In this study, we show previously unrecognized effects of MMP-9 on innate antitumor immunity. Adenoviral gene transfer of MMP-9 caused a dose-dependent massive infiltration of neutrophils into breast cancers, which resulted in decreased tumor growth and angiogenesis of breast cancer explants in nude mice and immune-competent mice bearing breast cancers. When the neutrophils were depleted by Ab treatment,the therapeutic effect of AdMMP-9 was abolished. In addition, AdMMP-9 treatment altered the cytokine profile of the stroma in vivo. One major component of the stroma was macrophages, and gene transfer of MMP-9 to cultured macrophages induced a similar cytokine profile, as we had detected from the stroma of the tumors. By using PAR-1 and PAR-2 inhibitors, the cytokine profile of the macrophages after AdMMP-9 exposure was to some extent altered, suggesting that these receptors may, at least in part, mediate the effects of MMP-9. AdMMP-9 exposure to cultured neutrophils altered the levels of released cytokines, suggesting that these cells may also contribute to the change in cytokine levels in the tumors. Gene transfer of TIMP-1 did not affect tumor growth, angiogenesis, or the immune cells.
MMPs have a complex role in cancer progression and may exert both pro- and antitumorigenic activities (3
). Although MMP expression generally has been associated with tumor progression in several cancer forms including breast cancer (36
), clinical trials with broad-spectrum MMP inhibitors have failed, and in some cases, patients treated with the inhibitors even showed significantly poorer survival than patients receiving placebo (3
). These results are further supported by recent data showing unfavorable effects of TIMP-1 in breast cancer patients (14
). Moreover, recent data, including MMP-9 knockout mice, has clearly shown MMP-9 may be involved in tumor regression (5
). In line with these data, it has recently been shown that gene transfer of MMP-9 in combination with oncolytic viruses leads to regression of prostate cancer growth (37
). One explanation may be that clinical investigations have been focused on immunohistochemistry or mRNA quantifications, which may reflect the intracellular content but not the enzymatic activity of the MMP, as these proteins are activated at a posttranslational level in the extracellular space. We, and others, have shown that increased expression of MMP-9 induces a potent and significant antitumor effect by increasing the release of antiangiogenic fragments such as endostatin (4
). Interestingly, in vitro data implicate MMP-9 as a potent enzyme for releasing soluble VEGF and thereby involved in the angiogenic switch during cancer progression (38
). Indeed, our in vivo data using microdialysis for sampling of free VEGF from the extracellular space in live tumor tissue suggest an increase of free VEGF by MMP-9; however, this effect was not significant and did not result in increased angiogenesis. This may be explained by a simultaneous increase of released endostatin, leading to a net result of antiangiogenesis in the tissue.
The MMP-9–treated tumors exhibited profound changes in the tumor stroma with high numbers of infiltrating immune cells. The secreted cytokine pattern of cells in vivo are difficult to explore with traditional methods such as immunohistochemistry and/or flow sorting of whole tumor tissue not differentiating between intracellular/extracellular molecules. Therefore, we performed microdialysis, which we previously have shown to be an excellent tool for sampling of extracellular proteins in vivo (39
). In the tumor model used in this study with human cancer cells in a murine stroma, we were able not only to sample cytokines in vivo, but also to distinguish from which compartment of the tumor microenvironment the secreted cytokines originated. In the AdMMP-9–treated tumors, we found increased levels of murine IL-1β, IL-1Ra, and IL-6 and decreased levels of IL-10. One major component of the stroma in our tumors was macrophages. TAMs may affect tumor progression and metastasis formation (20
). However, TAMs may be polarized into different phenotypes that are either beneficial or detrimental for cancer growth (45
). Classically, macrophages may be polarized into M1 and M2 phenotypes depending on their cytokine profile. In a simplistic manner, M1 macrophages secrete low levels of arginase-1 and IL-10 and high levels of IL-1β, IL-6, TNF-α, and IL-12, whereas M2 macrophages secrete high levels of arginase-1, IL-10, and IL-1Ra and low levels of IL-12, IL-1β, IL-6, and TNF-α (45
). However, great heterogeneity and plasticity of the macrophage skewing has been found, and the typical M1 and M2 state has been considered extreme variants, and these two phenotypes may also coexist in the tumor microenvironment (45
). Moreover, in the case of IL-1, this may also be a paradox because the M1 cytokine IL-1β has been shown to promote tumor angiogenesis and metastasis that may be counteracted by the M2 cytokine IL-Ra, an antagonist of IL-1 without any agonist effect (46
). To further investigate if the change of cytokine levels found in vivo after MMP-9 therapy could be attributable to macrophages, we cultured this cell type and exposed them to AdMMP-9. Indeed, we found that AdMMP-9 exposure to murine macrophage-like cells and freshly isolated human macrophages induced a similar cytokine profile as we had detected in the in vivo microdialysates. TNF-α was not detectable in microdialysates, but in cell culture, the macrophages responded with a prompted increase of TNF-α after AdMMP-9 therapy. This is in line with a recent study showing that two other MMPs, namely MMP-1 and MMP-3, induced a TNF-α release from macrophages (49
). By blocking the PAR-1 and PAR-2 receptors in vitro, the cytokine profile was reversed, suggesting that the MMP-9 effect on the macrophages was mediated via these receptors.
