Tumor Vascular Density Research Articles

Secreted protein, acidic and rich in cysteine (SPARC) expression in astrocytic tumour cells negatively correlates with proliferation, while vascular SPARC expression is associated with patient survival

Secreted protein, acidic and rich in cysteine (SPARC) is a regulator of cell-matrix interaction and has been associated with tumour stage and patient survival in various malignancies. As no large-scale study has yet been undertaken, we investigated human brain and astrocytomas for SPARC expression and associations with tumour grade, proliferation, vascular density and patient survival. A spectrum of 188 WHO grade I-IV astrocytic tumours and 24 autopsy cases were studied by immunohistochemistry for SPARC, MIB-1 proliferation index and CD31-positive vessels. SPARC protein expression was confirmed by quantitative real-time polymerase chain reaction and Western blot in 13 cases. In normal brain, SPARC is expressed in cortical marginal glia, cerebellar Bergmann glia and focally in white matter but is absent in neurones or vessels. High SPARC expression levels in the cytoplasm of astrocytic tumour cells decreased with the grade of malignancy but showed an increase with grade of malignancy in tumour vessels. SPARC negatively correlated with tumour proliferation but not with vascular density. While cytoplasmic SPARC staining was not associated with survival, vascular SPARC showed a significant association in the group of grade II-IV tumours (P = 0.02) and also in grade II astrocytomas alone (P = 0.01) with vascular SPARC associated with worse prognosis. SPARC is highly expressed in astrocytomas and decreases with tumour progression. We confirm an association of increased SPARC expression and decreased proliferation. While there is no association between the level of SPARC in the tumour cells and patient survival, increased tumour vascular SPARC expression is associated with decreased patient survival.

The Antiangiogenic Activity in Xenograft Models of Brivanib, a Dual Inhibitor of Vascular Endothelial Growth Factor Receptor-2 and Fibroblast Growth Factor Receptor-1 Kinases

Tumor angiogenesis is a complex and tightly regulated network mediated by various proangiogenic factors. The fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) family of growth factors, and associated tyrosine kinase receptors have a major influence in tumor growth and dissemination and may work synergistically to promote angiogenesis. Brivanib alaninate is the orally active prodrug of brivanib, a selective dual inhibitor of FGF and VEGF signaling. Here, we show that brivanib demonstrates antitumor activity in a broad range of xenograft models over multiple dose levels and that brivanib alaninate shows dose-dependent efficacy equivalent to brivanib in L2987 human tumor xenografts. Brivanib alaninate (107 mg/kg) reduced tumor cell proliferation as determined by a 76% reduction in Ki-67 staining and reduced tumor vascular density as determined by a 76% reduction in anti-CD34 endothelial cell staining. Furthermore, Matrigel plug assays in athymic mice showed that brivanib alaninate inhibited angiogenesis driven by VEGF or basic FGF alone, or combined. Dynamic contrast-enhanced magnetic resonance imaging, used to assess the effects of brivanib alaninate on tumor microcirculation, showed a marked decrease in gadopentetate dimeglumine contrast agent uptake at 107 mg/kg dose, with a reduction in area under the plasma concentration-time curve from time 0 to 60 minutes at 24 and 48 hours of 54% and 64%, respectively. These results show that brivanib alaninate is an effective antitumor agent in preclinical models across a range of doses, and that efficacy is accompanied by changes in cellular and vascular activities.

