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. 2012 Jan 15;72(2):420-9.
doi: 10.1158/0008-5472.CAN-10-4627. Epub 2011 Nov 28.

Interleukin-10 ablation promotes tumor development, growth, and metastasis

Affiliations

Interleukin-10 ablation promotes tumor development, growth, and metastasis

Takashi Tanikawa et al. Cancer Res. .

Abstract

Interleukin-10 (IL-10) is a broadly acting immune inhibitory cytokine that is generally thought to support tumor growth. Here we challenge this view with evidence that genetic ablation of IL-10 in the mouse significantly heightens sensitivity to chemical carcinogenesis, growth of transplanted tumors, and formation of metastases. Tumor growth in IL-10-deficient (IL-10(-/-)) mice was associated with an increased level of myeloid-derived suppressor cells (MDSC) and CD4(+)Foxp3(+) regulatory T (Treg) cells in both the tumor microenvironment and the tumor-draining lymph nodes. IL-10(-/-) MDSCs express high levels of MHC and IL-1, and they efficiently induced formation of Treg cells. IL-1 signaling blockade reduced tumor growth mediated by IL-10 deficiency, associated with a partial rescue of tumor infiltration and function of effector T cells and a decrease in tumor angiogenesis and tumor infiltration by Treg cells. Taken together, our findings establish that endogenous IL-10 inhibits inflammatory cytokine production and hampers the development of Treg cells and MDSCs, two key components of the immunosuppressive tumor microenvironment, thereby inhibiting tumor development, growth, and metastasis.

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Conflict of interest statement

