In this study, we show the crucial role played by the ECM degrading enzyme MMP-9 provided by BM-derived CD11b+ myelomonocytic cells in allowing tumors to grow in irradiated normal tissues of the mice. We used the irradiated tumor bed model to abrogate local angiogenesis so as to examine the role of BM-derived cells in tumor growth. We demonstrate that CD11b+ myelomonocytic cells are recruited into irradiated tumors and into tumors growing in pre-irradiated tissues, and these cells restored tumor growth in MMP-9 KO mice by allowing immature blood vessels to develop. Further, when MMP-9 or a major source of MMP-9 expressing cells are genetically absent, ablated, or chemically inhibited, tumors in pre-irradiated tissues fail to grow beyond a very small size and are composed of normal, mature blood vessels.
Given the role of MMP-9 in degrading and remodeling the ECM, CD11b+ myelomonocytic cells expressing MMP-9 are likely to be important in reorganizing stromal compartments of tumors. In particular, by degrading the ECM they could provide a means for endothelial cells to enter or migrate to the tumor when the existing endothelial cells in and adjacent to the tumor cannot proliferate because of the local irradiation they have received. MMP-9 is involved in cleaving fibrillar type I collagen, the major constituent of the extracellular matrix to which endothelial cells are exposed in an injured tissue, allowing growth-factor induced angiogenesis to occur in chick chorioallantoic membrane assay (Seandel et al., 2001
). Moreover MMP-9 provided by BM-derived macrophages has been shown to be essential in capillary branching in ischemia-induced revascularization of normal tissues (Johnson et al., 2004
). MMP-9 may also enhance local angiogenesis in a spatiotemporal manner due to its ability to cleave membrane-bound VEGF thereby increasing bioavailable levels of VEGF (Bergers et al., 2000
), a growth factor critical for survival and growth of endothelial cells (Ferrara et al., 2003
). Macrophages themselves can also express VEGF and these cells are observed in poorly vascularized areas of human breast carcinoma (Lewis et al., 2000
). Other functions of macrophages such as inducible nitric oxide synthase, arginase, and cyclooxygenase-2 have been reported to be important in growth of irradiated tumors in mice (Tsai et al., 2007
CD11b+ myelomonocytic cells, including a subset of CD11b+
myeloid suppressor cells, are increasingly recognized for their roles in promoting tumor progression. Studies have shown that these cells enhance tumor angiogenesis (Yang et al., 2004
), prepare the pre-metastatic niche in the lung (Hiratsuka et al., 2006
), and are responsible for the refractoriness of tumors to anti-VEGF treatment (Shojaei et al., 2007
). Our study is consistent with these reports by showing that they promote tumor growth in pre-irradiated tissues of mice when local angiogenesis is inhibited.
Increased leukocyte infiltration, especially by CD68 positive macrophages, is observed in biopsy samples of rectal cancer patients after high-dose (and short term) and low-dose (and long term) fractionated radiotherapy (Baeten et al., 2006
). The authors of this study proposed that an increased expression of adhesion molecules such as intracellular adhesion molecule-1, vascular cell adhesion molecule, and E-selectin on tumor endothelium after radiotherapy is responsible for stimulating leukocyte infiltration in the tumors. However, other factors including VEGF and stromal-cell derived factor-1 (SDF-1) have been also reported to be essential in recruiting BM-derived myelomonocytic cells to tumors (Grunewald et al., 2006
; Petit et al., 2007
). VEGF and SDF-1 are downstream targets of hypoxia-inducible factor -1 (HIF-1), a transcription factor induced by hypoxia due to stabilization under hypoxic conditions (Ceradini et al., 2004
; Semenza, 2003
). HIF-1 levels are likely to increase in tumors regrowing after irradiation or tumors grown in pre-irradiated tissues due to the increased levels of hypoxia (Kim et al., 1993
; Teicher et al., 1994
). Furthermore, recent studies have shown that irradiation increases HIF-1 activity in tumors (Moeller et al., 2004
) and this occurs by recruited macrophages in irradiated tumors producing nitric oxide, which in turn nitrosylates cysteine residues of the oxygen-dependent degradation domain of HIF-1α thereby stabilizing it (Li et al., 2007
Overall, there is strong evidence that BM-derived myelomonocytic cells promote tumor growth and they do so by forming a positive feedback loop with many other components of the tumor microenvironment. Indeed several investigators have reported that targeting TAMs produces significant antitumor activity (Allavena et al., 2005
; Lin et al., 2001
; Luo et al., 2006
; Zeisberger et al., 2006
). However, it is evident that tumors show large variations in recruiting and utilizing these BM-derived cells, and our study demonstrates that the pattern of BM-derived infiltrates and reliance for the source of MMP-9 varies widely between RIF and MT1A2 tumors. Hence prospective knowledge of tumor cytokines and their role in the recruitment of BM-derived cells will be important to derive maximum antitumor activity from therapies targeting these cells.
We observed a minimal contribution of BM-derived EPCs to the vasculature of tumors grown in pre-irradiated tissues. This raises the question as to the source of the additional endothelial cells. Studies have shown that there are mature circulating endothelial cells derived from vessel wall turnover and these cells are increased in patients with some types of cancer (Bertolini et al., 2006
). Recently, Aicher and colleagues reported that there are non-BM-derived circulating progenitor cells from organs such as the small intestine and liver and that these cells incorporate into sites of neovascularization (Aicher et al., 2007
). However further studies are needed to determine the exact source of those new endothelial cells that promote tumor growth following high dose radiotherapy.
Our results also suggest that BM-derived cells expressing MMP-9 are sufficient but not essential for tumor vasculogenesis. This is evident from the similar tumor growth in pre-irradiated tissues of WT mice + MMP-9
KO BM compared to WT mice + WT BM. These data indicate that non-BM cells of the host that are still proficient in MMP-9, such as fibroblasts and smooth muscle cells, can compensate for the deficiency of MMP-9 from BM cells. MMP-9 in fibroblasts has been shown to promote mitogenic induction of breast cancer cells by enhancing endothelial cell survival and function in an in vitro
co-culture model (Shekhar et al., 2001
). This supports our observation that other sources of MMP-9 could also play a role in promoting tumor growth and angiogenesis. It also strengthens the rationale that MMP-9 is an important target for adjunct therapy to radiotherapy.
Clinical trials with MMP inhibitors have been uniformly disappointing. Although MMP-9 expression has been shown to correlate with tumor response in patients (Unsal et al., 2007
), MMP inhibitors when given alone or in combination with cytotoxic agents showed no gain in clinical efficacy (Coussens et al., 2002
). However, none of these trials have been performed in conjunction with radiotherapy, a therapy that can selectively inhibit local angiogenesis and make tumor growth dependent on vasculogenesis. Even though currently available MMP-9 inhibitors lack specificity for MMP-9 by also inhibiting the closely related MMP-2, a recent pre-clinical study showed that the MMP-2 and MMP-9 inhibitor Metastat significantly potentiated the antitumor efficacy of irradiation (Kaliski et al., 2005
), supporting the rationale of combining MMP inhibitors with radiotherapy. We believe that our data point the way to further studies of MMP-9 inhibitors with radiation.