Intracranial Microenvironment Reveals Independent Opposing Functions of Host αVβ3 Expression on Glioma Growth and Angiogenesis

αVβ3 integrins are overexpressed in the host-derived vasculature of glioblastoma multiform (GBM) and are believed to contribute to angiogenesis and tumor growth. To directly address the role of host αVβ3 expression in GBM growth and behavior, we intracranially implanted integrin β3-expressing GBM cells into β3 wild type (WT) or β3 knock out (KO) mice and monitored angiogenesis and growth. GBM in β3 WT animals had a vessel density greater than that in β3 KO animals, consistent with a pro-angiogenic, pro-tumorigenic view of host integrin function. GBM in β3 WT animals, however, were no larger than those in β3 KO animals, because GBM in β3WT animals were infiltrated with a higher number of tumor necrosis factor α-secreting, apoptosis-inducing macrophages than the tumors in the corresponding β3 KO animals. The tumor-suppressive effects of host β3 expression could be reversed by macrophage depletion or by transplantation of bone marrow from β3 KO animals into β3 WT animals, both of which significantly increased tumor growth independently of tumor vessel density. Taken together, these results show that host αVβ3 integrin expression has opposing actions in the intracranial setting, enhancing tumor vascularization and growth while independently enhancing macrophage-mediated tumor elimination. Appropriate management of these functions could lead to enhanced efficacy of anti-integrin based therapies for glioma. αVβ3 integrins are overexpressed in the host-derived vasculature of glioblastoma multiform (GBM) and are believed to contribute to angiogenesis and tumor growth. To directly address the role of host αVβ3 expression in GBM growth and behavior, we intracranially implanted integrin β3-expressing GBM cells into β3 wild type (WT) or β3 knock out (KO) mice and monitored angiogenesis and growth. GBM in β3 WT animals had a vessel density greater than that in β3 KO animals, consistent with a pro-angiogenic, pro-tumorigenic view of host integrin function. GBM in β3 WT animals, however, were no larger than those in β3 KO animals, because GBM in β3WT animals were infiltrated with a higher number of tumor necrosis factor α-secreting, apoptosis-inducing macrophages than the tumors in the corresponding β3 KO animals. The tumor-suppressive effects of host β3 expression could be reversed by macrophage depletion or by transplantation of bone marrow from β3 KO animals into β3 WT animals, both of which significantly increased tumor growth independently of tumor vessel density. Taken together, these results show that host αVβ3 integrin expression has opposing actions in the intracranial setting, enhancing tumor vascularization and growth while independently enhancing macrophage-mediated tumor elimination. Appropriate management of these functions could lead to enhanced efficacy of anti-integrin based therapies for glioma. Brain tumors affect ∼100,000 Americans per year and remain among the most difficult of human tumors to successfully treat. Individuals with glioblastoma multiform (GBM), 3The abbreviations used are: GBM, glioblastoma multiform; VEGF, vascular endothelial growth factor; TNF, tumor necrosis factor; KO, knock out; BMT, bone marrow (BM) transplant; FITC, fluorescein isothiocyanate; DIG, digoxigenin; PBS, phosphate-buffered saline; WT, wild type; HDMEC, human dermal microvascular endothelial cell line; vWF, von Willebrand factor; DAPI, 4′, 6-diamidino-2-phenylindole; TUNEL, terminal deoxynucleotidyltransferase-mediated nick end labeling. . a highly aggressive and angiogenic form of glioma, have a particularly poor prognosis, with an average survival of <2 years (1Ludgate C. Douglas B.G. Dixon P.F. Steinbok P. Jackson S.M. Goodman G.B. Int. J. Radiat. Oncol. Biol. Phys. 1988; 15: 1091-1095Abstract Full Text PDF PubMed Scopus (39) Google Scholar, 3US CBTR Statistical Report: Primary Brain Tumors in the United States, 1992-1997. Central Brain Tumors Registry of the United States, Hinsdale, IL2000Google Scholar). Although surgical resection followed by radiation and chemotherapy have been shown to slightly increase the lifespan of individuals with GBM, new approaches that can selectively