William Leenders

Antiangiogenic therapy of cerebral melanoma metastases results in sustained tumor progression via vessel co-option

Purpose: In the brain, tumors may grow without inducing angiogenesis, via co-option of the dense pre-existent capillary bed. The purpose of this study was to investigate how this phenomenon influences the outcome of antiangiogenic therapy. Experimental Design: Mice carrying brain metastases of the human, highly angiogenic melanoma cell line Mel57-VEGF-A were either or not treated with different dosages of ZD6474, a vascular endothelial growth factor (VEGF) receptor 2 tyrosine kinase inhibitor with additional activity against epidermal growth factor receptor. Effect of treatment was evaluated using contrast-enhanced magnetic resonance imaging (CE- MRI) and (immuno)morphologic analysis. Results: Placebo-treated Mel57-VEGF-A brain metastases evoked an angiogenic response and were highlighted in CE-MRI. After treatment with ZD6474 (100 mg/kg), CE-MRI failed to detect tumors in either prevention or therapeutic treatment regimens. However, (immuno)histologic analysis revealed the presence of numerous, small, nonangiogenic lesions. Treatment with 25 mg/kg ZD6474 also resulted in efficient blockade of vessel formation, but it did not fully inhibit vascular leakage, thereby still allowing visualization in CE-MRI scans. Conclusions: Our data show that, although angiogenesis can be effectively blocked by ZD6474, in vessel-dense organs this may result in sustained tumor progression via co-option, rather than in tumor dormancy. Importantly, blocking VEGF-A may result in undetectability of tumors in CE-MRI scans, leading to erroneous conclusions about therapeutic efficacy during magnetic resonance imaging follow-up. The maintenance of VEGF-A-induced vessel leakage in the absence of neovascularization at lower ZD6474 doses may be exploited to improve delivery of chemotherapeutic agents in combined treatment regimens of antiangiogenic and chemotherapeutic compounds.

Concerns about anti-angiogenic treatment in patients with glioblastoma multiforme

BackgroundThe relevance of angiogenesis inhibition in the treatment of glioblastoma multiforme (GBM) should be considered in the unique context of malignant brain tumours. Although patients benefit greatly from reduced cerebral oedema and intracranial pressure, this important clinical improvement on its own may not be considered as an anti-tumour effect.DiscussionGBM can be roughly separated into an angiogenic component, and an invasive or migratory component. Although this latter component seems inert to anti-angiogenic therapy, it is of major importance for disease progression and survival. We reviewed all relevant literature. Published data support that clinical symptoms are tempered by anti-angiogenic treatment, but that tumour invasion continues. Unfortunately, current imaging modalities are affected by anti-angiogenic treatment too, making it even harder to define tumour margins. To illustrate this we present MRI, biopsy and autopsy specimens from bevacizumab-treated patients.Moreover, while treatment of other tumour types may be improved by combining chemotherapy with anti-angiogenic drugs, inhibiting angiogenesis in GBM may antagonise the efficacy of chemotherapeutic drugs by normalising the blood-brain barrier function.SummaryAlthough angiogenesis inhibition is of considerable value for symptom reduction in GBM patients, lack of proof of a true anti-tumour effect raises concerns about the place of this type of therapy in the treatment of GBM.

Vessel Co-Option: How Tumors Obtain Blood Supply in the Absence of Sprouting Angiogenesis

The hypothesis that solid tumors are dependent on angiogenesis, the formation of new vessels, for outgrowth and metastasis has acquired a central position in cancer research and has since inspired many scientists for several decades. Among the various angiogenic stimuli that are secreted by tumor cells, members of the Vascular Endothelial Growth Factor (VEGF) family are most prominent. More recently it has become clear, however, that tumors may use alternative ways to obtain blood supply. Vessel co-option, the use of pre-existent vessels, was described first in the brain, one of the most densely vascularized organs in the body. Thus, brain tumors may develop without the need of an angiogenic switch to occur. Obviously, this way of blood supply will not be affected by angiogenesis inhibition. In addition, it is predicted that tumors with this type of behavior will be less visible in contrast-enhanced MRI. In this article we present our recently developed mouse brain model of vessel co-option in melanoma. The effects of expression of VEGF on tumor vascularity, and on MRI visualization of these brain lesions are described. Possible consequences of anti-angiogenesis therapy are discussed.

The endogenous anti-angiogenic VEGF isoform, VEGF165b inhibits human tumour growth in mice.