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. IL-10 deficiency increased tumor incidence, growth, and foci formation
(A). Chemically-induced tumor incidence in IL-10+/+ and IL-10−/− mice. IL-10+/+ and IL-10−/− mice were given no treatment, DSS, or DSS and AOM as described in Material and Methods. Numbers of colon polyps were recorded. Results are expressed as the mean of colon polyps +/− SEM. 6 mice per genotype. *, P < 0.05. (B–D). Tumor growth in IL-10+/+ and IL-10−/− mice. MC38 cells were inoculated subcutaneously into the left flank of IL-10+/+ and IL-10−/− mice. B. Tumor volume was monitored and recorded. 10 mice per genotype. *, P < 0.05. C. Tumors excised at each time point were weighed. 5 mice per time point per genotype. *, P < 0.05. D. Six actual tumors are shown. (E). Tumor lung foci in IL-10+/+ and IL-10−/− mice. MCA310 cells were injected intravenously into IL-10+/+ and IL-10−/− mice. The numbers of lung tumor foci were counted two weeks after tumor inoculation. 6 mice per genotype. *, P < 0.05.
Figure 2
Figure 2. IL-10 deficiency increased Treg cells in tumor bearing mice
(A–E). Single-cell suspensions were made from tumor tissues, spleen, and tumor-draining lymph nodes (TDLN). These cells were stained with anti-CD3, CD45, CD4, and FoxP3. Antigen and cytokine expression was analyzed by FACS. A. Treg cells were quantified as the percentage of Foxp3+ cells in CD4+CD3+ cells isolated from tumor tissue. 5 mice per time point. *, P < 0.05. B. Representative FACS dot plots from data in A. C. Correlation between tumor weight and Treg percentage. Cells were analyzed as in A and the percentage of Treg cells in CD45+ cells was plotted against corresponding tumor weight. Treg cells versus tumor weight in IL-10−/−(n=30) and IL-10+/+ (n=30) mice. Quadratic regression analysis between IL-10−/− and IL-10+/+ mice across tumor volumes (P <0.05). D. Treg cells were quantified as the percentage of FoxP3+ cells in CD4+CD3+ cells isolated from TDLN. 5 mice per time point. *, P < 0.05. E. Tregs were quantified as the percentage of FoxP3+ cells in CD4+CD3+ cells isolated from spleens in tumor-bearing mice. 5 mice per time point. *, P < 0.05.
Figure 3
Figure 3. IL-10 deficiency increased MDSCs in tumor bearing mice
(A, B). Single-cell suspensions were made from tumor tissues and stained with anti-CD3, CD45, CD4, CD8, Gr-1, and CD11b for membrane antigen expression. The expression of antigens was analyzed by FACS. A. Increased MDSCs in tumor-bearing IL-10−/− mice. Gr-1+CD11b+ MDSCs were analyzed in MC38 tumors of IL-10−/− and IL-10+/+ mice. 5 mice per genotype per time point. *P < 0.05, IL-10−/− versus IL-10+/+ mice. B. Tumor MDSC levels were higher in IL-10−/− mice than IL-10+/+ mice. Cells were analyzed as in A and the percentage of MDSCs in CD45+ cells was plotted against corresponding tumor weight. Quadratic regression analysis between IL-10−/− (n=20) and IL-10+/+ (n=20)mice across tumor volumes (P <0.05). C. Tumor-associated IL-10−/− MDSCs mediate immune suppression in vitro. MDSCs were sorted from MC38 tumors. MDSCs were cultured with naïve T cells in different ratios as detailed in Materials and Methods. T cell proliferation was determined by thymidine incorporation. Results are expressed as the mean of CPM +/− SD in triplicates. Empty bars: IL-10+/+. Filled bars: IL-10−/−. One of three experiments is shown. *P < 0.01 compared to groups with MDSCs. D. MDSCs suppressed antitumor immunity in vivo. IL-10+/+ mice were lethally irradiated (2 × 600 rad, three hours apart, for a total of 1200 rad) and given an infusion of IL-10+/+ or IL-10−/− whole spleen cells or spleen cells depleted of MDSCs from tumor bearing mice. On the same day, mice were injected subcutaneously with MC38. Tumor growth was measured over a period of two weeks. 3–4 mice per group. *P < 0.05, Day 9–15: IL-10−/− vs. IL-10+/+, IL-10−/− vs. MDSC-depleted IL-10−/−; and Day 12–15: IL-10+/+ vs. MDSC-depleted IL-10+/+. E. Tumor-derived IL-10−/− MDSCs were superior to IL-10+/+ MDSCs in Treg cell induction. MDSCs were isolated from MC38 tumors and co-cultured with IL-10+/+ CD4+ T cells for 6 days. CD4+ FoxP3+ cells were analyzed via FACS in cultured T cells. One of three experiments with triplicates per experiment is shown. IL-10+/+ vs. IL-10−/− MDSC in 3 independent experiments, *P<0.05.
Figure 4
Figure 4. IL-1 blockade reduced tumor growth in IL-10−/− mice
(A). IL-10−/− myeloid derived cells expressed high levels of IL-1. Myeloid derived cells were isolated from spleens in tumor-free IL-10−/− and IL-10+/+ mice and not stimulated or stimulated with LPS for 8 hours. Real-time PCR was performed to determine the expression of IL-1α and IL-1β. One of 3 experiments is shown. (B, C). Anakinra treatment reduced tumor burden in IL-10−/− mice but had no significant effect on tumor burden in IL-10+/+ mice. MC38 was injected subcutaneously into IL-10−/− and IL-10+/+ mice. The mice were treated with Anakinra or vehicle as described in Materials and Methods. Tumor volume was measured every 3 days. Results are shown as the mean values of tumor volume +/− SEM. N = 4–5 mice per group. *, P < 0.05, Anakinra versus control in IL-10−/− mice. (D). Reduced tumor growth in IL-1R−/− mice. MC38 was injected subcutaneously into IL-1R−/− and IL-1R+/+ mice. Tumor volumes were measured every 3 days. Results are shown as the mean values of tumor volume +/− SEM in IL-10−/− mice. N = 3–4 mice per group. *, P < 0.05, IL-1R−/− versus IL-1R+/+ mice.
Figure 5
Figure 5. IL-1 blockade altered immune phenotype and tumor vascularization in IL-10−/− mice
(A). Increased tumor-infiltrating CD8+ T cells in Anakinra-treated IL-10−/− mice. Immunofluorescent staining was performed on tumor tissue sections. The absolute numbers of CD8+ T cells were counted. Results are expressed as the mean values +/− SEM per 15 high-powered fields (HPF). N = 5 mice per group. *P < 0.05, Anakinra versus control. (B). Increased tumor-infiltrating IFNγ+CD8+ T cells in Anakinra-treated IL-10−/− mice. Single-cell suspensions were made from tumor tissues. The cells were stained for CD8 and intracellular IFNγ. Results are expressed as the mean percent of IFNγ+CD8+ T cells in CD8+ cells +/− SEM. N = 4–5 mice per group. *, P < 0.05, Anakinra versus control. (C). Reduced tumor-infiltrating Treg cells in Anakinra-treated IL-10−/− mice. Immunofluorescent staining was performed on tumor tissue sections. The absolute numbers of Foxp3+ T cells were counted. Results are expressed as the mean values +/− SEM per 15 high-powered fields (HPF). N = 5 mice per group. *, P < 0.05, Anakinra versus control. (D). Reduced tumor vascularization in Anakinra-treated IL-10−/− mice. Immunofluorescent staining was performed to analyze the expression of CD31 in tumor tissue sections. The areas of CD31+ vessels and the size of CD31+ vessels were analyzed as detailed in Materials and Methods. Results are expressed as the mean area or size of vessels +/− SEM in 10 HPF. N = 4–5 mice per group. *, P < 0.05, Anakinra versus control.

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