target the tumor are needed. The search for agents that can selectively target glioma cells has led to an interest in the role of integrins in glioma biology. Integrins are transmembrane glycoprotein complexes of noncovalently linked α and β subunits. There are 8 known β subunits that combine with 18 α subunits in a defined manner to create more than 24 unique αβ heterodimers. Although some β subunits combine with a large number of α subunits, others exhibit greater selectivity and combine with single α subunits (4Hynes R.O. Cell. 2000; 110: 673-687Abstract Full Text Full Text PDF Scopus (6955) Google Scholar). As an example, the β3 subunit combines exclusively with the αV subunit in most tissues except in platelets, where it also exhibits a pro-clotting interaction with the αIIb integrin subunit (5Milner R. Campbell I.L. J. Neurosci. Res. 2002; 69: 286-291Crossref PubMed Scopus (184) Google Scholar). Integrin heterodimers each recognize a specific range of ligands in the extracellular matrix or on neighboring cells, and integrin binding plays a key role in adhesion of integrinexpressing cells to the extracellular matrix. Integrin-mediated cellular anchorage, however, is also accompanied by linkage of activated integrins to the actin cytoskeleton and the triggering of a large number of signal transduction pathways that can influence nearly every aspect of cellular behavior (6Christofori G. EMBO J. 2003; 22: 2318-2323Crossref PubMed Scopus (246) Google Scholar, 8Schwartz M.A. Ginsberg M.H. Nat. Cell Biol. 2002; 4: 65-68Crossref PubMed Scopus (682) Google Scholar). Because integrins play a key role in processes requiring cell-cell interaction such as cell migration and angiogenesis, they also appear to play a key role in tumorigenesis. αVβ3 integrin complexes in particular are overexpressed in the proliferating vascular endothelial cells surrounding the tumor (9Albeda S.M. Mette S.A. Elder D.E. Stewart R. Damjanovich L. Herlyn M. Buck C.A. Cancer Res. 1990; 50: 6757-6764PubMed Google Scholar, 11Natali P.G. Hamby C.V. Felding-Habermann B. Liang B. Nicotra M.R. Di Filippo F. Giannarelli D. Temponi M. Ferrone S. Cancer Res. 1997; 57: 1554-1560PubMed Google Scholar). This is especially true in GBM, in which αVβ3 is overexpressed at the invasive, highly angiogenic edges of the tumor (12Bello L. Francolini M. Marthyn P. Zhang J. Carroll R.S. Nikas D.C. Strasser J.F. Villani R. Cheresh D.A. Black P.M. Neurosurgery. 2001; 49: 380-389PubMed Google Scholar). These observations have led to the suggestion that αVβ3 integrins may play a role in the angiogenesis and/or growth and invasion of GBM. This concept has been further supported by the demonstration that monoclonal antibodies and small cyclic arginine-glycine-aspartate peptide ligands of αVβ3, both of which function presumably as αVβ3 antagonists, led to tumor regression in animal models of GBM (13Chatterjee S. Matsumura A. Schradermeier J. Gillespie G.Y. J. Neurooncol. 2000; 46: 135-144Crossref PubMed Scopus (79) Google Scholar). These studies as a whole have defined αVβ3 as a promising target for tumor-selective therapy of GBM. More recent detailed studies, however, have questioned the established role of tumor and host αVβ3 expression in glioma growth. We previously showed that overexpression of αVβ3in intracranially implanted GBM cells did not increase the blood vessel density or size of the subsequent tumors but, rather, suppressed both tumor oxygenation and growth (14Kanamori M. Vanden Berg S.R. Bergers G. Berger M.S. Pieper R.O. Cancer Res. 2004; 64: 2751-2758Crossref PubMed Scopus (52) Google Scholar). Because the growth suppressive effects of β3 expression in the tumor could be overcome by genetic alterations (Akt activation, VEGF overexpression) common to GBM, however, these studies suggested that αVβ3 expression in the host, rather than in the tumor, may play a more important role in modulating glioma growth. The effects of host αVβ3 expression on tumor growth have been examined, and in these studies host αVβ3 expression surprisingly suppressed rather than enhanced the growth of subcutaneously implanted human tumor cells in association with suppression of angiogenesis (15Reynolds L.E. Wyder L. Lively J.C. Taverna D. Robinson S.D. Huang