Tumor-associated neutrophils contribute to neovascularization by supplying matrix metalloproteinase-9 (MMP-9), a protease that has been genetically and biochemically linked to induction of angiogenesis. Specific roles of inflammatory neutrophils and their distinct proMMP-9 in the coordinate regulation of tumor angiogenesis and tumor cell dissemination, however, have not been addressed. We demonstrate that the primary tumors formed by highly disseminating variants of human fibrosarcoma and prostate carcinoma recruit elevated levels of infiltrating MMP-9-positive neutrophils and concomitantly exhibit enhanced levels of angiogenesis and intravasation. Specific inhibition of neutrophil influx by interleukin 8 (IL-8) neutralization resulted in the coordinated diminishment of tumor angiogenesis and intravasation, both of which were rescued by purified neutrophil proMMP-9. However, if neutrophil proMMP-9, naturally devoid of tissue inhibitor of metalloproteinases (TIMP), was delivered in complex with TIMP-1 or in a mixture with TIMP-2, the protease failed to rescue the inhibitory effects of anti-IL8 therapy, indicating that the TIMP-free status of proMMP-9 is critical for facilitating tumor angiogenesis and intravasation. Our findings directly link tumor-associated neutrophils and their TIMP-free proMMP-9 with the ability of aggressive tumor cells to induce the formation of new blood vessels that serve as conduits for tumor cell dissemination. Thus, treatment of cancers associated with neutrophil infiltration may benefit from specific targeting of neutrophil MMP-9 at early stages to prevent ensuing tumor angiogenesis and tumor metastasis.

Key Points• Acquisition of high angiogenesis-inducing capacity by human and murine macrophages requires their polarization toward the M2 phenotype.• M2-polarized macrophages shutdown their TIMP1 gene expression and initiate production of highly angiogenic TIMP-deficient proMMP-9.A proangiogenic function of tissue-infiltrating monocytes/macrophages has long been attributed to their matrix metalloproteinase-9 zymogen (proMMP-9). Herein, we evaluated the capacity of human monocytes, mature M0 macrophages, and M1-and M2-polarized macrophages to induce proMMP-9-mediated angiogenesis. Only M2 macrophages induced angiogenesis at levels comparable with highly angiogenic neutrophils previously shown to release their proMMP-9 in a unique form, free of tissue inhibitor of metalloproteinases-1 (TIMP-1). Macrophage differentiation was accompanied by induction of low-angiogenic, TIMP-1-encumbered proMMP-9. However, polarization toward the M2, but not the M1 phenotype, caused a substantial downregulation of TIMP-1 expression, resulting in production of angiogenic, TIMP-deficient proMMP-9. Correspondingly, the angiogenic potency of M2 proMMP-9 was lost after its complexing with TIMP-1, whereas TIMP-1 silencing in M0/M1 macrophages rendered them both angiogenic. Similar to human cells, murine bone marrow-derived M2 macrophages also shut down their TIMP-1 expression and produced proMMP-9 unencumbered by TIMP-1. Providing proof that angiogenic capacity of murine M2 macrophages depended on their TIMP-free proMMP-9, Mmp9-null M2 macrophages were nonangiogenic, although their TIMP-1 was severely downregulated. Our study provides a unifying molecular mechanism for high angiogenic capacity of TIMP-free proMMP-9 that would be uniquely produced in a pathophysiological microenvironment by influxing neutrophils and/or M2 polarized macrophages. (Blood. 2013;122(25):4054-4067) Introduction A strong link has been established between infiltrating leukocytes and various pathophysiological conditions involving tissue remodeling and transformation. [1][2][3] The leukocyte infiltrate can be represented by hematopoietic cells of different lineages, including lymphocytes, granulocytes, and macrophages. Tumor-associated macrophages (TAMs) have been implicated in cancer progression, 4,5 and high numbers of TAMs have been linked to poor prognosis in certain human malignancies. 6,7 A remarkable plasticity of macrophages allows them to acquire functionally distinct phenotypes. 8,9 Two major alternative phenotypes, namely M1 and M2, have been ascribed to tumor-suppressing and tumor-promoting TAMs, respectively, although a spectrum of activation states has been demonstrated in several settings. [10][11][12][13] In general, M1 macrophages are associated with an induction of strong immune response and tumoricidal activity. In contrast, M2 macrophages appear to suppress immune surveillance and enhance neovascularization.Macrophage-induced angiogenesis involves an angiogenic switch, 14,15 which is triggered by proteolytic release of directacting angiogenic ...

According to established notion, one of the major angiogenesis-inducing factors, pro-matrix metalloproteinase-9 (proMMP-9), is supplied to the tumor microenvironment by tumor-associated macrophages (TAMs). Accumulated evidence, however, indicates that tumor-associated neutrophils (TANs) are also critically important for proMMP-9 delivery, especially at early stages of tumor development. To clarify how much angiogenic proMMP-9 is actually contributed by TAMs and TANs, we quantitatively evaluated TAMs and TANs from different tumor types, including human xenografts and syngeneic murine tumors grown in wild-type and Mmp9-knockout mice. Whereas host MMP-9 competence was required for full angiogenic potential of both normal and tumor-associated leukocytes, direct comparisons of neutrophils versus macrophages and TANs versus TAMs demonstrated that macrophages and TAMs secrete 40- to 50-fold less proMMP-9 than the same numbers of neutrophils or TANs. Correspondingly, the levels of MMP-9–mediated in vivo angiogenesis induced by neutrophils and TANs substantially exceeded those induced by macrophages and TAMs. MMP-9–delivering TANs were also required for development of metastasis-supporting intratumoral vasculature, characterized by ≥ 11-μm size lumens and partial coverage with stabilizing pericytes. Importantly, MMP-9–producing TAMs exhibit M2-skewed phenotype but do not express tissue inhibitor of metalloproteinases-1 (TIMP-1), a novel characteristic allowing them to secrete TIMP-1–free, neutrophil-like MMP-9 zymogen unencumbered by its natural inhibitor. Together, our findings support the notion whereby TANs, capable of immediate release of their pre-stored cargo, are the major contributors of highly angiogenic MMP-9, whereas tumor-influxing precursors of macrophages require time to differentiate, polarize into M2-skewed TAMs, shut down their TIMP-1 expression, and only then, initiate relatively low-level production of TIMP-free MMP-9 zymogen.

RLIP76 (RALBP1) is a glutathione-conjugate transporter that is a critical component of clathrin-coated pit-mediated endocytosis, as well as in stress responses. In cultured cells, it provides protection from stressors including heat, oxidant chemicals, chemotherapeutic agents, UV irradiation, and X-irradiation. Here, we show marked reduction in glutathione conjugate transport capacity and stepwise increase in radiation sensitivity associated with heterozygous or homozygous loss of the RLIP76 gene in mice. Survival after radiation in homozygous knockout animals was significantly shorter than either the heterozygous knockouts or the wild type. Delivery of recombinant RLIP76 to mice lacking RLIP76 via a liposomal delivery system rescued radiation sensitivity. Furthermore, treatment of wild-type mice with RLIP76-containing liposomes conferred resistance to radiation. These findings suggest that inhibiting RLIP76 could be used for sensitization to radiation during cancer therapy and that RLIP76 liposomes could be radioprotective agents useful for treatment of iatrogenic or catastrophic radiation poisoning. (Cancer Res 2005; 65(14): 6022-8)

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