. Tumor volumes were measured every 3 days. Data (mean ± SEM) are representative of 2 independent experiments. *, p< 0.05; **, p<0.01.