Vascular endothelial growth factor-A is widely regarded as the principal stimulator of angiogenesis required for tumour growth. VEGF is generated as multiple isoforms of two families, the pro-angiogenic family generated by proximal splice site selection in the terminal exon, termed VEGFxxx, and the anti-angiogenic family formed by distal splice site selection in the terminal exon, termed VEGFxxxb, where xxx is the amino acid number. The most studied isoforms, VEGF165 and VEGF165b have been shown to be present in tumour and normal tissues respectively. VEGF165b has been shown to inhibit VEGF- and hypoxia-induced angiogenesis, and VEGF-induced cell migration and proliferation in vitro. Here we show that overexpression of VEGF165b by tumour cells inhibits the growth of prostate carcinoma, Ewings sarcoma and renal cell carcinoma in xenografted mouse tumour models. Moreover, VEGF165b overexpression inhibited tumour cell-mediated migration and proliferation of endothelial cells. These data show that overexpression of VEGF165b can inhibit growth of multiple tumour types in vivo indicating that VEGF165b has potential as an anti-angiogenic, anti-tumour strategy in a number of different tumour types, either by control of VEGF165b expression by regulation of splicing, overexpression of VEGF165b, or therapeutic delivery of VEGF165b to tumours.

Antiangiogenic compounds interfere with chemotherapy of brain tumors due to vessel normalization

Glioblastomas are highly aggressive primary brain tumors. Curative treatment by surgery and radiotherapy is generally impossible due to the presence of diffusely infiltrating tumor cells. Furthermore, the blood-brain barrier (BBB) in infiltrative tumor areas is largely intact, and this hampers chemotherapy as well. The occurrence of angiogenesis in these tumors makes these tumors attractive candidates for antiangiogenic therapies. Because antiangiogenic compounds have been shown to synergize with chemotherapeutic compounds in other tumor types, based on vessel normalization, there is a tendency toward such combination therapies for primary brain tumors also. However, vessel normalization in brain may result in restoration of the BBB with consequences for the efficacy of chemotherapeutic agents. In this study, we investigated this hypothesis. BALB/c nude mice with intracerebral xenografts of the human glioblastoma lines E98 or U87 were subjected to therapy with different dosages of vandetanib (an angiogenesis inhibitor), temozolomide (a DNA alkylating agent), or a combination (n > 8 in each group). Vandetanib selectively inhibited angiogenic growth aspects of glioma and restored the BBB. It did not notably affect diffuse infiltrative growth and survival. Furthermore, vandetanib antagonized the effects of temozolomide presumably by restoration of the BBB and obstruction of chemodistribution to tumor cells. The tumor microenvironment is an extremely important determinant for the response to antiangiogenic therapy. Particularly in brain, antiangiogenic compounds may have adverse effects when combined with chemotherapy. Thus, use of such compounds in neuro-oncology should be reconsidered. [Mol Cancer Ther 2008;7(1):71–8]

Vascular Endothelial Growth Factor and Semaphorin Induce Neuropilin-1 Endocytosis via Separate Pathways

The neuropilin (Nrp)1 receptor is essential for both nervous and vascular system development. Nrp1 is unusually versatile, because it transmits both chemoattractive and repulsive signals in response to vascular endothelial growth factor (VEGF)-A and class 3 semaphorins, respectively. Both Nrp1 and VEGF receptor 2 undergo ligand-dependent endocytosis. We sought to establish the endocytic pathway of Nrp1 and to determine whether uptake is required for its signaling. Whereas Nrp1 underwent clathrin-dependent endocytosis in response to VEGFA165 treatment, semaphorin 3C (sema3C) induced lipid raft–dependent endocytosis. The myosin VI PDZ (postsynaptic density 95, Disk large, Zona occludens-1) adaptor protein synectin was essential for Nrp1 trafficking. Sema3C failed to inhibit migration of synectin−/− endothelial cells, mirroring the lower migratory response of these cells to VEGFA165. These results show that the endocytic pathway of Nrp1 is determined by its ligand and that the trafficking of Nrp1 is essential for its signaling.

Vascular endothelial growth factor-A determines detectability of experimental melanoma brain metastasis in GD-DTPA-enhanced MRI.

We have previously shown that the dense vascular network in mouse brain allows for growth of human melanoma xenografts (Mel57) by co‐option of preexisting vessels. Overexpression of recombinant vascular endothelial growth factor‐A (VEGF‐A) by such xenografts induced functional and morphologic alterations of preexisting vessels. We now describe the effects of VEGF‐A expression on visualization of these brain tumors in mice by magnetic resonance imaging (MRI), using gadolinium diethylenetriaminepenta‐acetic acid (Gd‐DTPA) and ultra small paramagnetic iron oxide particles (USPIO) as contrast agents. Brain lesions derived from (mock‐transfected) Mel57 cells were undetectable in MRI after Gd‐DTPA injection. However, the majority of such lesions became visible after injection of USPIO, due to the lower vascular density in the lesions as compared to the surrounding parenchyma. In contrast, VEGF‐A‐expressing lesions were visualized using Gd‐DTPA‐enhanced MRI by a rapid circumferential enhancement, due to leaky peritumoral vasculature. USPIO‐enhanced MRI of these tumors corroborated the immunohistochemic finding that peritumorally located, highly irregular and dilated vessels were present, while intratumoral vessel density was low. Our study shows that VEGF‐A is a key factor in imaging of brain neoplasms. Our data also demonstrate that, at least in brain, blood‐pool agent‐enhanced MRI may be a valuable diagnostic tool to detect malignancies that are not visible on Gd‐DTPA‐enhanced MRI. Furthermore, the involvement of VEGF‐A in MRI visibility suggests that care must be taken with MRI‐based evaluation of antiangiogenic therapy, as anti‐VEGF treatment might revert a tumor to a co‐opting phenotype, resulting in loss of contrast enhancement in MRI.

