In this study, we observed that macrophages were present in high numbers in rat prostate tumors, particularly at the invasive zone but importantly also in the extratumoral normal prostate tissue. In addition, we showed that the tumor expressed numerous factors that stimulate monocyte recruitment, angiogenesis, and tissue remodeling. Furthermore, a reduction of monocytes/macrophages to the tumor and its surroundings with clodronate-liposomes resulted in inhibited tumor growth and repressed intratumoral and extratumoral angiogenesis and arteriogenesis.
Studies of macrophage infiltration to human prostate cancer have shown diverse results with both positive and negative associations to clinical outcome and cancer progression [6,7
]. Interestingly, TAMs in different tumor compartments apparently have opposing effects on prostate cancer progression [6,7
]. Two different polarization states have been described for macrophages: the M1 phenotype has antitumoral effects, whereas the M2 macrophage promotes angiogenesis, tumor growth, and metastasis [19
]. Tumor-associated macrophages generally exhibit an M2-like phenotype, although a mixed M1 and M2 phenotype is also described [19
]. In this prostate cancer model, macrophages in the extratumoral tissue seemed particularly important for vessel growth, whereas the role of macrophages inside the tumor was more unclear.We noted that both macrophage density and vascular proliferation were highest in the tumor-invasive zone where macrophages could promote invasion, tissue remodeling, and angiogenesis. Already existing vessels in the extratumoral tissue may also promote tumor vascularization through co-option, a process where existing vessels are surrounded by tumor cells and used to supply the tumor [20
]. Furthermore, macrophages infiltrating the extratumoral tissue were often seen in close contact with proliferating endothelial cells in capillaries and with smooth muscle cells in arterioles and venules. Reduced macrophage infiltration resulted in repressed proliferation in capillaries and in large vessels, and macrophages therefore seem to have a central role in both angiogenesis and arteriogenesis in this tumor model. Almost all studies on TAMs explore their roles within tumors, and their roles in the surrounding nonmalignant tissue and particularly for tumor- related arteriogenesis have surprisingly not been examined [17
]. However, as macrophages play an important role for the arteriogenesis and collateral artery growth in ischemia [21,22
], we suggest that they are probably also important for the expansion of larger vessels in the normal tissue surrounding tumors. Previous studies have shown that inhibiting the macrophage chemoattractant CCL-2 in a subcutaneous prostate xenograft model in nude mice reduced macrophage influx, intratumoral angiogenesis, and tumor growth [8
]. In a subcutaneous rat prostate tumor model, treatment with an antiangiogenic agent also inhibited macrophage function and impaired tumor growth [23
]. Agents used in the earlier studies have, beside the effects onmacrophages, direct effects on both tumor and endothelial cells. To reduce macrophage infiltration, we used intraperitoneal injections of clodronate-liposomes, which are phagocytosed by, and induce apoptosis in circulating phagocytic cells [11
]. This method has been used to deplete macrophages and thereby to reduce angiogenesis and tumor growth in a variety of tumor models [24–26
]. In some models such as glioma, depletion of macrophages, however, resulted in increased tumor growth, although a small reduction in vessel density was observed [27
]. Because the concentration of free clodronate in the prostate is unknown in this experimental setup, some of the effects seen might be due to direct effects of clodronate on tumor and endothelial cells. Direct effects are, however, most likely minor because clodronate-liposomes cannot cross-vascular barriers, and free clodronate released from dead phagocytic cells have a very short half-life in the circulation [11
]. Monocytes/macrophages that have phagocytosed clodronate-liposomes could also be inhibited in their normal functions. Circulating monocytes have been shown to be depleted, but as new monocytes are constantly entering the circulation from the bonemarrow, a continuous supply of clodronate-liposomes is needed to inhibit macrophage recruitment [28
]. This probably explains why tumors from clodronate-liposome-treated animals contained CD68-positive cells at day 10 (2 days after the last liposome injection). Notably, although the reduction in macrophage densities in tumor and extratumoral tissues was modest, we observed a significant inhibition in tumor growth, suggesting that only a moderate decline in macrophage infiltration is sufficient. Together, our report and a previous report [8,23
] strongly suggest the importance of macrophages for prostate cancer growth and angiogenesis. The specific effects of macrophage depletion during various phases of prostate tumor development and metastasis, however, need to be examined further.
Prostate tumor growth is angiogenesis-dependent, and the orthotopic rat prostate tumors expressed numerous factors important for angiogenesis. Several of these factors are apparently produced by tumor cells, whereas others are produced by host cells. Some of the factors produced, for example, VEGF, TGFβ, and ANG-2, have already been reported to be of major significance for angiogenesis in prostate cancer [29,30
]. Macrophages could be the source of many of the angiogenic factors because expressions of these factors, for example, CCL-2, IL-1β, FGF-2, TGFβ, and MMP-9, were highly increased in the tumor in vivo compared with the tumor cells in vitro
. To support this, MMP-9 and IL-1β staining was found in macrophages in the invasive zone. Matrix metalloproteinases, and MMP-9 in particular, are important for tissue remodeling and facilitate tumor growth, migration, invasion, and angiogenesis (for review, see [31,32
]). Matrix metalloproteinase 9 could thus be a key factor produced by macrophages in our tumor model. Interleukin 1β has been shown to alter androgen receptor function [33
] and promote tumor invasiveness and angiogenesis in prostate, breast, and melanoma tumor models [34
]. Macrophage accumulation in the normal tissue adjacent to tumors could thus explain why this tissue shows an altered early response to castration [9
]. It is also possible that factors upregulated in tumors in vivo versus in vitro
are produced by tumor cells. Hypoxia (chronic or intermittent) and other environmental factors in the prostate in vivo
may alter their expression profile. We could, however, not observe any major differences between cells exposed to hypoxia for 24 hours and controls at the mRNA level. Although this finding does not exclude the possibility that the local environment may alter expression in tumor cells, it suggests that the most likely reason to the large differences in expression profiles between tumors in vivo
and tumor cells in vitro
is expression in host cells.
Our clodronate-liposome-treated animals (examined 2 days after last injection) were unfortunately not suitable to prove macrophage versus tumor cell expressions of these factors because the reduction in tumor macrophage content was incomplete.
If macrophages are important for prostate tumor growth, it is important to elucidate the factors responsible for the macrophage accumulation. Chemokine (C-C) ligand 2, CSF-1, and VEGF are all important for monocyte recruitment to tumors [1
] and, together with other chemokines, were highly expressed by the AT-1 tumor in vivo
. Colony-stimulating factor 1 and VEGF were also expressed in vitro
suggesting that AT-1 cells attract macrophages by secreting these factors, and that macrophages arriving to the tumor enhance this process by secreting CCL-2 and VEGF. This suggests that inhibition of several chemotactic factors simultaneously might be needed to reduce macrophage infiltration.
In summary, macrophages accumulating in the normal tissue surrounding tumors could be one important source of angiogenic and arteriogenic factors in prostate cancer and decreasing macrophages could be used as an antivascular/antitumor treatment. Decreasing macrophages could also inhibit tissue remodeling and tumor invasion into the surrounding normal tissue by reducing the levels of MMP-9 and IL-1β. Nevertheless, combinatorial therapies directed against both tumor and nonmalignant cells and neutralizing many of the individual factors involved are probably needed to get a pronounced and sustained effect on tumor growth. Further studies are needed to test this hypothesis.