HIF1 Recruits Bone Marrow-Derived Cells to the Tumor Site
To test the hypothesis that GBMs promote BMDC-dependent neovascularization in a HIF-dependent manner, we transplanted bone marrow cells from β-actin-EGFP mice into lethally irradiated Rag1-deficient (Ragko) mice. Four weeks later, mice were implanted intracranially with HIF1α-proficient (WT-GBM) or HIF1α-deficient (HIFko GBM) GBM cells. We found that GFP+ BMDCs were randomly distributed within the tumor and represented about 15%–20% of all cells in WT-GBM tumors, whereas HIFko GBMs contained only about one-third this number (). Normal brain recruited virtually no GFP+ cells. These results indicate that HIF1α activity promotes recruitment of a substantial number of BMDCs in GBM.
HIF1 Recruits Bone Marrow-Derived Cells to Orthotopic Glioblastomas
We next assessed the BMDC types recruited by HIF1 by fluorescence-activated cell sorting (FACS) and immunohistochemical analyses of the tumor-derived GFP+ cell populations. WT-GBM elicited about three times more CD45+ monocytic cells and seven times more PDGFRβ+/Sca-1+ PPCs than HIFko GBM (). WT-GBM also contained nearly four times as many EPCs as HIFko GBM (). Importantly, GFP+ BMDCs were comprised of about 8% EPCs and 2% PPCs, respectively, while up to 90% (about 16% of total cells within the tumor) were positive for the panleukocyte marker CD45 (). The majority of the CD45+ BMDCs were reminiscent of CD11b+ monocytes/myeloid cells and F4/80+ macrophages, with ~5% comprised of Tie2+ expressing monocytes or VEGFR1+ hemangiocytes (). All CD45+ subpopulations were reduced 2- to 4-fold in HIFko GBM. These results indicate that HIF1 activation results in recruitment of heterogeneous populations of BMDC. Given that all of these cell types are diminished in HIFko GBM, our data suggest that a reduction in the number of BMDCs may be the limiting factor in promoting angiogenesis.
HIF1 Induces SDF1α in Tumor Cells
This result raises the question about the signaling events that recruit BMDC in a HIF-dependent manner. Both VEGF, which is implicated in the recruitment of vascular progenitor cells (Hattori et al., 2001
), and stromal-derived factor 1α (SDF1α/CXCL12), which signals through CXCR4 and may retain CXCR4+ BMDC in tumors (Grunewald et al., 2006
; Hattori et al., 2003
), are HIF1 target genes (Ceradini et al., 2004
; Shweiki et al., 1992
). Indeed, WT-GBM produced about 4-fold higher levels of VEGF and SDF1α than HIFko GBM (). While WT-GBM contained clusters of SDF1α+ cells, HIFko GBM contained only few single SDF1α+ cells within tumors (). Importantly, we found that WT-GBM cells, but not HIFko GBM cells, upregulated SDF1α mRNA under hypoxic conditions (), implying that GBMs, through HIF1 and its target SDF1α, recruit CXCR4+ BMDCs to the tumor site to facilitate new blood vessel growth.
HIF1 Recruits MMP-9-Positive Monocytic Cells from the Bone Marrow
If HIF1-induced BMDC influx regulates neovascularization, then which of the BMDC are functionally significant in this process and how do they achieve angiogenic initiation in GBM? Clearly, EPCs and PPCs could support new vessel formation by providing an additional source of endothelial cells and pericytes (Kopp et al., 2006
; Rajantie et al., 2004
; Song et al., 2005
). However, it is less obvious what roles the heterogeneous CD45+ myeloid cell populations play. Therefore, we used a candidate approach to identify proangiogenic molecules produced or regulated by vascular-modulating CD45+ BMDCs. Recent studies have shown that these cells express matrix metalloproteinase (MMP)-9 (Jodele et al., 2005
; Page-McCaw et al., 2007
; Yang et al., 2004
; Nozawa et al., 2006
). We had discovered that MMP-9, conveyed by inflammatory cells, enables an angiogenic switch by making sequestered VEGF bioavailable for its receptor VEGFR2 in pancreatic islet tumors (Bergers et al., 2000
; Nozawa et al., 2006
). In human astrocytomas, MMP-9 expressed in inflammatory cells and the invading tumor cell compartment correlates with tumor progression (Hormigo et al., 2006
; Kunishio et al., 2003
). We observed that the expression of MMP-9 in GBM was substantially reduced in the absence of HIF1 (). Similar to human GBMs, WT-GBM expressed MMP-9 in monocytic cells including F4/80+ macrophages and Tie2+, CD11b+, and VEGFR1+ myeloid populations (). We also detected MMP-9 in a small subset of tumor cells within the tumor mass and in single tumor cells infiltrating the brain parenchyma, as visualized by double positivity for SV40Tag and MMP-9, which were diminished in HIFko GBMs (). Substrate-gel zymography revealed that most of MMP-9 in the tumors was in an active form and reduced in HIFko GBM ().
