Experimental in vivo tumor models are essential for comprehending the dynamic process of human cancer progression, identifying therapeutic targets, and evaluating antitumor drugs. However, current rodent models are limited by high costs, long experimental duration, variability, restricted accessibility to the tumor, and major ethical concerns. To avoid these shortcomings, we investigated whether tumor growth on the chick chorio-allantoic membrane after human glioblastoma cell grafting would replicate characteristics of the human disease. Avascular tumors consistently formed within 2 days, then progressed through vascular endothelial growth factor receptor 2-dependent angiogenesis, associated with hemorrhage, necrosis, and peritumoral edema. Blocking of vascular endothelial growth factor receptor 2 and platelet-derived growth factor receptor signaling pathways by using small-molecule receptor tyrosine kinase inhibitors abrogated tumor development. Gene regulation during the angiogenic switch was analyzed by oligonucleotide microarrays. Defined sample selection for gene profiling permitted identification of regulated genes whose functions are associated mainly with tumor vascularization and growth. Furthermore, expression of known tumor progression genes identified in the screen (IL-6 and cysteine-rich angiogenic inducer 61) as well as potential regulators (lumican and F-box-only 6) follow similar patterns in patient glioma. The model reliably simulates key features of human glioma growth in a few days and thus could considerably increase the speed and efficacy of research on human tumor progression and preclinical drug screening. angiogenesis ͉ animal model alternatives ͉ glioblastoma

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Cancer cells have complex, unique characteristics that distinguish them from normal cells, such as increased growth rates and evasion of anti-proliferative signals. Global inhibition of class I and II histone deacetylases (HDACs) stops cancer cell proliferation in vitro and has proven effective against cancer in clinical trials, at least in part, through transcriptional reactivation of the p21 WAF1/Cip1 gene. The HDACs that regulate p21 WAF1/Cip1 are not fully identified. Using small interfering RNAs, we found that HDAC4 participates in the repression of p21 WAF1/Cip1 through Sp1/Sp3-, but not p53-binding sites. HDAC4 interacts with Sp1, binds and reduces histone H3 acetylation at the Sp1/Sp3 binding site-rich p21 WAF1/Cip1 proximal promoter, suggesting a key role for Sp1 in HDAC4-mediated repression of p21 WAF1/Cip1 . Induction of p21 WAF1/Cip1 mediated by silencing of HDAC4 arrested cancer cell growth in vitro and inhibited tumor growth in an in vivo human glioblastoma model. Thus, HDAC4 could be a useful target for new anti-cancer therapies based on selective inhibition of specific HDACs.

Vascular endothelial growth factor (VEGF) inhibitors are the most promising anti‐angiogenic agents used increasingly in the clinic. However, to be efficient, anti‐VEGF agents need to be associated with classic chemotherapy. Exploring gene regulation in tumor cells during anti‐angiogenesis might help to comprehend the molecular basis of response to treatment. To generate a defined anti‐angiogenic condition in vivo, we transfected human glioma cells with short‐interfering RNAs against VEGF‐A and implanted them on the chick chorio‐allantoic membrane. Gene regulation in avascular tumors was studied using human Affymetrix™ GeneChips. Potentially important genes were further studied in glioma patients. Despite strong VEGF inhibition, we observed recurrent formation of small, avascular tumors. CHI3L2, IL1B, PI3/elafin and CHI3L1, which encodes for YKL‐40, a putative prognosticator for various diseases, including cancer, were strongly up‐regulated in avascular glioma. In glioblastoma patients, these genes showed coregulation and their expression differed significantly from low‐grade glioma. Importantly, high levels of CHI3L1 (p = 0.036) and PI3/elafin mRNA (p = 0.0004) were significantly correlated with poor survival. Cox regression analysis further confirmed that PI3 and CHI3L1 levels are survival markers independent from patient age and sex. Elafin‐positive tumor cells were only found in glioblastoma, where they were clustered around necrotic areas. PI3/elafin is strongly induced by serum deprivation and hypoxia in U87 glioma cells in vitro. Our results indicate that anti‐angiogenesis in experimental glioma drives expression of critical genes which relate to disease aggressiveness in glioblastoma patients. In particular, CHI3L1 and PI3/elafin may be useful as new prognostic markers and new therapeutic targets. © 2007 Wiley‐Liss, Inc.

