Amino-biphosphonate-mediated MMP-9 inhibition breaks the tumor-bone marrow axis responsible for myeloid-derived suppressor cell expansion and macrophage infiltration in tumor stroma

Abstract

BALB-neuT mice expressing an activated rat c-erbB-2/neu transgene under the mouse mammary tumor virus long terminal repeat show enhanced hematopoiesis with hyperproduction of myeloid-derived suppressor cells (MDSC) because of vascular endothelial growth factor (VEGF) secreted by the tumor. Here, we show that both tumor and stromal cells express matrix metalloproteinase-9 (MMP-9), thereby increasing the levels of pro-MMP-9 in the sera of tumor-bearing mice. Treatment with amino-biphosphonates impaired tumor growth, significantly decreased MMP-9 expression and the number of macrophages in tumor stroma, and reduced MDSC expansion both in bone marrow and peripheral blood by dropping serum pro-MMP-9 and VEGF. We dissected the role of tumor-derived MMP-9 from that secreted by stromal leukocytes by transplanting bone marrow from MMP-9 knockout mice into BALB-neuT mice. Although bone marrow progenitor-derived MMP-9 had a major role in driving MDSC expansion, amino-biphosphonate treatment of bone marrow chimeras further reduced both myelopoiesis and the supportive tumor stroma, thus enhancing tumor necrosis. Moreover, by reducing MDSC, amino-biphosphonates overcome the tumor-induced immune suppression and improved the generation and maintenance of antitumor immune response induced by immunization against the p185/HER-2. Our data reveal that suppression of MMP-9 activity breaks the vicious loop linking tumor growth and myeloid cell expansion, thus reducing immunosuppression. Amino-biphosphonates disclose a specific MMP-9 inhibitory activity that may broaden their application above their current usage.

Figure 1

MMP-9 expression in BALB-neuT mice. A, pro–MMP-9 serum levels detected in single BALB-neuT by ELISA. Serum pro–MMP-9 increased along with age and tumor development in three representative females. Bars, SD. B, MMP-9 in a mammary tumor analyzed by immunohistochemistry. Both tumor cells and stromal macrophages produce MMP-9, although at different levels: tumor cells show a diffuse cytoplasmic positivity (bottom left, inset), whereas macrophages have a stronger and granular positivity (top left, inset; magnification, ×20; inset magnification, ×40).

Figure 2

Effect of amino-biphosphonate treatment in BALB-neuT mice. Groups of BALB-neuT mice were treated five times per week with zoledronate or pamidronate, or with saline (untreated) starting at ages 4, 7, or 12 wk. The experiment was repeated twice to thrice with five to six animals per group of treatment, and the results were combined. A, tumor growth, expressed as the sum of the single tumor volumes, was significantly inhibited in all treatment groups compared with untreated controls. *, P = 0.01; **, P = 0.005 at 28 wk; **, P = 0.003; ***, P = 0.0001 at 29 wk. Bars, SE. B, weekly comparison of tumor volumes among different treatment schedules. Treatment started at age 4 wk significantly impairs tumor growth compared with both treatments started later; *, P = 0.01; **, P < 0.007; ***, P = 0.0006. Data from mice treated with zoledronate and pamidronate are combined. Bars, SE.

Figure 3

Zoledronate treatment reduces tumor-secreted factors and alters cell composition and function of tumor stroma. A, pro–MMP-9 and VEGF evaluation in tumor-bearing BALB-neuT. Sera from treated (n = 13) and untreated (n = 12) BALB-neuT mice were analyzed for pro–MMP-9 and VEGF content by ELISA and compared with normal BALB/c serum levels. ***, P = 0.0009; **, P = 0.001. Bars, SD. B, cytofluorimetric analysis of tumor stroma. Single-cell suspensions from primary tumors (n = 16–19) were labeled with CD11b-PE and Gr-1-FITC or F4/80-FITC antibodies. The mean percent of double-positive cells showed a significant reduction of CD11b/Gr-1 (***, P = 0.0003; *, P = 0.033) and CD11b/F4/80 (*, P = 0.04) expressing macrophages in the stroma of treated versus control mice. Bars, SD. C, immunohistochemistry analysis of untreated (top) and treated (bottom) mammary tumors stained with F4/80-(a–d), Gr-1– (b–e), and MMP-9–specific (c–f) antibodies. Zoledronate decreased the number of F4/80-positive macrophages (d versus a) and Gr-1–positive cells (e versus b) in the tumor stroma and dramatically reduced the MMP-9 expression in tumor and stroma cells (f versus c). Magnification, ×10.

