Myeloid-derived suppressor cells in the development of lung cancer

Abstract

Myeloid-derived suppressor cells (MDSC) are widely implicated in immune suppression associated with tumor progression and chronic inflammation. However, very little is known about their possible role in tumor development. Here, we evaluated the role of MDSC in two experimental models of lung cancer: inflammation-associated lung cancer caused by chemical carcinogen urethane in combination with exposure to cigarette smoke; and a transgenic CC10Tg model not associated with inflammation. Exposure of mice to cigarette smoke alone resulted in significant accumulation in various organs of cells with typical MDSC phenotype (Gr-1(+)CD11b(+)). However, these cells lacked immunosuppressive activity and could not be defined as MDSC. When cigarette smoke was combined with a single dose of urethane, it led to the development of tumor lesions in lungs within 4 months. By that time, Gr-1(+)CD11b(+) cells accumulated in the spleen and lung and had potent immunosuppressive activity, and thus could be defined as MDSC. In the CC10Tg model, accumulation of immunosuppressive MDSC was observed only at 4 months of age, after the appearance of tumor lesions in the lungs. Accumulation of MDSC in both models was abrogated in S100A9 knockout mice. This resulted in a dramatic improvement in survival of mice in both models. Thus, cigarette smoke results in the expansion of immature myeloid cells lacking suppressive activity. Accumulation of bona fide MDSC in both models was observed only after the development of tumor lesions. However, MDSC played a major role in tumor progression and survival, which suggests that their targeting may provide clinical benefits in lung cancer.

Figure 1. The accumulation of myeloid cells in spontaneous lung cancer model

A. Lung tissues from CC10Tg mice at 4 months of age were harvested and stained with H&E. Images (mag.×20) are representative of lung tissues from 5 mice. B–D. Percentages of Gr-1⁺CD11b⁺ cells in peripheral blood (B), spleen (C) and lymph nodes (D) from CC10Tg mice and wild-type (WT) mice at indicated ages. Representative FACS data are shown in top panels. E, F. The proportions of MDSC (E) and macrophages (F) among CD45⁺ hematopoietic cells isolated from lungs. In all panels Mean and SEM from 3–6 mice per group are shown. * - p<0.05 between CC10Tg and WT mice.

Figure 2. Evaluation of myeloid cell suppressive activity

Gr-1⁺CD11b⁺ cells were isolated from the spleens of CC10Tg mice at 3 months of age and co-cultured with CFSE-labeled splenocytes from wild-type CD45.1⁺ congenic C57BL/6 mice stimulated with anti-CD3/CD28 antibody (A) or with CFSE-labeled CD45.2⁺ OT-I T cells stimulated with the specific peptide SIINFEKL (B). Irradiated CD45.1⁺ splenocytes were used as antigen presenting cells. After 3 days of cultures, T cell proliferation was analyzed by CFSE dilution. Typical examples of 3 performed experiments are shown.

Figure 3. Effect of S100A9 deletion on the survival of CC10Tg mice

A. The percentages of Gr-1⁺CD11b⁺ cells in the blood from CC10Tg mice (WT) and CC10Tg x S100A9KO. Analyses were performed at the indicated time points. Each group included 3 mice. B. Survival of CC10Tg (n=8) and CC10Tg x S100A9 (n=9) mice.

Figure 4. The proportion of myeloid cells in the peripheral blood of mice exposed to CS

FVB/N mice were exposed to CS for one (A) and three (B) months. The proportions of the indicated population of myeloid cells were evaluated. Each group included 3 mice. P values were calculated in 2-tailed Student t-test. ns-p>0.05

Figure 5. The proportion of myeloid cells in different organs of mice exposed to CS

Mice were exposed to CS for 4 months and the proportions of the indicated populations of myeloid cells were evaluated in different organs. Each group included 3–6 mice. The proportions of cells isolated from the lungs were calculated among CD45⁺ hematopoietic cells. P values were calculated in 2-tailed Student t-test. ns-p>0.05

Figure 6. Gr-1⁺CD11b⁺ myeloid cells from mice exposed to CS do not suppress T cell proliferation

A. Splenocytes from naïve FVB/N mice were stimulated with CD3- and CD28-specific antibodies in the presence of Gr-1⁺CD11b⁺ IMC purified from control mice and mice exposed to CS at the indicated IMC:splenocyte ratios. T cell proliferation was measured in triplicated by [³H] thymidine incorporation. Each group included 3 mice. B. Purified IMCs from the spleens or lungs of smoking or control FVB/N mice were added at the indicated ratios to a mixed lymphocyte reaction, which was set up using splenocytes from BALB/C mice as responders and irradiated splenocytes from FVB/N mice as stimulators mixed at 1:1 ratio. T cell proliferation was measured in triplicates by [³H] thymidine incorporation. Mean and SEM from 3 experiments are shown.

Figure 7. The effect of S100A9 deletion on tumor progression in mice exposed to urethane and CS

WT and S100A9KO FVB/N mice were treated with a single injection of urethane (1.5g/kg) and 4 months of CS. A. Typical example of lung tissue stained with H&E. Bars = 1 μm. B. The number of tumor lesions per lung. The differences were not significant (p>0.1). C. Immune suppressive activity of Gr-1⁺CD11b⁺ cells isolated from the spleens of mice assessed in allogeneic mixed leukocyte reaction. Each group included 3 mice. Mean and SEM are shown. The differences between groups: ** - p<0.01; *** - p<0.001. The differences from “No MDSC” control were significant (p<0.05) in all groups except at 1:4 ratio. D. The number of indicated myeloid populations in the spleens and lungs of mice. * - p<0.05 between the groups. Each group included 4 mice. E. Survival of mice of WT (n=6) and S100A9KO (n=7) mice treated with urethane and CS.