MMP-9 Is Expressed in Various Subpopulations of CD45+ BMDC and in Infiltrating Tumor Cells
Given that HIFko GBM do not undergo vascular remodeling, we hypothesized that the nonangiogenic behavior of HIFko GBM is mainly caused by the substantial reduction of MMP-9- expressing BMDCs in the tumors. The reduced levels of MMP-9 would then keep VEGF in a predominantly sequestered state. Indeed, when we ectopically expressed active MMP-9 in HIFko-GBM cells (HIFko MMP-9+) to increase intratumoral MMP-9 levels (), tumors became hemorrhagic () and, to a certain extent, exhibited a tortuous, irregularly shaped and hyperdilated vasculature indicative of vascular remodeling (), keeping with the observation that an early indication of VEGF activity is vascular hyperdilation (Dvorak, 2000
). Since the VEGF levels in HIFko GBM are about 3.5-fold less than in WT-GBM (), the magnitude of these responses would be expected to be limited due to the rather low VEGF levels. Thus, although VEGF is liberated from the matrix in the presence of MMP-9, the total VEGF levels are not sufficient to produce the vigorous angiogenesis seen in WT-GBM. Indeed, we confirmed that the soluble VEGF fraction was substantially higher in HIFko MMP-9+ GBM compared to mock transfectants (). Our data suggest that HIF1 regulates two steps of VEGF activity: it both induces VEGF levels and regulates its mobilization from the ECM by recruiting monocytes that transport MMP-9 to the tumor site.
MMP-9 Deficiency Impedes Vascular Remodeling and Neovascularization in GBM
If MMP-9 is a critical downstream factor of HIF1α regulating angiogenesis, then MMP-9 deficiency should give rise to tumors that, like HIFko GBMs, are nonangiogenic. We, therefore, generated MMP-9ko GBMs in the same manner we produced WT-GBM and HIFko GBMs (Blouw et al., 2003
). Both MMP-9ko GBM and WT-GBM cells gave rise to similar numbers of soft agar colonies (Figure S1
). MMP-9ko GBMs injected into MMP-9ko mice exhibited typical features of high-grade astrocytomas/GBMs, and although they did not differ from WT-GBM in proliferation, these mice exhibited modestly longer survival times than wild-type mice bearing WT-GBM (Figure S1
We found that the majority of tumor vessels from MMP-9ko tumors were slim, elongated, and regularly shaped as visualized by fluorescent angiography, similar to blood vessels in normal brain, and in contrast to the distorted and angiogenic tumor vasculature from WT-GBM (). Congruent with the morphologic changes of an angiogenic tumor vasculature, we observed VEGF-VEGFR2-complex formation, which marks the activated, angiogenic state on endothelial cells (Bergers et al., 2000
; Brekken et al., 2000
) (), and RGS-5 expression (), a marker of activated pericytes (Berger et al., 2005
; Bondjers et al., 2003
), in WT-GBMs but not in MMP-9ko tumors (). These results indicate that tumor vessels in the absence of MMP-9 did not undergo vascular remodeling.
MMP-9-Deficient GBM Do Not Undergo Vascular Remodeling and Neovascularization
Notably, the inability of VEGF to bind to its receptor in MMP-9ko tumors was not caused by a severe reduction in VEGF or its receptor VEGFR2 () because VEGF levels were comparable to those of WT-GBM (Figure S2
). However, most of the VEGF-164, the predominant VEGF isoform in GBM, was bound to the extracellular matrix and cell surface of MMP-9ko tumor cells and, unlike WT-GBM, was not present in the supernatant (). These data confirm our observation in HIFko MMP-9+ tumors that MMP-9 is an important initiator of angiogenesis in GBM by releasing sequestered VEGF and making it bioavailable to its receptor VEGFR2.