D. R.O. Nat. 2002; Scopus Google Scholar, D. D. L. A. R.O. S. A. 2004; PubMed Scopus Google Scholar). These studies, however, were in the not address the role of host αVβ3 integrin expression in the highly vascular intracranial that is to GBM. Because angiogenesis and tumor growth are highly both of which are by the tumor B. J. R.S. Bergers G. Cancer Cell. 2003; 4: Full Text Full Text PDF PubMed Scopus Google Scholar, P. L. S. G.B. M. Huang Cancer Res. 2001; Google we the that effects of host αVβ3 expression be and in the intracranial the We that effects of host αVβ3 expression are highly on tumor and that in the intracranial host αVβ3 expression glioma vessel density while independently glioma growth by enhancing macrophage into the of these opposing functions of αVβ3 expression functions could lead to enhanced efficacy of therapies for glioma. Cell an established human GBM cell from the of Brain a GBM cell by intracranial of a in a animal J. by of at were as in a of with human dermal microvascular endothelial cell by Bergers of cells were in with and cells were at in a of for cell were from Cell and integrin knock out (KO) (15Reynolds L.E. Wyder L. Lively J.C. Taverna D. Robinson S.D. Huang D. R.O. Nat. 2002; Scopus Google mice were with mice the to KO mice with or β3 integrin expression The were by the as previously D.A. D. M. S. R.O. J. PubMed Scopus Google Scholar). of and cells into mice were as previously (14Kanamori M. Vanden Berg S.R. Bergers G. Berger M.S. Pieper R.O. Cancer Res. 2004; 64: 2751-2758Crossref PubMed Scopus (52) Google Scholar, Berger M.S. Pieper R.O. Cancer Res. 2001; Google Scholar). cells were into of and mice (14Kanamori M. Vanden Berg S.R. Bergers G. Berger M.S. Pieper R.O. Cancer Res. 2004; 64: 2751-2758Crossref PubMed Scopus (52) Google Scholar, M. Berger M.S. Pieper R.O. Cancer Res. 2003; Google Scholar). were were at the of cellular and were and the and of the tumor were To of were into and every for and the studies, cells were into the and the the M. L. J. G. C. M. L. A. Cancer Res. 2002; Google Scholar, M. S. S. C. M. R. J. Biol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). KO mice with or β3 integrin expression were with at a and by of cells from mice from the of animals and with the genetic These mice were with for the mice were used for further or were used for Primary antibodies used for were Willebrand factor and were used at except which used at or antibodies were used at the of These were by and with density by the number of or in tumor at with the of an (14Kanamori M. Vanden Berg S.R. Bergers G. Berger M.S. Pieper R.O. Cancer Res. 2004; 64: 2751-2758Crossref PubMed Scopus (52) Google Scholar). density as the S.D. with as A. D. L. M. M. Neurosurgery. 2001; 49: Google Scholar). by a tumor were by and with for at at in a of of of of of of and of The To the were for which in monoclonal antibodies were used to or cells in the blood of were with a of for at with cell R.O. Cell. Full Text Full Text PDF PubMed Scopus Google with phosphate-buffered in PBS, and in a with of macrophages from β3 WT and KO mice were and for further from β3 WT or KO mice were activated by with The in with at for 24 the end of the were into and at for further The from the macrophage used as a macrophages were and as previously (14Kanamori M. Vanden Berg S.R. Bergers G. Berger M.S. Pieper R.O. Cancer Res. 2004; 64: 2751-2758Crossref PubMed Scopus (52) Google Scholar). cell to and an The in and with antibodies or at with of Cell were and in and in at The cells were with followed by in and for at in the were on with used to cell The binding an to the with macrophage cells were with and were and in binding with to the cell and cells were on with cell deoxynucleotidyltransferase-mediated nick end by the to the were with were by of an and to the tumor and at as and and a animal these antibodies that significantly the number of macrophages while not of As a used in the in macrophage we migration D. J. Cell Biol. PubMed Scopus Google Scholar). were with for from the and at were on the in of were to to on for The macrophages from β3 KO mice were to and of cell were per into the of the and the were into the to at the the of the and the to for at which the number of cells that had into