Doxorubicin eliminates myeloid-derived suppressor cells and enhances the efficacy of adoptive T-cell transfer in breast cancer

Abstract

Myeloid-derived suppressor cells (MDSC) expand in tumor-bearing hosts and play a central role in cancer immune evasion by inhibiting adaptive and innate immunity. They therefore represent a major obstacle for successful cancer immunotherapy. Different strategies have thus been explored to deplete and/or inactivate MDSC in vivo. Using a murine mammary cancer model, we demonstrated that doxorubicin selectively eliminates MDSC in the spleen, blood, and tumor beds. Furthermore, residual MDSC from doxorubicin-treated mice exhibited impaired suppressive function. Importantly, the frequency of CD4(+) and CD8(+) T lymphocytes and consequently the effector lymphocytes or natural killer (NK) to suppressive MDSC ratios were significantly increased following doxorubicin treatment of tumor-bearing mice. In addition, the proportion of NK and cytotoxic T cell (CTL) expressing perforin and granzyme B and of CTL producing IFN-γ was augmented by doxorubicin administration. Of therapeutic relevance, this drug efficiently combined with Th1 or Th17 lymphocytes to suppress tumor development and metastatic disease. MDSC isolated from patients with different types of cancer were also sensitive to doxorubicin-mediated cytotoxicity in vitro. These results thus indicate that doxorubicin may be used not only as a direct cytotoxic drug against tumor cells, but also as a potent immunomodulatory agent that selectively impairs MDSC-induced immunosuppression, thereby fostering the efficacy of T-cell-based immunotherapy.

Figure 1. Doxorubicin eliminates tumor-induced MDSC

A, Schematic of the experimental design followed to evaluate the effects of doxorubicin on MDSC in the 4T1 breast cancer model. Mice were injected orthotopically (mammary fat pad) with 4T1 tumor cells (1×10⁶). Doxorubicin (2.5 and 5 mg/kg) was administered intravenously on day 7 and 12 post-tumor injection. Spleen and blood samples were harvested and evaluated on days 14, 17 and 23. B, Proportion of MDSC (CD11b⁺Gr-1⁺) in the spleen of 4T1 tumor-bearing mice post-doxorubicin treatment (right panel) and representative flow cytometry analysis 17 days post-tumor injection (left panel). C, Absolute number of MDSC in tumor-bearing mice treated or not with doxorubicin. D, Proportion of MDSC in the blood of tumor-bearing mice after doxorubicin treatment. E, Confocal microscopy analysis of CD11b⁺Gr-1⁺ cells in the spleens from untreated or doxorubicin-treated mice 17 days after tumor injection (5 days after the last doxorubicin treatment). CD11b (green), Gr-1 (red) and sytox orange nuclear staining (Nuc, blue). Scale bar 20 μm. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. n=4 mice per group. Data represent one of 3 experiments performed and analyzed independently.

Figure 2. Doxorubicin increases the frequency, proliferation and cytotoxic activity of effector T lymphocytes and NK

A similar experimental design as described in figure 1 was followed. A, Frequency of CD4⁺ T cells in the spleen (left panel) and blood (right panel) of tumor-bearing mice after doxorubicin treatment. B, Proportion of CD8⁺ T cells in the spleen (left panel) and blood (right panel) of tumor-bearing mice after doxorubicin treatment. C, NK cell frequency in the spleen (left panel) and blood (right panel) of doxorubicin-treated mice. D, Analysis of Ki67 expression after gating on CD4⁺, CD8⁺ T lymphocytes or NK (DX5⁺) cells as indicated (left panel) and related mean fluorescent intensity (MFI) (right panel) (day 17). E, Percent of CD8⁺ T and NK cells expressing granzyme B or perforin in the spleen of doxorubicin-treated mice 17 days post tumor cell injection. F, Percent of CD3⁺, CD4⁺ and CD8⁺ T lymphocytes expressing IFNγ in the spleen of tumor-bearing mice treated or not with doxorubicin (day 17). *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. n=4 mice per group. Data represent one of 3 experiments performed and analyzed independently.