Bone Marrow-Derived MMP-9+ Cells Are Sufficient to Initiate the Angiogenic Switch in GBM
To determine whether MMP-9 in tumor cells or host cells is critical for the angiogenic phenotype, we implanted WT-GBM cells into MMP-9ko mice (), and MMP-9ko GBMs into wild-type (WT) hosts (). In both instances, tumor vessels were irregularly shaped and tortuous indicative of vascular remodeling and showed VEGF/VEGFR2 binding, albeit to a lesser extent than WT-GBM in WT hosts, indicating that as long as a critical threshold of MMP-9 exists in tumors, angiogenesis can be initiated. In both instances, MMP-9+ cell numbers were reduced by 50% when compared to the WT situation (). Interestingly, when we injected MMP-9ko GBMs into WT mice whose bone marrow was reconstituted with MMP-9ko bone marrow, MMP-9+ cells in the tumors dropped significantly by 3- to 4-fold compared to WT-GBM, while blood vessels were more elongated and slim and did not exhibit VEGF:VEGFR2 activation, mimicking the phenotype of MMP-9ko tumor blood vessels in a MMP-9ko host (). Similarly, when we reconstituted the bone marrow of MMP-9ko mice harboring MMP-9ko GBM with WT bone marrow, the number of MMP-9+ cells increased while blood vessels became enlarged and distorted, exhibiting typical features of vascular remodeling concomitant with VEGF binding to its receptor ().
MMP-9+ BMDCs Contribute to the Angiogenic Switch in GBM
These data support the notion that a specific MMP-9 threshold is required for the angiogenic switch and that, indeed, BMDCs expressing MMP-9 have a significant effect on GBM angiogenesis.
MMP-9 Enables Recruitment of EPC and PPC to the GBM Vasculature
To elucidate how MMP-9+ BMDCs promote GBM angiogenesis, we analyzed whether lack of MMP-9 in bone marrow cells impacts recruitment of vascular progenitor cells and CD45+ myeloid vascular support cells. Transplant experiments with actin-GFP bone marrow cells revealed a 4-fold reduction of VEGFR2+ GFP+ EPC incorporated into tumor vessels when compared to WT-GBM (). Using two different pericyte markers (desmin and α-smooth muscle actin [α–SMA]) to detect pericytes (), we also observed about 50% less pericyte coverage in the absence of MMP-9. Upon transplantation of MMP-9ko GBM-bearing MMP-9ko mice with WT bone marrow, pericyte coverage increased, approximating the WT situation (). In contrast, when we reconstituted bone marrow of WT tumor-bearing mice with MMP-9ko bone marrow, we lowered pericyte coverage, indicating that a subpopulation of pericytes is recruited from the bone marrow in a MMP-9-dependent manner ().
CD45+ MMP-9+ BMDCs Are Sufficient to Initiate Angiogenesis in GBM
Recruitment of CD45+ Cells Is Independent of MMP-9 but Dependent on SDF1α
We then asked whether MMP-9 is also required for recruitment of the CD45+ population of BMDC in GBM. We observed similar numbers of CD45+ cells in MMP-9ko GBM and WT-GBM tumors (). By contrast, we observed an ~3-fold reduction in the number of CD45+ and F4/80+ cells in HIFko GBMs when compared to WT tumors (). Similarly, while WT-GBM and MMP-9ko GBM expressed comparable levels of SDF1α, HIFko GBM expressed severely reduced levels of SDF1α, reflective of the differing CD45+ cell influx in the respective tumors (). These data suggest that GBMs recruit CXCR4+ BMDC to the tumor site through HIF1 and its target SDF1α to facilitate new blood vessel growth.
To demonstrate the functional significance of SDF1α in neovascularization, we treated WT-GBM bearing mice with the CXCR4 inhibitor AMD3100 (Petit et al., 2007
). AMD3100 substantially reduced the recruitment of CXCR4+/CD45+ BMDC (), and tumor vessels appeared slimmer and “normalized” as observed when angiogenesis is inhibited ().
