Type I IFN system influences carcinogenesis and tumor growth (59
) and is used as a therapeutic agent for several tumors (11
). One of the reasons for this phenomenon could be that endogenous IFN-β affects tumor angiogenesis. The role of IFN-β in this event became obvious when B16F10 tumors were transplanted s.c. into mice that were deficient for this cytokine. Tumors grew faster and reached significantly larger size in Ifnb1–/–
mice compared with controls, which correlated with enhanced angiogenic processes in the tumor, resulting in more and better-developed blood vessels.
Enhanced tumor growth and angiogenesis in Ifnb1–/– mice appeared to be caused by CD11b+Gr1+ neutrophils. Notably, more of these cells could be found in blood, in tumors, and to a small extent already in the bone marrow of such mice. Depletion of neutrophils significantly reduced tumor growth to equal levels in both types of mice, thus abolishing the differential effect of IFN-β deficiency. These findings confirm the involvement of CD11b+Gr1+ neutrophils in the induction and maintenance of tumor angiogenesis.
Further corroboration of the essential role of neutrophils in tumor angiogenesis was obtained by coinjecting neutrophils and tumor cells. Tumors coinjected with WT neutrophils grew faster than tumor cells alone. However, a dramatic increase in growth rate was observed when the neutrophils could not respond to endogenous type I IFN due to their deficiency in IFNAR. This compellingly shows that neutrophils are the major if not the sole cell population with tumor angiogenic activity, which is strongly controlled by endogenous type I IFN.
The cells that provide endogenous IFN-β are radio resistant and therefore most likely of nonhematopoietic origin. Thus, endothelial cells could be one of the sources of IFN-β. On the other hand, bone marrow stroma cells were also shown to produce type I IFN, hence influencing B cell development (62
). These cells could also be responsible for the phenomenon observed here.
In the absence of endogenous IFN-β, CD11b+
neutrophils expressed higher levels of molecules such as CXCR4, VEGF, and MMP9 that are potent factors involved in mobility, tumor homing, and stimulation of angiogenesis (63
). In addition, other regulatory molecules that are known to increase tumor homing, such as c-myc
and STAT3 (44
), were also found at significantly higher expression levels in CD11b+
neutrophils from tumors of mice that lack IFN.
A direct effect of IFN-β on the regulation of tumor homing and angiogenesis molecules could indeed be observed in vitro. When CD11b+
neutrophils from tumors of Ifnb1–/–
mice were exposed to low levels of recombinant IFN-β, genes such as Vegf
, and Cxcr4
and their molecular regulators were downregulated to levels found in controls. This is consistent with the hypothesis that low levels of IFN-β are constitutively produced by cells like stroma cells (65
). Such cells may imprint neutrophils to inhibit tumor angiogenesis. Although only small differences in CXCR4 expression on CD11b+
neutrophils could be observed in bone marrow of Ifnb1–/–
compared with normal mice, it is possible that such cells immediately leave this anatomical niche upon maturation. Hence, differences might not become apparent.
It is generally accepted that angiogenesis is augmented by hypoxia. Hypoxic regions can be found in tumors of a certain size. Therefore, one could argue that the lack of IFN-β leads to a faster initial growth of tumors. This would result in larger hypoxic regions and in turn would induce enhanced angiogenesis. Enhancement of angiogenesis in tumors of mice that lack IFN-β would thus result from the initial increased tumor growth and development of large hypoxic areas and would therefore not be due to inhibition of angiogenesis by endogenous IFN-β. We think we can exclude this argument: (a) tumors of comparable size from both types of mice with presumably similar hypoxic regions were compared. Nevertheless differential angiogenesis was observed. (b) A direct effect of IFN-β on molecules involved in angiogenesis induction by CD11b+Gr1+ neutrophils could be demonstrated. (c) Coinjection of neutrophils resulted in a dramatic increase in tumor growth rate. At the time of tumor injection, necrosis and hypoxia could not have been different for both groups of tumors. (d) Augmented metastasis formation in mice lacking endogenous IFN-β could also be shown, as well as increased angiogenesis in the Matrigel model. These conditions should be identical in both mice, deficient or sufficient for IFN-β. No difference in hypoxia should exist either in the lungs or in Matrigel pads. Nevertheless, increased metastasis growth and angiogenesis is observed in Ifnb1–/– mice. Thus, we believe that enhanced growth of tumors in Ifnb1–/– mice is indeed due to a direct effect of IFN-β produced by radio-resistant cells, such as bone marrow stroma cells, on migratory and maturation capacities of CD11b+Gr1+ neutrophils that are involved in tumor angiogenesis.
This interpretation is complementary to recent findings on tumor editing by type I IFNs (22
). Tumor editing by type I IFNs was shown to act on hematopoietic cells. It was believed that this is due to direct effects on T cells. On the other hand, only part of the tumors induced in mice that lacked a functional type I IFN system were highly immunogenic, i.e., had escaped immune editing by IFN. Here, we describe the complementing explanation. We interpret these findings now to mean that some of the tumors could establish themselves due to the uncontrolled angiogenesis in the absence of an endogenous type I IFN system. The differential growth rates of B16F10 in RAG2-deficient and RAG2 IFN-β double-deficient mice are in line with our interpretation. Thus, we conclude that type I IFN could either support an adaptive T cell–dependent tumor immune defense or block tumor angiogenesis via an innate mechanism.
Inhibition of angiogenesis by type I IFNs has been suggested before (66
). Many patients suffering from SLE also exhibit vascular diseases such as premature atherosclerosis (67
). This has been attributed to high amounts of type I IFNs in the serum of such patients. In vitro experiments confirmed the antiangiogenesis potential of type I IFNs. However, this phenotype is observed under inflammatory conditions, with high amounts of IFNs present. In contrast, in a tumor situation, inflammation should be significantly lower than in SLE. Consistently, downregulation of proangiogenic molecules required low to very low amounts of IFN-β (data not shown).
Certainly, one could argue that the transplantation of the tumor also initiates an inflammatory reaction. To avoid this problem, we carried out the analysis of tumor-infiltrating CD11b+Gr1+ neutrophils 14 days after tumor implantation. At that time, the inflammatory phase of tumor transplantation should have ceased already. Nevertheless, a clear difference in number and angiogenic properties between neutrophils from control and Ifnb1–/– mice could be observed. Therefore, we believe that the low amounts of constitutively produced endogenous IFN-β are sufficient to restrict tumor angiogenesis.
Interestingly, no difference was observed in the number of neutrophils in blood of both types of mice not bearing tumors. Possibly the cytokine milieu that is induced in the host organism by the tumor is a prerequisite for the phenotype to become apparent.
Taken together, our findings demonstrate the importance of the type I IFN system in biological processes apart from its essential role in host defense. Similar to other processes of host response, in which the type I IFN system is involved, the activity of IFN-β appears to be pleiotropic and highly complex. Simultaneously, our findings underscore the therapeutic potential of this system when applied appropriately.