Identifying and overcoming glioblastoma resistance to treatments targeting vascular endothelial growth factor
Despite advances in surgery, chemotherapy, and radiation therapy, the median survival for patients with glioblastoma remains poor, unchanged at 12-15 months over the past decade. Indeed, over the past 35 years, there have been only two chemotherapies, implantable carmustine-containing Gliadel wafers and temozolomide, that have been FDA approved for the treatment of glioblastoma, and these two agents only showed modest survival benefits in phase III clinical trials. This lack of progress underscores the importance of developing a better understanding of the unique biology of glioblastoma and designing novel therapeutic regimens based on this unique biology.
Recognition of the role of rich vascularity in glioblastoma growth and resistance to treatments has led to phase II clinical trials of agents targeting vascular endothelial growth factor (VEGF) or its receptors in order to reduce the number of glioma blood vessels. VEGF is an appealing therapeutic target because VEGF is a growth factor secreted by tumor cells that has a nourishing effect on endothelial cells, which form the inner lining of blood vessels. The phase II clinical trials of these VEGF-targeting drugs have shown brief initial responses in some patients followed by resumption of tumor growth consistent with acquired resistance to these VEGF-targeting agents.
In attempting to identify mechanisms of glioblastoma resistance to VEGF-targeting agents, it must be recognized that tumors including glioblastomas have recently been shown by us1 and others2"4 to form blood vessels by two means: (1) angiogenesis, the usurping of local vessels that are then rerouted towards the tumor; or (2) vasculogenesis, the derivation of new blood vessels by recruiting circulating bone marrow-derived endothelial progenitor cells (EPCs) into the tumor, where they differentiate into new mature endothelium, leading to the formation of new blood vessels. We have shown that glioblastoma secretion of stromal derived factor-1 (SDF-1) leads to the recruitment of circulating EPCs from the circulation into the tumor, a key step in vasculogenesis.1
We hypothesize that therapies that target VEGF only target angiogenesis, and that glioblastomas that develop resistance to these VEGF targeting agents do so by upregulating their use of vasculogenesis to form blood vessels, enabling them to maintain the vasculature they need to overcome VEGF-targeting treatments.
Testing of our hypothesis will be done through both retrospective and prospective analysis of surgically resected glioblastomas that have recurred after developing resistance to avastin, a VEGF-neutralizing antibody that has undergone phase II clinical trials in patients with recurrent glioblastoma and has been used to treat 60 patients with recurrent glioblastoma at our institution.
In our first specific aim, we will retrospectively analyze glioblastomas that underwent a first surgery to resect glioblastoma, then were treated with avastin and demonstrated an initial response, followed by glioblastoma regrowth suggestive of avastin resistance, leading to a second surgery for glioblastoma re-resection. We have identified 20 such cases, each with blood and glioblastoma tissue from both surgeries, before and after the development of avastin resistance, stored in the UCSF Brain Tumor Research Center (BTRC) for research purposes under informed consent. We will begin by comparing the blood of these patients before and after the development of avastin resistance, looking for biomarkers predictive of glioblastoma resistance to avastin such as increased numbers of circulating EPCs or increased plasma SDF-1. While the ultimate goal of this proposal is to develop a therapeutic regimen capable of causing glioblastoma regression, identifying these serum biomarkers predictive of avastin resistance may also eventually enable clinicians to determine whether glioblastoma patients being treated with VEGF-targeted therapy have developed resistance to these therapies before they develop radiographic and clinical findings of glioblastoma growth, enabling the implementation of different therapies in these patients in a timely fashion. After a thorough genomic and transcriptional comparison of each of the 20 paired pre- and post-avastin treated ghoblastomas, we will then identify changes in these ghoblastomas that could lead to avastin resistance, with a particular emphasis on changes that could lead to increased vasculogenesis.
In our second specific aim, we will prospectively identify ghoblastomas undergoing resection after developing avastin resistance. Fresh glioblastoma tissue taken from these cases, and from recurrent ghoblastomas not treated with avastin, will be analyzed in vitro
and in vivo
. The in vitro
analysis will determine if avastin resistance is associated with a higher fraction of stem cells, the recently identified self renewing cells that can recapitulate the original glioblastoma5 and have been shown in our unpublished data to secrete more SDF-1, a stimulus of the vasculogenesis that we hypothesize causes VEGF-targeted therapy resistance, than mature glioblastoma cells. The in vivo
analysis will involve implanting the fresh glioblastoma tissue into the brains of immunodeficient mice in which the bone marrow-derived cells will selectively express the marker green fluorescent protein (GFP) due to a bone marrow transplant from transgenic mice expressing GFP in all tissues. The portion of blood vessels whose endothelium expresses GFP will then be measured to assess vasculogenesis in these tumors. We hypothesize that the avastin resistant ghoblastomas will exhibit more vasculogenesis, as indicated by a larger portion of GFP expressing endothelium, than non-avastin treated ghoblastomas.
Finally, in our third specific aim, we will determine if combining avastin with AMD3100, a small molecule inhibitor of the SDF-1 receptor that we have shown inhibits tumor vasculogenesis,1 inhibits the growth of avastin-resistant ghoblastomas implanted intracranially into immunodeficient mice by inhibiting both angiogenesis and vasculogenesis.
Thus, this proposal draws on the unique strengths of the principal investigator as both a neurosurgeon and scientist. In particular, the first two specific aims will transition from the bedside to the bench by taking glioblastoma tissue from the operating room to the laboratory in order to enhance our understanding of glioblastoma biology, especially the mechanisms by which ghoblastomas form blood vessels and how this biology leads to resistance to drugs that attempt to reduce blood vessel formation by targeting VEGF. The third specific aim will then begin the transition back from the bench to the bedside by investigating the efficacy in vivo of a novel glioblastoma therapeutic regimen that we believe will cause glioblastoma devascularization and regression by inhibiting both angiogenesis and vasculogenesis.
1. Aghi M, Cohen KS, Klein RJ, et al: Tumor stromal-derived factor-1 recruits vascular progenitors to mitotic neovasculature, where microenvironment influences their differentiated phenotypes. Cancer Res 66:9054-9064, 2006
2. Garcia-Barros M, Paris F, Cordon-Cardo C, et al: Tumor response to radiotherapy regulated by endothelial cell apoptosis. Science 300:1155-1159, 2003
3. Lyden D, Hattori K, Dias S, et al: Impaired recruitment of bone-marrow-derived endothelial and hematopoietic precursor cells blocks tumor angiogenesis and growth. Nat Med 7:1194-1201, 2001
4. Ruzinova MB, Schoer RA, Gerald W, et al: Effect of angiogenesis inhibition by Id loss and the contribution of bone-marrow-derived endothelial cells in spontaneous murine tumors. Cancer Cell 4:277-289, 2003
5. Singh SK, Hawkins C, Clarke ID, et al: Identification of human brain tumour initiating cells. Nature 432:396-401, 2004