Reduced inflammation in the tumor microenvironment delays the accumulation of myeloid-derived suppressor cells and limits tumor progression

Abstract

Chronic inflammation is frequently associated with malignant growth and is thought to promote and enhance tumor progression, although the mechanisms which regulate this relationship remain elusive. We reported previously that interleukin (IL)-1beta promoted tumor progression by enhancing the accumulation of myeloid-derived suppressor cells (MDSC), and hypothesized that inflammation leads to cancer through the production of MDSC which inhibit tumor immunity. If inflammation-induced MDSC promote tumor progression by blocking antitumor immunity, then a reduction in inflammation should reduce MDSC levels and delay tumor progression, whereas an increase in inflammation should increase MDSC levels and hasten tumor progression. We have tested this hypothesis using the 4T1 mammary carcinoma and IL-1 receptor (IL-1R)-deficient mice which have a reduced potential for inflammation, and IL-1R antagonist-deficient mice, which have an increased potential for inflammation. Consistent with our hypothesis, IL-1R-deficient mice have a delayed accumulation of MDSC and reduced primary and metastatic tumor progression. Accumulation of MDSC and tumor progression are partially restored by IL-6, indicating that IL-6 is a downstream mediator of the IL-1beta-induced expansion of MDSC. In contrast, excessive inflammation in IL-1R antagonist-deficient mice promotes the accumulation of MDSC and produces MDSC with enhanced suppressive activity. These results show that immune suppression by MDSC and tumor growth are regulated by the inflammatory milieu and support the hypothesis that the induction of suppressor cells which down-regulate tumor immunity is one of the mechanisms linking inflammation and cancer.

Figures

Figure 1

Inflammation-associated cytokines are higher in 4T1 primary tumors of BALB/c mice than in 4T1 tumors of IL-1R–deficient mice. BALB/c and IL-1R−/− mice were inoculated with 7,000 4T1 tumor cells in the abdominal mammary gland on day 0, and primary tumors were surgically removed when their diameters reached 8 to 10 mm. The resulting tumor tissue and control mammary tissue from tumor-free mice was minced, incubated at 37°C for 24 h, and the resulting supernatants were assayed for cytokines by multiplex analysis. A, 4T1 tumor tissue versus normal mammary tissue from BALB/c mice. B, 4T1 tumor tissue from BALB/c mice compared with 4T1 tumor tissue from IL-1R−/− mice. Columns, average of triplicates of individual mice; bars, SD.

Figure 2

Tumor progression is delayed in IL-1R−/− mice but not in IL-1Ra−/− mice. A, BALB/c and IL-1R−/− mice were inoculated with 7,000 4T1 tumor cells in the abdominal mammary gland and monitored for primary tumor growth. Tumor progression was delayed in IL-1R−/− mice relative to BALB/c mice (P < 0.05). B, 4T1 tumor growth did not differ in BALB/c and IL-1Ra−/− mice. C, BALB/c, IL-1R−/−, and BALB/c → IL-1R−/− and IL-1R−/− → BALB/c bone marrow chimeras were inoculated with 4T1 tumor cells and monitored for primary tumor growth. Tumor progression in both chimeras was delayed compared with BALB/c mice (P < 0.05). Points, averages of 10 to 15, 10 to 15, and 7 to 15 mice per group (for A, B, and C, respectively); bars, SD. Data are pooled from two to three experiments.

Figure 3

IL-1R−/−, but not IL-1Ra−/−, mice have reduced lung metastases. BALB/c, IL-1R−/−, and IL-1Ra−/− mice were inoculated with 7,000 4T1 tumor cells in the abdominal mammary gland on day 0, and their lungs harvested on days 35 to 40, when BALB/c mice were moribund. Metastatic cells were quantified using the clonogenic assay. Lung metastases are reduced in IL-1R−/− mice relative to BALB/c mice (P < 0.05). ●, number of metastatic cells in the lungs of individual mice. Data are pooled from two to three experiments.

Figure 4

IL-1β or the absence of the IL-1Ra enhances MDSC accumulation, whereas the absence of the IL-1R delays MDSC accumulation. BALB/c, IL-1R−/−, and IL-1Ra−/− mice were inoculated with 7,000 4T1 or 4T1/IL-1β tumor cells in the abdominal mammary gland on day 0. Mice were tail-bled at the indicated time points and the percentage of CD11b+Gr1+ MDSC was quantified by flow cytometry. MDSC are elevated in BALB/c mice with 4T1/IL-1β tumors and in IL-1Ra−/− mice with 4T1 tumors by day 8, and remain elevated up to day 16 (P = 0.05 and P < 0.05, respectively for days 8–10; P < 0.01 for both groups on days 14–16). MDSC are reduced in IL-1R−/− mice (P < 0.01). Columns, average of 5 to 15 mice per group and are pooled from two experiments; bars, SD.

Figure 5

MDSC from IL-1Ra−/− mice are more suppressive towards CD4+ and CD8+ T cells than MDSC from BALB/c mice. BALB/c, IL-1R−/−, and IL-1Ra−/− mice were inoculated with 7,000 4T1 tumor cells in the abdominal mammary gland, and tail-bled to test for CD11b+Gr1+ MDSC. When MDSC were >90% of the WBCs, mice were sacrificed, blood was collected and RBCs were lysed. The resulting MDSC were used in T cell proliferation assays. CD4+ TS1 or CD8+ clone 4 transgenic splenocytes were stimulated with HA110–119 or HA518–526 peptides, respectively, and cocultured in the presence or absence of graded doses of blood MDSC. Inhibitors of arginase (nor-NOHA) or nitric oxide (L-NMMA) were added to some of the wells. T cell proliferation was measured as counts per minute of [3H]thymidine. A, BALB/c and IL-1R−/− MDSC were equally suppressive. B and C, IL-1Ra−/− MDSC were more suppressive than BALB/c MDSC towards CD4+ T cells (P < 0.05; B) and CD8+ T cells (P < 0.05; C). Data are from one of two to five independent experiments.

Figure 6

IL-6 compensates for tumor progression and MDSC accumulation in IL-1R−/− mice. BALB/c and IL-1R−/− mice were inoculated with 7,000 4T1 or 4T1/IL-1β tumor cells in the abdominal mammary gland on day 0 and monitored for primary tumor growth, lung metastases, and blood MDSC. A, tumor diameter was measured at the indicated time points. Points, averages of 15 to 17 mice per group; bars, SD. B, mice were sacrificed and lungs were harvested when BALB/c mice were moribund (days 35–40). Metastatic cells (●) in individual mice were quantified using the clonogenic assay. C, the percentage of blood CD11b+Gr1+ MDSC was measured by flow cytometry at the indicated time points. Columns, averages of 15 to 17 mice per group and are pooled from three independent experiments; bars, SD. D, blood CD11b+Gr1+ MDSC from tumor-bearing BALB/c or IL-1R−/− mice with 4T1 or 4T1/IL-1β were stained with IL-6R antibody or isotype control antibody. 4T1 tumor cells and BALB/c splenocytes were stained as negative and positive controls, respectively. All MDSC express the IL-6R. Data are pooled from two to three experiments.