Funded Grants

Increasing the radiocurability of GBM by inhibition of tumor vasculogenesis

We propose to test a novel hypothesis that we believe will improve the cure rate of glioblastoma multiforme (GBM). Radiotherapy is a proven treatment mode for this tumor but despite efforts to increase the dose to the tumor or to sensitize the tumor cells to radiation, failure leading to patient death is common. Significantly most of the recurrences occur within the radiation field (1), At first sight it is difficult to understand how the large doses given locally to brain tumors do not result in cures because even though all of the tumor cells may not be sterilized by radiation, it is unlikely that any of the endothelial cells comprising the blood vessels in the tumor could survive the large radiation doses given. However, it is now well established that tumor blood vessel growth occurs by two means: 1) by the classical mechanism of angiogenesis involving sprouting and in-growth from adjacent normal vessels, and 2) by vasculogenesis produced by recruitment of bone marrow cells into the tumor that are capable of growing blood vessels. We hypothesize that the large radiation doses given to GBM and the immediately surrounding normal brain will severely restrict and probably eliminate the first of these mechanisms because, in general, the normal tissues immediately surrounding the tumor receive a dose similar to that of the tumor. This dose would be expected to kill essentially all the endothelial cells in and around the tumor and thus eliminate angiogenesis. Thus, we hypothesize that tumor regrowth following large doses of radiation will depend largely, if not entirely, on vasculogesis from bone marrow derived cells, and that eliminating this pathway will markedly increase tumor curability by radiation. Importantly this could only be true for radiotherapy (as opposed to surgery or chemotherapy) because of the massive local cell killing by radiotherapy, which is likely to sterilize the vasculature in and immediately surrounding the tumor. Data in the literature and our own preliminary studies suggest that the bone marrow derived cells that infiltrate tumors following radiation are in the monocyte/macrophage lineage, and that they contribute to post-irradiation survival and growth of tumor cells by promoting the tumor vasculature. Our preliminary studies with several human tumor xenografts including U251 GBM and mouse GBM have shown the following:
  1. Depletion of monocytes from the bone marrow of bone marrow transplanted mice both inhibits growth of endothelial cells in vitro and markedly enhances the radiocurability of transplanted tumors in those mice.
  2. Irradiation doses of 15-35 Gy produce a rapid and massive influx of bone marrow derived host cells of the monocyte/macrophage lineage, and these cells express large amounts of the pro-angiogenic cytokine MMP-9 in the tumors.
  3. It is known that mobilization and trafficking of bone marrow derived cells to sites of tissue injury is regulated by low oxygen levels (hypoxia) and the induction of the hypoxia inducible factor, HIF-1(2). Importantly we have now shown that local tumor irradiation of HIF-1 knockout GBM implanted intracranially have a much greater prolongation of life than do HIF-1 wild-type tumors. This is consistent with our hypothesis that HIF-1 controls vasculogenesis and that inhibiting this will sensitize tumors to radiation.
To test our hypothesis we propose to use both a mouse derived GBM tumor (transformed astocytes) and a human GBM (U251) orthotopically transplanted in the brains of immunodeficient (nu/nu) mice. As we have established both these models with fractionated irradiation protocols that mimic both clinical practice and therapeutic outcome, we anticipate no technical problems in performing these studies. We will evaluate the contribution of MMP-9 to tumor radiosensitivity and host cell infiltration using combinations of tumor cells with and without MMP-9 transplanted into hosts with and without MMP-9 with transplantation of bone marrow from mice with and without MMP-9. We will also evaluate the contribution to tumor response to fractionated irradiation of the cytokine stromal derived factor 1 (SDF-1) which has been shown to be crucial in the homing and maintenance of bone marrow derived cells in tumor vascular (3, 4). We will do this using appropriate neutralizing antibodies and inhibitors.

Importantly we will evaluate clinically relevant strategies for inhibition of MMP-9 and SDF-1 using the inhibitors zoledronic acid and AMD3100 respectively, both of which are approved for clinical use, applied during and following fractionated local irradiation to orthotopic GBM in mice. Targeting both the local tumor by irradiation and vasculogenesis by inhibiting MMP-9 and/or SDF-1 is a novel paradigm and could lead to a major increase in the curability of GBM by radiotherapy.

1. Sneed PK, Gutin PH, Larson DA, et al. Patterns of recurrence of glioblastoma multiforme after external irradiation followed by implant boost. Int J Radiat Oncol Biol Phys 1994;29(4):719- 27.
2. Ceradini DJ, Kulkarni AR, Callaghan MJ, et al. Progenitor cell trafficking is regulated by hypoxic gradients through HIF-1 induction of SDF-1. Nat Med 2004;10(8):858-64.
3. Aghi M, Cohen KS, Klein RJ, Scadden DT, Chiocca EA. Tumor stromal-derived factor-1 recruits vascular progenitors to mitotic neovasculature, where microenvironment influences their differentiated phenotypes. Cancer Res 2006;66(18):9054-64.
4. Jin DK, Shido K, Kopp HG, et al. Cytokine-mediated deployment of SDF-1 induces revascularization through recruitment of CXCR4+ hemangiocytes. Nat Med 2006;12(5):557-67.