the studies, anti-integrin β3 to a of The were on a for at the cells were in and in of This to per each the number of cells that had as the of cells in Statistical were the with defined as Integrin β3 on studies have shown that host αVβ3 expression suppressed rather than enhanced the growth of human tumor cells in association with suppression of angiogenesis (15Reynolds L.E. Wyder L. Lively J.C. Taverna D. Robinson S.D. Huang D. R.O. Nat. 2002; Scopus Google Scholar, D. D. L. A. R.O. S. A. 2004; PubMed Scopus Google these studies were in the To host β3 expression contribute to tumor in a tumor we implanted human GBM cells subcutaneously and intracranially into β3 KO mice and monitored the tumor vessel density and size of the β3 expression suppressed the growth of cells and also the density of blood in the tumor as by antibodies the endothelial cell or These results are consistent with host suppression of the growth and angiogenesis of subcutaneously implanted human tumor cell (15Reynolds L.E. Wyder L. Lively J.C. Taverna D. Robinson S.D. Huang D. R.O. Nat. 2002; Scopus Google Scholar, D. D. L. A. R.O. S. A. 2004; PubMed Scopus Google Scholar). the intracranial setting, however, effects of host β3 expression on tumor growth and were GBM cells intracranially implanted into β3 WT animals tumors that were no in size from those that in β3 KO animals an of vessel density showed that in the setting, host β3 expression enhanced rather than tumor vessel density with the particularly in the the of the tumors These were unique to the GBM cell to the of human cells into because GBM cells were intracranially implanted into the animals used for the cell studies, the cells tumors of size in animals as in studies human GBM the tumors that in the β3 WT animals had a greater vessel density than those in the β3 KO animals These results that host β3 expression tumor behavior on the of tumor β3 in a for host β3 expression enhanced intracranial tumor tumor size is that host β3 expression may have actions as as as to these growth suppressive actions be the that the intracranial GBM in β3 WT animals, in to highly also had significantly more of macrophages than in the vascular tumors in β3 KO animals Although macrophages are known to contribute to tumor J. Neurooncol. 2002; 57: PubMed Scopus Google could not the of the infiltrated cells to tumor the influence of β3 expression on macrophage or the of these To to these we studies in which β3 animals were and with the bone marrow from β3 KO animals. GBM cells were intracranially implanted into animals with bone which effects of β3 expression in the bone marrow on tumor growth and were As shown in the size of intracranial tumors in β3 WT animals that did not no from that in β3 WT animals that had β3WT bone marrow with that from a β3 WT animal with in these tumors were also of a size to those that in β3 KO animals, which no or were with marrow The transplantation of β3 WT marrow into however, suppressed the growth of intracranially implanted cells the of β3 WT marrow with marrow enhanced tumor growth These results that in to a pro-angiogenic, host β3 expression also a previously by in the bone of intracranial GBM tumor size and vessel density β3 WT or mice were and with β3 WT or β3 KO marrow from BMT, cells were tumor animals were the and tumor and as a of tumor growth in mice with β3 KO marrow each were with and by a were in tumor at with the of an vessel density higher in β3 WT animals than in β3 KO animals of the β3 of the host are as the S.D. or in a β3 integrin of the host bone marrow could influence intracranial tumor growth in The function of and is known to be on αVβ3 expression M.A. M. P. D. M. D. C. S. C. J. Biol. 2003; Full Text Full Text PDF PubMed Scopus Google and of β3 expression could and to enhanced glioma growth. in however, were in KO animals that and a role for these The bone marrow has also been suggested to contribute cells to the vasculature of tumors Zhang L. M. Res. 2002; PubMed Scopus Google Scholar, A. M. R. B. G. 1997; PubMed Scopus Google and it is that host β3 expression could tumor growth by The alterations in tumor growth by BMT, however, were to vessel and as shown in tumor vessel density to β3 of the host to β3 of the host bone with no of