AdMMP-9–treated tumors also secreted very high levels of the murine inflammatory cytokines KC and MIP-2, corresponding to human IL-8. These chemokines originated from the macrophages and neutrophils in the stroma, at least in part, as our in vitro–cultured monocytes secreted significantly increased levels of MIP-2 from murine cells and IL-8 from human cells after AdMMP-9 gene transfer. In the tumors, the high levels of KC and MIP-2 were functional, as the tumor stroma exhibited massive infiltration of neutrophils. The role of neutrophils and IL-8 in cancer progression is to some extent contradictory in previous published data. The proangiogenic IL-8 and neutrophil infiltration have been described as important mediators of transforming benign conditions into malignant lesions (44
); in contrast, massive infiltration of neutrophils and very high levels of IL-8 have been shown to be effective tumor-killing modalities (51
). One explanation may be a dose-dependent effect, as it has been demonstrated that increasing levels of transduced IL-8 into melanoma cells caused increased tumor formation, but at very high levels of IL-8, tumor growth was impaired, dependent on massive neutrophil infiltration (26
). The mechanistic explanation of such findings may be an increased production of hydrogen peroxide by the neutrophils, leading to tumor killing as recently described (52
). Another recent study has also revealed that neutrophils may be activated/differentiated into an antitumorigenic or a protumorigenic phenotype (25
). Depletion of these antitumor neutrophils augments tumor growth (25
). This is in line with our present data in which depletion of neutrophils counteracted the therapeutic effects of AdMMP-9. One strength of our experiments is that we used a highly neutrophil-specific mAb (1A8) that specifically depletes neutrophils, whereas other studies have used the RB6-8C5 Ab, which has been shown to also target a second epitope expressed on many other cell types (54
). During gene transfer into tumor tissue, all cells present in the tumor at this time will overexpress the transgene, including macrophages and neutrophils. However, infiltrating cells after gene transfer will not be subjected to the viruses loaded with the transgene—in this case, MMP-9. Although neutrophils may produce MMP-9 per se, the production of MMP-9 of the incoming neutrophils after gene transfer was not high enough to sustain increased MMP-9 levels throughout the tumors, as the intensity of staining did not differ between tumors with or without depletion of neutrophils.
Although the nude mouse model gives the advantage of investigating the innate immune system exclusively, it may be argued that the antitumor effects may not be present in species with intact immunity. However, our data from the immune competent PyMT model clearly show that AdMMP-9 also exerts an antitumor activity when an intact immune system is present.
In summary, we have demonstrated several mechanisms by which AdMMP-9 exerts antitumorgenic properties in both immune-deficient and immune-competent mouse models of human breast cancer. As previously shown, AdMMP-9 increased the release of the antiangiogenic fragment endostatin. AdMMP-9 also increased the levels of KC and MIP-2 by the stroma, leading to increased influx of neutrophils into the tumor tissue. By selective depletion of neutrophils, the AdMMP-9 effects were abolished, demonstrating that these neutrophils exerted an antitumorigenic action. A large part of the tumor stroma, ~10% of the whole tumor area, consisted of macrophages in our model. Previous data have shown that the phenotype of macrophages in cancerous tissue may be reversible (55
). In this study, we add MMP-9 to the complex network of molecules with abilities to activate macrophages into different functional states. Although MMP-9 did not activate the macrophages into a classic M1 tumor-killing phenotype, the cytokine profile suggested a polarization toward M1-like macrophages. Moreover, we also show that neutrophils changed their phenotype by MMP-9. Clearly, the role of MMP-9 in tumor growth is diverse, and inhibition of its activity may cause unexpected tumor response.