Matrix metalloprotease type 9 (MMP-9) has been functionally implicated in VEGF activation, the induction and maintenance of chronic angiogenesis, and early stage tumor growth in a number of mouse models of cancer. In this article, we have identified two inflammatory cell types that are major sources of MMP-9 in the angiogenic stages of pancreatic islet carcinogenesis that unfold in RIP1-Tag2 transgenic mice. MMP-9-expressing neutrophils were predominantly found inside angiogenic islet dysplasias and tumors, whereas MMP-9-expressing macrophages were localized along the periphery of such lesions. Transient depletion of neutrophils significantly suppressed VEGF:VEGF-receptor association, a signature of MMP-9 activity, and markedly reduced the frequency of initial angiogenic switching in dysplasias. Thus infiltrating neutrophils can play a crucial role in activating angiogenesis in a previously quiescent tissue vasculature during the early stages of carcinogenesis.VEGF ͉ macrophage ͉ granulocyte colony-stimulating factor (G-CSF) ͉ angiogenesis ͉ matrix metalloprotease type 9 T umors and their neoplastic progenitors are composed not only of transformed ''cancer cells'' but also of other cell types constituting the stroma. These stromal cells include cancerassociated fibroblasts, endothelial cells, pericytes, and a variable representation of leukocytes, including macrophages, neutrophils, mast cells, and B or T lymphocytes (1, 2). Increasing evidence indicates that leukocytic infiltration can either antagonize tumor formation and growth (immune surveillance) (2, 3) or, alternatively, promote tumor phenotypes, such as angiogenesis, growth, and invasion (immune enhancement) (1, 4, 5). Historically, tumor-infiltrating leukocytes have been considered to be manifestations of an intrinsic defense mechanism against developing tumors (2). However, most solid tumors are largely recognized as self and do not evoke effective immune responses capable of killing or antagonizing tumor formation, growth, and progression (3, 6). In contrast, accumulating clinical data for solid tumors show a correlation between high-density leukocytic infiltration into tumors and poor outcome of patients (1). In regard to the innate immune system, despite its capability to stimulate acquired immune responses, the weight of the evidence indicates that infiltration by innate immune cell types is tumorpromoting in many organs and tumorigenesis pathways (1,7,8). In addition to protumorigenic activities of tumor associated macrophages (9-11), neutrophils also have been shown to enhance the in vitro invasive and in vivo metastatic potential of syngeneic tumor cells by facilitating invasion into basement membrane (7).Genetically engineered mouse models of cancer are proving instructive about the immunobiology of tumors, revealing both enhancing and antagonizing roles played by the adaptive and innate immune system (4,5,(12)(13)(14). RIP1-Tag2 transgenic mice constitute a well characterized prototypical model for multistep carcinogenesis, involving the pancr...

Normally growing cells promptly cease DNA synthesis when exposed to genotoxic stresses, such as radiation, and this cell-cycle arrest prevents the accumulation of mutations. The transcription factor interferon regulatory factor (IRF)-1 is essential for the regulation of the interferon system, inhibits cell growth, and manifests tumour-suppressor activities. Here we show that mouse embryonic fibroblasts (EFs) lacking IRF-1 are deficient in their ability to undergo DNA-damage-induced cell-cycle arrest. A similar phenotype has been observed in EFs lacking the tumour suppressor p53 (refs 8, 9), although the expression of IRF-1 and p53 are independent of one another. Furthermore, we show that transcriptional induction of the gene encoding p21 (WAF1, CIP1), a cell-cycle inhibitor, by gamma-irradiation is dependent on both p53 and IRF-1, and that the p21 promoter is activated, either directly or indirectly, by both in a transient cotransfection assay. These two tumour-suppressor transcription factors therefore converge functionally to regulate the cell cycle through the activation of a common target gene.

Previous studies in the K14-HPV/E(2) mouse model of cervical carcinogenesis demonstrated that infiltrating macrophages are the major source of matrix metalloproteinase 9 (MMP-9), a metalloprotease important for tumor angiogenesis and progression. We observed increased expression of the macrophage chemoattractant, CCL2, and its receptor, CCR2, concomitant with macrophage influx and MMP-9 expression. To study the role of CCL2-CCR2 signaling in cervical tumorigenesis, we generated CCR2-deficient K14-HPV/E(2) mice. Cervixes of CCR2-null mice contained significantly fewer macrophages. Surprisingly, there was only a modest delay in time to progression from dysplasia to carcinoma in the CCR2-deficient mice, and no difference in end-stage tumor incidence or burden. Moreover, there was an unexpected persistence of MMP-9 activity, associated with increased abundance of MMP-9(+) neutrophils in tumors from CCR2-null mice. In vitro bioassays revealed that macrophages produce soluble factor(s) that can suppress neutrophil dynamics, as evidenced by reduced chemotaxis in response to CXCL8, and impaired invasion into three-dimensional tumor masses grown in vitro. Our data suggest a mechanism whereby CCL2 attracts proangiogenic CCR2(+) macrophages with the ancillary capability to limit infiltration by neutrophils. If such tumor-promoting macrophages are suppressed, MMP-9(+) neutrophils are then recruited, providing alternative paracrine support for tumor angiogenesis and progression.

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