Figure 4

Zoledronate inhibits tumor-enhanced bone marrow (BM) hematopoiesis. A, the expansion of Gr-1/CD11b double-positive cells during tumor progression was significantly reduced in the PB of treated versus control BALB-neuT. *, P = 0.01; **, P = 0.003; bars, SE. B, the treatment modified the ratio between the number of circulating Gr-1/CD11b–positive cells and the overall tumor volume. The tumor mass required to induce similar number of circulating MDSC is larger in treated than in untreated mice. Bars, SD. C, the total number of bone marrow colonies obtained from BALB-neuT in methylcellulose (CFU-GEMM, CFU-GM, and BFU-E) was significantly reduced by zoledronate and similar to that of tumor-free BALB/c. **, P = 0.0019; ***, P = 0.0005. Bars, mean. D, chronic zoledronate treatment showed no toxic effect on normal bone marrow hematopoiesis of BALB/c mice, as measured by the sum of bone marrow colonies obtained in methylcellulose. Bars, mean.

Figure 5

Dissection of tumor and stroma MMP-9 functions and response to zoledronate in bone marrow chimeras. A, lethally irradiated transgenic mice transplanted with bone marrow from MMP-9^−/− or MMP-9^+/− donors were treated with zoledronate or saline (untreated) and monitored for tumor growth. The experiment was repeated twice with six mice per group, and results were combined. MMP-9 expression by stroma cells of bone marrow origin was irrelevant for tumor onset and growth (open symbols), and zoledronate inhibited tumor growth in both bone marrow chimeras (filled symbols). Bars, SE. B, tumor outgrowth was associated with increased hematopoiesis in mice transplanted with MMP-9^+/− (P = 0.005) but not with MMP-9^−/− bone marrow, as shown by the total number of bone marrow colonies in methylcellulose. Zoledronate treatment significantly reduced the number of bone marrow colonies in both chimeras, normalizing hematopoiesis in tumor bearers. **, P = 0.002 to 0.003. Bars, mean. C and D, levels of pro–MMP-9 and VEGF detected by ELISA in sera of tumor-bearing bone marrow chimeras treated with zoledronate or saline (untreated). Zoledronate reduced the level of pro–MMP-9 and VEGF in mice chimeras receiving MMP-9^+/− but not MMP-9^−/− donor bone marrow. *, P = 0.01. Bars, mean.

Figure 6

Zoledronate adjuvant effects to antitumor immunotherapy. (FVB×BALB-neuT)F1females underwent four cycles of DNA vaccination (DNA vax) with or without zoledronate treatment. Tumor growth, MDSC expansion, and anti–r-HER-2 antibody response was monitored and compared with untreated controls. A, at advanced tumor stages, between ages 18 and 22 wk, mice vaccinated and treated with zoledronate had a smaller tumor mass than mice treated with vaccination alone. Bars, median. B, cumulative tumor volume analyzed in treated mice bearing the same number of transformed mammary glands. The addition of zoledronate to DNA vaccination significantly reduced the overall tumor size. *, P = 0.01 to 0.04; **, P = 0.003 to 0.005; bars, SE. C, the correlation between tumor volume and percentage of Gr-1/CD11b–positive cells in the PB highlighted the significant reduction of MDSC in mice treated with zoledronate and vaccination compared with mice vaccinated and untreated controls. **, P = 0.006 to 0.008. Bars, SD. D, antibody-mediated immune response to r-HER-2, measured as serum-binding potential to r-p185/HER-2–positive tumor cell line. Zoledronate-treated and vaccinated mice had significantly higher titer of anti–r-HER-2 antibody than mice treated with vaccination alone or untreated. Bars, SD.