Abstract B12: The antitumor and antiangiogenic activity of brivanib, a dual inhibitor of VEGFR-2 and FGFR-1 kinases

Abstract Angiogenesis plays a central supporting role in tumorigenesis in a wide variety of different cancer types. This complex and highly regulated process has been shown to be driven by various proangiogenic factors. The vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) family of growth factors in conjunction with their associated tyrosine kinase receptors exert a major influence on tumor growth and dissemination, and potentially work synergistically to promote angiogenesis. Brivanib alaninate (BMS-582664) which is currently in phase III clinical trails is the orally active prodrug of brivanib (BMS-540215), a selective dual inhibitor of VEGF and FGF signaling. We demonstrate here that in vivo brivanib provides antitumor activity in a broad range of xenograft models over multiple dose levels, and that brivanib alaninate demonstrates dose-dependent efficacy equivalent to brivanib. Brivanib alaninate also reduces tumor cell proliferation as determined by a significant reduction in Ki-67 staining as well as a decrease in tumor vascular density as reflected by a reduction in CD34 staining, a cellsurface marker of endothelial cells. Additionally, using Matrigel™ plug assays in athymic mice we demonstrated that brivanib alaninate inhibits angiogenesis driven by VEGF or bFGF alone as well as when both of these cytokines are combined to drive angiogenesis in this model. Moreover, brivanib was more effective in inhibiting angiogenesis in this model system under all these conditions when compared to either bevacizumab, an antibody directed at the human VEGF cytokine or DC101, an antibody directed at the murine form of VEGF receptor-2. Dynamic contrast-enhanced magnetic resonance imaging, used to assess the effects of brivanib alaninate on tumor microcirculation, demonstrated a marked decrease in contrast agent uptake at an efficacious dose level, with a reduction in area under the plasma concentration-time curve from time 0 to 60 min (AUC60) at 24 h and 48 h of 54% and 64%, respectively. These results demonstrate that brivanib alaninate is an effective antitumor agent in preclinical tumor and angiogenesis models across a range of doses, and that efficacy is accompanied by changes in cellular and vascular activities. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B12.

Heparanase expression by Barrett's epithelium and during esophageal carcinoma progression

Enzymatic activity responsible for the cleavage of heparan sulfate, commonly known as heparanase, is abundant in tumor-derived cells. Heparanase cleaves heparan sulfate side chains, presumably at sites of low sulfation, thus facilitating structural alterations of the extracellular matrix and basement membrane underlying epithelial and endothelial cells. Traditionally, heparanase activity was correlated with the metastatic potential of tumor-derived cells, attributed to enhanced cell dissemination as a consequence of heparan sulfate cleavage and remodeling of the extracellular matrix barrier. More recently, heparanase upregulation was documented in an increasing number of human carcinomas and hematological malignancies, correlating with increased tumor metastasis, vascular density, and shorter post-operative survival of cancer patients. Although heparanase upregulation and its pro-malignant features are well documented, the instance of its induction in the course of tumor development was less investigated. Here, we used immunohistochemical analysis to investigate heparanase expression in normal esophagus, Barrett's esophagus without dysplasia, Barrett's esophagus with low-grade dysplasia, Barrett's esophagus with high-grade dysplasia, and adenocarcinoma of the esophagus. We report that heparanase expression is already induced in Barrett's epithelium without dysplasia, and is further increased during progression through distinct pathological stages, namely, low-grade dysplasia, high-grade dysplasia, and adenocarcinoma. Notably, heparanase induction correlated with increased cell proliferation index revealed by Ki-67 staining. These findings suggest that heparanase function is not limited to the process of tumor metastasis, but rather is engaged at the early stages of esophagus carcinoma initiation and progression.

Sonographic Assessment of Tumor Response

The advent of antiangiogenic drugs in cancer therapy necessitates an imaging modality that can longitudinally assess posttreatment changes in tumor vasculature. In this regard, microbubble contrast-enhanced ultrasonography (CEUS) offers several advantages over conventional imaging modalities. The small size of microbubbles (approximately 2-3 mum) permits their retention in the intravascular compartment and travel through the tortuous tumor vasculature. Mathematical models applied to signal intensity versus time depicting the kinetics of microbubble flow through the tumor are used to characterize tumor vascular density, blood flow velocity, and perfusion. In vivo studies using CEUS have demonstrated its comparability to dynamic contrast-enhanced magnetic resonance imaging and fluorodeoxyglucose positron emission tomography in distinguishing between diseased or malignant tissue and normal tissue. Moreover, CEUS has great potential for other novel clinical applications such as improved cancer diagnosis, enhanced medication delivery, and early antiangiogenic cancer treatment response evaluation. This review discusses the principles and potential clinical applications of CEUS in determining tumor response and its promising role in enhancing medication delivery in certain tumors.