The tumour suppressor p53, involved in DNA repair, cell cycle arrest and apoptosis, also inhibits blood vessel formation, that is, angiogenesis, a process strongly contributing to tumour development. The p53 gene expresses 12 different proteins (isoforms), including TAp53 (p53 (or p53a), p53b and p53g) and D133p53 isoforms (D133p53a, D133p53b and D133p53g). The D133p53a isoform was shown to modulate p53 transcriptional activity and is overexpressed in various human tumours. However, its role in tumour progression is still unexplored. In the present study, we examined the involvement of D133p53 isoforms in tumoural angiogenesis and tumour growth in the highly angiogenic human glioblastoma U87. Our data show that conditioned media from U87 cells depleted for D133p53 isoforms block endothelial cell migration and tubulogenesis without affecting endothelial cell proliferation in vitro. The D133p53 depletion in U2OS osteosarcoma cells resulted in a similar angiogenesis blockade. Furthermore, using conditioned media from U87 cells ectopically expressing each D133p53 isoform, we determined that D133p53a and D133p53g but not D133p53b, stimulate angiogenesis. Our in vivo data using the chicken chorio-allantoic membrane and mice xenografts establish that angiogenesis and growth of glioblastoma U87 tumours are inhibited upon depletion of D133p53 isoforms. By TaqMan low-density array, we show that alteration of expression ratio of D133p53 and TAp53 isoforms differentially regulates angiogenic gene expression with D133p53 isoforms inducing pro-angiogenic gene expression and repressing anti-angiogenic gene expression.

Biological activities of vascular endothelial growth factor (VEGF) have been studied extensively in endothelial cells (ECs), but few data are available regarding its effects on pericytes. In murine embryoid body cultures, VEGF-induced expression of desmin and ␣-smooth muscle actin (␣-SMA) in CD-31 ϩ cells. The number of CD-31 ϩ /desmin ϩ vascular chords increased with VEGF treatment time and peaked during a differentiation window between 6 and 9 days after plating. In vivo, VEGF-induced elongation and migration of desmin-positive pericytes and coverage of angiogenic capillaries, as revealed by analysis of Sambucus nigra lectin-stained vascular beds of the chick chorioallantoic membrane. VEGF also caused significant decrease of intercapillary spaces, an indicator for intussusceptive vascular growth. These VEGF-mediated effects point at a more intricate interaction between ECs and pericytes cells than previously demonstrated and suggest that pericytes may be derived from EC progenitors in vitro and not only stabilize capillaries but also participate in vascular remodeling in vivo.

Fibroblast growth factors (FGFs) are potent stimulators of angiogenesis in vitro and in vivo. However, the precise role of FGFs and vascular development in normal and pathological tissue has long remained ill defined. Recently, substantial progress has been made toward a better understanding of their role. Genetic studies in mice or in culture systems indicate a role for FGFs in vessel assembly and sprouting. FGFs also stimulate blood vessel branching and lymphangiogenesis. The molecular mechanisms by which FGFs mediate angiogenesis are also better understood. Finally, the FGF/FGF-receptor system has become a focus for the development of novel therapeutic strategies for the treatment of angiogenesis-related diseases such as tissue ischemia.

Interleukin‐6 (IL6) and vascular endothelial growth factor (VEGFA) are abundantly produced by glioma cells and contribute to malignancy by promoting angiogenesis, cell proliferation and resistance to apoptosis. We compared the effect of inhibiting IL6 and VEGF on U87‐derived experimental glioma grown on the chick chorio‐allantoic membrane (CAM) or in the brain of xenografted mice. Tumor growth was monitored by biomicroscopy and immunohistology. In vitro, IL6 knockdown had no effect on proliferation but substantially enhanced invasion. In the CAM experimental glioma, IL6 or VEGF knockdown reduced growth and vascularization of the tumors with a comparable efficiency, but increased invasion of residual tumor cells. In contrast, combined IL6/VEGF knockdown not only showed enhanced reduction of tumor growth and angiogenesis but also significantly prevented invasion of residual tumor cells. In mice, combining IL6 knockdown and Avastin™ treatment completely abrogated tumor development and infiltration. Molecular response of tumor cells to single or combined treatment was studied by transcriptomic profiling. Many cell cycle promoting genes and chromatin components were silenced in the double knockdown. In addition, specific migratory signatures detected in tumors under single IL6 or VEGF knockdown were partially erased in combined IL6/VEGF knockdown tumors. Our results show that treatment with a combination of IL6 and VEGF inhibitors brings synergistic antitumoral benefit and reduces global activity of major pathways of cell survival, proliferation and invasiveness in remaining tumor cells that may be induced by using VEGF or IL6 inhibitors alone. © 2009 UICC