cells from the bone marrow of by in were with the tumor vasculature that the bone marrow did not contribute in a manner to the of tumor vasculature or to host β3 effects on blood vessel density. is that bone cells in KO mice cells, could directly or intracranial tumor growth. Because more macrophages were with intracranial tumors in β3 WT animals than in β3 KO animals, we the that β3 WT animals more tumor-suppressive macrophages than animals. of of macrophages in β3 WT and β3 KO animals by however, showed that of cells were not significantly in β3 KO animals the of of cells into the and the of β3 KO animals with or animals examined, no or bone cells were in the As shown in and however, of cells were in the of β3 WT mice that β3 WT bone and of these and cells were also bone The number of cells in the of β3 WT mice with β3 WT marrow no from that in β3 KO mice that β3 WT marrow significantly greater than that in β3 KO animals with β3 KO marrow and The of macrophages to the tumor by alterations in the in migration of these these studies were to on which macrophages from β3WT or β3 KO mice were on of the and for to As shown in β3 KO macrophages had than β3 WT and the of the β3 WT macrophages by with integrin antibodies To further that β3 WT, bone macrophages were for the glioma growth suppression in β3 WT animals, we used a to selectively animals of which the effects on glioma growth were of β3 WT or β3 KO animals with a in a depletion of macrophages in both to macrophage in animals with an and nearly macrophage into tumors in both while not the of cells Although macrophage depletion did not significantly the growth of cells intracranially implanted into β3 KO animals macrophage depletion significantly increased the growth of cells intracranially implanted into β3 WT animals and As a these results that macrophages from β3 KO bone marrow can in the appear to be in to out of the vasculature and into the tumor and to the growth of intracranially implanted GBM β3 WT and with β3 WT macrophages to intracranial tumors more than β3 KO the by which they suppressed tumor growth at the did not appear to the growth of tumor cells in a because the tumors that in of infiltrated β3 WT macrophages had a tumor growth no from that of larger tumors that in the of β3 WT macrophages tumors from β3 KO animals β3 KO bone marrow for of As shown in the larger tumors that in animals with β3 KO bone marrow WT or with β3 KO bone were of a number of cells the tumors that in animals with β3 WT bone marrow WT or with β3 WT bone cells were particularly at the edges of the tumor where the of bone macrophages were also Because macrophages can by of J. Neurooncol. 2002; 57: PubMed Scopus Google we also tumors from β3 KO animals β3 KO bone marrow for the of As shown in macrophages were in the larger tumors that in animals with β3 KO bone no macrophages were of however, were in the of the tumors that in animals with β3 WT bone The of macrophages from β3 KO animals to also by alterations in in these studies macrophages from β3 KO mice were and to into the which the were to cells, and the of and cell of cells with a were As shown in the of β3 KO macrophages than β3 WT The from β3 KO macrophages also significantly to in target cells than from β3 WT macrophages WT macrophages and in glioma from β3 WT or KO mice were activated by with which the were for by or to cell by cell and with that in cells with from β3 KO with cells a to be are as the S.D. These results as a whole that β3 WT macrophages have a greater to to the tumor, and to in the cell in the These tumor-suppressive effects in appear to the enhanced vascular density by marrow cells and to the effects of host β3 expression in the intracranial and Integrins have been of a role to the vascularization and growth of glioma J. PubMed Scopus Google Scholar, L. Francolini M. Marthyn P. Zhang J. Carroll R.S. Nikas D.C. Strasser J.F. Villani R. Cheresh D.A. Black P.M. Neurosurgery. 