Figure 3. Doxorubicin selectively induces MDSC apoptosis

A similar experimental design as described in figure 1 was followed. Spleens were collected 5 days after the last doxorubicin administration. A, Spleen samples were labeled for MDSC (CD11b⁺Gr-1⁺) and Annexin V and PI. Representative flow cytometry analysis (left panel, gated on C11b⁺Gr1⁺ cells), and proportion of apoptotic and secondary necrotic MDSC (right panel). B, Detection of caspase-3 cleavage in MDSC isolated from mice treated or not with doxorubicin 17 days after tumor injection. C, Spleen samples were labeled with anti-CD4 or anti-CD8 and Annexin V and PI. Gated CD4⁺ or CD8⁺ T lymphocytes were then analyzed for their Annexin V and PI status. Percent of apoptotic or secondary necrotic CD4⁺ (upper panels) and CD8⁺ (lower panels) T lymphocytes in doxorubicin-treated or untreated mice. Similar experimental design as in A. D, Effects of doxorubicin used at the indicated concentrations on MDSC isolated from 4T1 tumor-bearing mouse spleens (or on the MDSC-depleted cell population) determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays. Cells were cultured in quadruplicate for 30 hr with or without NAC (5 mM). % cell survival=(OD₅₆₀[treated cells at the indicated doxorubicin concentration]/OD₅₆₀[untreated cells] × 100). E, Analysis of ROS production by MDSC isolated from tumor-bearing mice and treated in vitro with the indicated concentrations of doxorubicin and for the indicated period of time. Cells were incubated with Dichlorodihydrofluorescein diacetate (DCFDA) and analyzed by flow cytometry. The mean fluorescent intensity representing ROS levels in MDSC is shown. F, Same as in D, but with MDSC isolated from EL4 tumor-bearing wild-type of gp91^−/− mice. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. n=4 mice per group. Data represent one of 3 experiments performed and analyzed independently.

Figure 4. Doxorubicin impairs MDSC immunosuppressive function

A–B, MDSC isolated from untreated or doxorubicin-treated mice were incubated for 4 days with cell trace violet-labeled naïve T cells (MDSC: T cell ratio=1:2). A, Effects of MDSC from the indicated groups of mice on the proliferation of CD4⁺ (left panel) or CD8⁺ (right panel) T lymphocytes assessed by flow cytometry. B, Effects of MDSC on CD25 expression by gated CD4⁺ (upper panels) or CD8⁺ (lower panels) T lymphocytes. C, MDSC isolated from tumor-bearing mice were treated or not in vitro with doxorubicin (100 ng/ml, 24 hr) and their ability to impair the proliferation of cell trace violet-labeled naïve CD4⁺ T cells induced by anti-CD3 and anti-CD28-coated activation beads was evaluated by flow cytometry. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. n=4 mice per group. Data represent one of 2 experiments performed and analyzed independently.

Figure 5. Doxorubicin decreases ROS production and arginase-1 and IDO expression by MDSC

A similar experimental design as described in figure 1 was followed. Spleens were harvested five days after the last doxorubicin treatment (day 17). A, Analysis of ROS production by MDSC in tumor-free (Tumor-free) or in tumor-bearing (Tumor Bearing) mice treated with the indicated concentration of doxorubicin. Cells were incubated with Dichlorodihydrofluorescein diacetate (DCFDA). Representative flow cytometry analysis of gated CD11b⁺Gr-1⁺ cells positive for DCFDA (upper panels). Percent of MDSC positive for DCFDA (left bottom panel). Mean fluorescent intensity representing ROS level in MDSC from the indicated groups (right bottom panel). B, C, Western blot analysis depicting expression of arginase-1 (B) or IDO (C) in MDSC isolated from doxorubicin treated or untreated mice. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. n=4 mice per group. Data represent one of 3 experiments performed and analyzed independently.

Figure 6. The combination of doxorubicin and Th1 or Th17 impairs 4T1 tumor development

Mice were injected orthotopically (mammary fat pad) with 4T1 tumor cells (1×10⁶). Doxorubicin (5 mg/kg) was injected intravenously on day 7 and 12 post-tumor cell injection. Th1 or Th17 lymphocytes were administered on day 9 and 14 post-tumor cell injection, intravenously (1×10⁶) and intratumoraly (2×10⁶). Tumor volume and number of metastatic nodules were evaluated on day 19 post-tumor injection. A, Schematic of the experimental design. B, Number of metastatic nodules (left panel) and tumor volume (right panel). C, Representative flow cytometry analysis of MDSC frequency in mice administered with the indicated therapies. D, Proportion of MDSC, CD4⁺ and CD8⁺ T cells in mice receiving the indicated therapies; n=8 mice per group. Data are representative of 3 independent experiments. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.

Figure 7. Doxorubicin selectively kill MDSC isolated from cancer patients

CD33⁺ cells were isolated from cancer patient PBMCs by magnetic cell sorting. A, Phenotypic analysis of the isolated cells. Representative results of n=10 patients. B, Ability of the CD33⁺ purified cells to impair the proliferation of cell trace violet-labeled T lymphocytes induced with anti-CD3 and anti-CD28-conjugated microbeads (at the indicated MDSC to T cell ratios). PI, proliferation index. C-D, Purified CD33⁺ MDSC or CD33⁻ cells were exposed to the indicated concentrations of doxorubicin for 24 hrs and stained with Annexin V and PI. (% dead cells = % of PI⁺ + % AnnexinV⁺PI⁻ cells). A total of n=10 patients were analyzed. E, Representative dot plots obtained with CD33⁺ MDSC or CD33⁻ cells isolated from a cancer patient. (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001).