Plexin D1 Expression Is Induced on Tumor Vasculature and Tumor Cells: A Novel Target for Diagnosis and Therapy?

We previously reported that during mouse embryogenesis, plexin D1 (plxnD1) is expressed on neuronal and endothelial cells. Endothelial cells gradually loose plxnD1 expression during development. Here we describe, using in situ hybridization, that endothelial plxnD1 expression is regained during tumor angiogenesis in a mouse model of brain metastasis. Importantly, we found PLXND1 expression also in a number of human brain tumors, both of primary and metastatic origin. Apart from the tumor vasculature, abundant expression was also found on tumor cells. Via panning of a phage display library, we isolated two phages that carry single-domain antibodies with specific affinity towards a PLXND1-specific peptide. Immunohistochemistry with these single-domain antibodies on the same tumors that were used for in situ hybridization confirmed PLXND1 expression on the protein level. Furthermore, both these phages and the derived antibodies specifically homed to vessels in brain lesions of angiogenic melanoma in mice after i.v. injection. These results show that PLXND1 is a clinically relevant marker of tumor vasculature that can be targeted via i.v. injections.

Phenotypic and genotypic characterization of orthotopic human glioma models and its relevance for the study of anti-glioma therapy.

Most human gliomas are characterized by diffuse infiltrative growth in the brain parenchyma. Partly because of this characteristic growth pattern, gliomas are notorious for their poor response to current therapies. Many animal models for human gliomas, however, do not display this diffuse infiltrative growth pattern. Furthermore, there is a need for glioma models that represent adequate genocopies of different subsets of human gliomas (e.g., oligodendrogliomas). Here, we assessed the intracerebral growth patterns and copy number changes [using multiplex ligation‐dependent probe amplification (MLPA)/comparative genomic hybridization (CGH)] of 15 human glioma lines in nude mice. Most xenografts present with compact growing lesions intracerebrally. Only the E98 and, to a lesser degree, E106 xenograft lines (propagated through subcutaneous growth) consistently produced intracerebral tumors, displaying diffuse infiltrative growth in the brain parenchyma. In contrast, four xenograft lines (E434, E468, E473 and E478), established by direct intracerebral inoculation of human glioma cells and serially propagated intracerebrally, consistently showed extensive diffuse infiltration throughout the brain. After several passages, the neoplastic cells still carry typical chromosomal aberrations [(−1p/−19q in oligodendroglioma, +7/−10 in glioblastoma multiforme (GBM)]. Especially these latter four models and the E98 line thus represent adequate geno‐ and phenocopies of human gliomas and form an attractive platform to investigate different therapeutic approaches in a preclinical setting.

Specific imaging of VEGF-A expression with radiolabeled anti-VEGF monoclonal antibody.

Vascular endothelial growth factor‐A (VEGF‐A) is one of the most important angiogenic factors. Here, we studied in a nude mouse model whether the expression of VEGF‐A in a tumor could be imaged with a radiolabeled anti‐VEGF antibody. The humanized anti‐VEGF‐A antibody A.4.6.1. (bevacizumab), which is reactive with all VEGF‐A isoforms, was radiolabeled with In‐111 or with I‐125. The accumulation of the radiolabeled antibodies in VEGF‐A expressing tumors (LS174T) in nude mice was examined in biodistribution studies and by gamma camera imaging. The uptake of the In‐111‐bevacizumab in the tumor at 3 days p.i. was significantly higher than that of I‐125‐bevacizumab (19.4 ± 7.0 %ID/g vs. 9.6 ± 3.3 %ID/g, p = 0.04). Coinjection of an excess unlabeled antibody resulted in a significant decrease in radioactivity concentration in the tumor (<2.9 ± 1.9 %ID/g, p < 0.005), indicating VEGF‐mediated antibody uptake. Highest uptake in the tumor was observed at relatively low antibody protein doses (<3 μg) (20–25 %ID/g). VEGF‐A‐expressing tumors could be clearly visualized on planar scintigraphic images from 24‐hr post injection onwards. In conclusion, VEGF‐A expression in tumors can be visualized specifically with radiolabeled anti‐VEGF‐A‐mAb.