BMD-CD45+ Cells Are Sufficient to Initiate Neovascularization in GBM
Since AMD3100 also blocks CXCR4+ EPC, we sought to investigate whether the CXCR4+ monocytic population is functionally significant and sufficient in promoting the angiogenic switch. While both EPC and PPC populations were reduced in MMP-9ko GBM, we found that MMP-9ko GBM and WT-GBM contained similar numbers of CD45+ BMDC (), indicating that recruitment of these monocytic cells is independent of MMP-9 in the bone marrow. This suggested that MMP-9ko tumors were nonangiogenic, despite the unaltered levels of monocytic cells, because CD45+ cells lacked MMP-9 necessary to initiate neovascularization, whereas HIFko tumors were nonangiogenic because they had reduced numbers of CD45+ cells and, thus, insufficient MMP-9 to commence neovascularization. To assess the functional significance of MMP-9+ CD45 cells in this process, we isolated MMP-9-proficient GFP+ CD45+ cells from bone marrow of WT mice and injected them intravenously into MMP-9ko mice bearing MMP-9ko GBM (). We used MMP-9ko GFP+ CD45+ cells as controls. We confirmed by zymogram analysis that injections of MMP-9+ CD45+ cells led to MMP-9 activity in the tumor, whereas injection of MMP-9ko CD45+ cells did not (), and that tumors recruited GFP+ CD45+ MMP-9-proficient or -deficient cells at similar levels (). The tumors that attracted CD45+ MMP-9+ BMDC exhibited hyperdilated and more irregularly shaped vessels, whereas GBM that attracted CD45+ MMP-9ko cells did not alter their slim vascular anatomy (). Notably, we often detected MMP-9+ cells encircling enlarged tumor vessels (), suggesting that MMP-9 may preferentially facilitate local “activation” of sequestered VEGF. We further noticed that VEGFR2 activation occurred in tumors undergoing vascular remodeling, but not in tumors that received CD45+MMP-9ko cells (). Taken together with the results in and , these data reveal that MMP-9 expressed and secreted by a heterogeneous group of CD45+ cells from the bone marrow is sufficient to initiate angiogenesis by making sequestered VEGF bioavailable to its receptor VEGFR2.
VEGF Is a Direct and Negative Regulator of Perivascular Tumor Invasion
In view of our previous observation that genetic ablation of HIF1α or VEGF (Blouw et al., 2003
), which blocks the ability of GBM to initiate VEGF-dependent neovascularization, results in a more invasive phenotype, we reevaluated the paradigm of angiogenesis as being key to tumor progression. It is important to note that the induced invasive mode in the absence of these angiogenic factors was distinct from the invasive mode in WT-GBM because tumor cells predominantly moved along blood vessels in the brain parenchyma (Blouw et al., 2003
; present paper; white arrows). In contrast, WT-GBMs have a more infiltrative behavior in which tumor cells percolate as single cells through the brain parenchyma (, yellow arrows). Interestingly, we found that MMP-9 was expressed in a subset of these infiltrating cells, while MMP-9 was not detected in HIFko GBMs migrating along blood vessels (). These data support the hypothesis that MMP-9 facilitates infiltration of tumor cells directly but is not implicated in the perivascular invasive mode. Because MMP-9ko GBMs do not undergo vascular remodeling, we predicted that they would display a more perivascular invasive phenotype. We observed that GBM in the complete absence of MMP-9 grew more diffusively into the brain parenchyma than WT-GBM (). Notably, while WT-GBMs in a WT host and WT-GBMs in an MMP-9ko host both preferentially infiltrated into the brain parenchyma, loss of MMP-9 in the tumor cell compartment alone was sufficient to tip the balance to a more perivascular invasive mode, which was even more exaggerated in the complete absence of MMP-9 and comparable to the phenotype observed in HIFko GBM tumors (). Notably, regardless of whether HIF1α or MMP9 was completely ablated, disabling angiogenesis resulted in a perivascular invasive phenotype.
VEGF Directly Inhibits Perivascular Tumor Invasion
These results point to a specific adaptation mechanism for GBM when deprived of key angiogenic factors that drive VEGF-dependent neovascularization and suggest that the mechanisms of tumor cell infiltration and perivascular tumor cell invasion are very distinct. Since perivascular tumor invasion is negatively correlated with angiogenesis, we then tested the hypothesis that VEGF is the negative regulatory factor. Indeed, by ectopically expressing VEGF-164, the major VEGF isoform expressed in GBM, we were able to convert HIFko GBMs, which are highly invasive and produce low levels of VEGF, into noninvasive tumors with smooth borders ().
How does VEGF-induced angiogenesis block perivascular invasion? Based on several reports that tumor cells can express VEGF receptors (Lesslie et al., 2006
), we asked whether VEGF has a direct effect on the invasive behavior of GBM. Both WT-GBM and HIFko GBM cell lines expressed VEGFR1 and -2 receptors to varying extents, as well as the coreceptors neuropilin-1 (NP-1) and neuropilin-2 (NP-2) (). We then tested the ability of VEGF to affect invasive behavior of GBM cells directly in vitro in a Boyden chamber assay. Both WT-GBM and HIFko GBM cells were highly invasive in response to HGF, a stimulator of GBM invasion (Abounader and Laterra, 2005
; Eckerich et al., 2007
), but surprisingly, VEGF reduced their invasive behavior significantly (). Similarly, HIFko GBM cells over-expressing VEGF were less invasive than their parental counterparts (). VEGF alone did not stimulate or inhibit invasiveness of GBM cells. In summary, these results reveal that VEGF acts as a direct negative regulator of perivascular GBM tumor cell invasion.