Dr. Bergers presented work on two topics. The first dealt with the recruitment of pericyte progenitor cells from bone marrow and their differentiation into more mature pericytes in tumor blood vessels. By way of background, the normal microvasculature is comprised of two types of cells, endothelial cells (the cells lining the blood vasculature), and perictyes, cells that surround and tightly envelop endothelial cells. Pericytes are thought to be necessary for the health of endothelial cells and so of the blood microvasculature [21
]. Tumors, however, include many blood vessels that lack, or have a reduced coating of pericytes, e.g. mother vessels (see Dvorak presentation and ). Further, treating mice bearing the Rip1Tag2 tumor at later stages of growth with imatinib, a tyrosine kinase inhibitor that binds to the PDGF receptor (PDGFR) succeeded in regressing tumor vessels by causing pericytes to detach. These data emphasize the importance of pericytes in maintaining the tumor vasculature. However, the nature and source of tumor vessel pericytes has been little investigated. To address this question, the Bergers lab observed that, as in the developing vasculature, PDGFRβ+cells enveloped the blood vessels of Rip1Tag2 tumors and that the PDGF ligands B and D for PDGFRβ were expressed by tumor endothelial cells [23
]. This suggested that a paracrine communication pathway between pericytes and endothelial cells might be operative in tumors, as in normal vascular development. Mature pericytes are known to express additional markers, namely, NG2, αSMA, and desmin. However, in tumors, not all tumor PDGFRβ vessel enveloping cells bore these mature pericyte markers. Flow cytometry and immunohistochemical studies revealed three distinct types of such cells: PDGFRβ+ NG2− cells, PDGFRβ+ NG2+ cells, and PDGFRβ−+ NG2+ cells. Thus, in Rip1Tag2 tumors, only a subset of PDGFRβ+ pericytes expressed mature pericyte markers and a subset of mature pericytes did not express detectable levels of PDGFRβ likely reflecting distinct differentiation stages. Indeed, further work demonstrated that PDGFRβ NG2− cells represent a population of progenitor pericytes, some of which are recruited from the bone marrow and bear markers (e.g., Sca1, CD45) characteristic of hematopoietic stem cells [23
]. When PDGFRβ cells were mixed with endothelial cells in three-dimensional cultures, endothelial cells formed tubes with pericytes attaching, particularly at branch points. In addition, these pericytes differentiated, acquiring markers of mature pericytes (NG2, αSMA and desmin). These studies demonstrated that tumor blood vessel pericytes derive, at least in part from hematopoietic bone marrow progenitors and that progenitor pericytes undergo maturation when apposed to endothelial cells. Part of the maturation effect, but only that leading to αSMA expression, could be replicated by exposing percyte progenitors to TGF-β.
A second series of studies demonstrated that VEGF is a negative regulator of cell invasion in the case of glioblastoma multiforme (GBM), rapidly growing and highly aggressive grade IV astrocytomas (brain tumors) [24
]. GBM are characterized by zones of necrosis and hypoxia and, as a result, typically express high levels of HIF-1 and downstream angiogenic factors such as VEGF, angiopoietin-2 and SDF-1α [25
]. They disrupt the blood-brain barrier and are characterized by extensive edema and an influx of inflammatory cells. GBM spread with several distinct dispersion patterns: subpial spread, white matter infiltration, perivascular spread, and ventricular spread.
To investigate the functional significance of hypoxia and angiogenesis in astrocytoma progression, initial studies were performed with transformed astrocytes genetically engineered from murine primary culture astrocytes in which the hypoxia-responsive transcription factor HIF-1α or its target gene, the angiogenic factor VEGF, was deleted [28
]. Genetic deletion of VEGF blocked tumor angiogenesis and increased vascular cell apoptosis, but, paradoxically, increased tumor invasion. When HIF-1α was knocked out in GBM cells, the new vessels that formed remained slim and regularly shaped, more closely resembling those of the normal brain vasculature. However, HIF-1α k/o GBM adapted to their inability to grow new blood vessels by co-opting and moving along preexisting blood vessels, a phenomenon described as perivascular spread, and were actually more invasive than wild type GBM cells [28
Matrix metalloproteases (MMP), a large family of zinc-dependent endopeptidases, have been implicated in many aspects of tumor growth and progression. Of these, MMP-2 has been particularly implicated in tumor angiogenesis. To investigate the role of MMP-2 in GBM tumor cell survival and invasion, genetically engineered MMP-2 knockout GBM cells were prepared and their growth properties studied in MMP-2 knockout (k/o) mice [29
]. Wild type GBM cells grew as invasive, highly angiogenic tumors with a leaky, tortuous vasculature and with hypoxic centers. In contrast, GBM-MMP-2k/o cells developed a markedly increased vasculature. Paradoxically, however, the tumor cells grew slower and were more prone to apoptosis and the mice exhibited a longer mean survival time. Apparently the dense and highly branched network of tumor blood vessels induced by these cells were not able to support tumor growth. In support of this hypothesis, tumor vessels exhibited substantially less VEGFR-2, pericytes were greatly reduced, and vessels were poorly perfused. Also, the pattern of tumor cell invasion was different in the absence of MMP-2. MMP-2 k/o GBM grew more diffusely by migrating along preexisting blood vessels into the brain parenchyma, a pattern also observed when both HIF-1 and VEGF deficient GBM were implanted in brain (such tumors did not induce proper neovascularization). Apparently, MMP-2 acts as a negative regulator of vascular patterning and angiogenesis in GBM. Investigating the mechanisms for these findings, HIF-1α expressed by GBM cells was found to induce SDF1α, and, in this way recruit bone marrow-derived CD45+ myeloid cells containing Tie2+, VEGFR1+, CD11b+, and F4/80+ subpopulations, as well as endothelial and pericyte progenitor cells to promote neovascularization [29
]. Further, MMP-9 activity of bone marrow derived CD45+ cells was found to be sufficient and essential to initiate angiogenesis by increasing VEGF bioavailability (releasing it from its bound state to matrix or cells). Conversely, in the absence of HIF-1α, SDF1α levels decreased and fewer bone-marrow derived cells were recruited to tumors, thus decreasing MMP-9 and mobilization of VEGF.
Finally, it was found that VEGF is a direct and negative regulator of tumor cell invasion [29
]. VEGF reduced the ability of VEGFR-expressing GBM cells to migrate and invade in vitro and in vivo. When VEGF activity was impaired, tumor cells invaded deep into the brain in the perivascular compartment. Further support for this finding has come from studies in which tumors were subjected to anti-angiogenesis therapies aimed at targeting the VEGF pathway. GBM and Rip1Tag2 tumors treated with tyrosine kinase inhibitors targeting VEGFR-2 showed initial vascular dropout and tumor stasis, only to be followed by a tumor adaptive-evasive response, mediated by other growth factors such as fibroblast growth factor and leading to augmented invasion and, in some cases, dissemination and distant metastasis [30
]. Obviously these findings in animal models have implications for treating cancer patients with anti-angiogenic drugs, and particularly those targeting the VEGF pathway.