2001; 49: 380-389PubMed Google Scholar). The overexpression of αVβ3 in glioma cells and the vasculature as the of αVβ3 to glioma growth has further supported the that αVβ3 integrin complexes are in the of The recent demonstration that β3 overexpression rather than the growth of some glioma (14Kanamori M. Vanden Berg S.R. Bergers G. Berger M.S. Pieper R.O. Cancer Res. 2004; 64: 2751-2758Crossref PubMed Scopus (52) Google as as the demonstration that host β3 expression the growth of tumors (15Reynolds L.E. Wyder L. Lively J.C. Taverna D. Robinson S.D. Huang D. R.O. Nat. 2002; Scopus Google have a of the role host αVβ3 integrins may play in glioma. the we have shown that host αVβ3 integrin expression play a role in glioma growth and angiogenesis that role is highly on the We also show that host β3 expression has a of effects on glioma behavior in the intracranial setting, enhancing both tumor and growth while at the enhancing the of macrophages to and in the suppression of glioma growth. These results a more view of the function of host αVβ3 expression than previously Although host β3 expression has been shown to play a and role in the angiogenesis and growth of a of implanted tumor cell (14Kanamori M. Vanden Berg S.R. Bergers G. Berger M.S. Pieper R.O. Cancer Res. 2004; 64: 2751-2758Crossref PubMed Scopus (52) Google Scholar, L.E. Wyder L. Lively J.C. Taverna D. Robinson S.D. Huang D. R.O. Nat. 2002; Scopus Google studies to have been in the setting, the of integrin function. The in both as as the of on integrin β3 function. The that subcutaneously implanted GBM cells, previously subcutaneously implanted and cells (15Reynolds L.E. Wyder L. Lively J.C. Taverna D. Robinson S.D. Huang D. R.O. Nat. 2002; Scopus Google exhibit tumor growth and angiogenesis in β3WT than in β3 KO however, the previously effects of host β3 expression were reversed in the intracranial The effects of host β3 expression in the were to of VEGF that in a in β3 KO host endothelial cells not in β3 WT endothelial cells L.E. Lively J.C. Robinson S.D. Cancer Res. 2004; 64: PubMed Scopus Google Scholar). we that expression of VEGF is significantly higher in tissues of β3 KO animals than in the tissues from β3 WT animals. The however, of VEGF and these were not by β3 is that endothelial cells in most to the of β3 expression by VEGF and and blood vessel endothelial cells are of and not angiogenesis in to of β3 is the however, by which β3 expression rather than of β3 also intracranial tumor vessel density. to the in β3 effects on angiogenesis, the studies that β3 expression a suppressive on tumor growth. As in the setting, host β3 expression suppressed the growth of intracranial by the pro-tumorigenic effects of host β3 expression on in the suggested that the effects of host β3 expression were by bone cells (15Reynolds L.E. Wyder L. Lively J.C. Taverna D. Robinson S.D. Huang D. R.O. Nat. 2002; Scopus Google the of these cells not the we show that the of host β3 expression to intracranial tumor growth with the bone marrow and with which in β3 WT animals were to and into the tumor, where linked to and tumor cell The function of αVβ3 in macrophages is not integrins appear to be not in the for macrophage and migration also for the and of tumor cells D. J. Cell Biol. PubMed Scopus Google Scholar, J. Neurooncol. 2002; 57: PubMed Scopus Google Scholar). The of macrophages to a of endothelial cells in as as to to the tumor in that the of β3 expression with macrophage function at not with the of the macrophages macrophage function linked to the effects of β3 on tumor growth and macrophage depletion studies were shown to selectively macrophages and not cells, the studies out the that bone cells such as may also contribute to host β3 effects of glioma growth and More detailed studies of the effects of these cells are to be of Although the the role of host β3 expression in the of tumor growth and angiogenesis, it also has for tumor the setting, β3 expression to contribute to tumor and of β3 expression to tumor growth and type of appear to be for a true β3 that function as β3 as has been suggested for R.O. Nat. 2002; PubMed Scopus Google be to be of the more intracranial glioma setting, host β3 expression has a more function. the intracranial setting, β3 could the of the tumor by the at the of increased angiogenesis and growth of the β3 tumor angiogenesis also tumor elimination. that of defined integrin agents could the effects of the while enhancing the growth suppressive effects of integrin The of the of host β3 expression in the intracranial is a in with