PET of EGFR Antibody Distribution in Head and Neck Squamous Cell Carcinoma Models

Epidermal growth factor receptor (EGFR) is a well-characterized protooncogene that has been shown to promote tumor progression in solid cancers. Clinical results for EGFR targeting with specific monoclonal antibodies (mAbs) such as cetuximab and panitumumab are promising; however, most studies indicate that only a subgroup of patients receiving the mAbs benefit from the immunotherapy, independent of EGFR expression level. To understand the in vivo kinetics of antibody delivery and localization, we performed small-animal PET studies with (64)Cu-labeled panitumumab in xenografts derived from 3 cell lines of human head and neck squamous cell carcinoma (HNSCC). Nude mice bearing HNSCC tumors with different levels of EGFR expression were imaged with small-animal PET using (64)Cu-1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA)-panitumumab. Antibody distribution in the tumors was confirmed by ex vivo immunostaining using panitumumab and fluorescein 5(6)-isothiocyanate (FITC) panitumumab. CD31 immunostaining and Evans blue assay were also performed to assess the tumor vascular density and permeability. Among these 3 tumor models, UM-SCC-22B tumors with the lowest EGFR protein expression showed the highest (64)Cu-DOTA-panitumumab accumulation, whereas SQB20 tumors with the highest EGFR expression showed the lowest (64)Cu-DOTA-panitumumab accumulation. Ex vivo staining demonstrated that SQB20 cells still had extremely high EGFR expression after forming tumors in nude mice, indicating that the low uptake of (64)Cu-DOTA-panitumumab in SQB20 tumors was not due to the loss of EGFR expression. The results from CD31 immunostaining and Evans blue permeability assay suggest that the low vessel density, poor vascular permeability, and binding site barrier are likely responsible for the overall low tumor uptake of the highly EGFR-expressing SQB20 tumors. The results from this study provide a possible explanation for the lack of an observed correlation between therapeutic efficacy of cetuximab and panitumumab and EGFR expression level as determined by immunohistochemistry or fluorescent in situ hybridization and may shed new light on the complications of anti-EGFR mAb therapy for HNSCC and other malignancies.

D004 Pharmacological blockade of angiotensin II type 2 receptor inhibits tumor growth decreasing cell proliferation and tumor vascularization

Angiotensin II type 2 receptor (AT2R) stimulation is thought to induce vasodilation and apoptosis. Nevertheless, its role remains controversial. In hypertension and aging, AT2R might promote vasoconstriction and cell growth. In cancer cells, AT2R is highly expressed but its role remains poorly understood. Thus, we investigated the role of AT2R in tumor growth, hypothesizing that AT2R affects cell proliferation and/or tumor vascularization. Tumor induction by 3-methylcholanthrene was significantly delayed in AT2R knockout mice, suggesting a role for AT2R in tumor initiation. Tumor growth after subcutaneous injection of LL/2 cancer cells in C57/BL6 mice treated with PD123,319, an AT2R antagonist, was decreased when PD123,319 was given at an early stage, suggesting that AT2R is also involved in tumor promotion. In vitro proliferation of LL/2 cells was reduced by PD123,319 with a significant decrease in Ki67 expression and ERK1/2 phosphorylation. In addition, tumor vascular density, measured by CD31 labeling and angiogenesis using the aortic ring assay on Matrigel®, were reduced by PD123,319 and in AT2R-null mice. Therefore, we uncovered a novel mechanism by which AT2R promotes tumor development favoring both malignant cancer cell proliferation and tumor angiogenesis. Thus, blocking AT2R could be a new approach in tumor therapy.

Vasculostatin Inhibits Intracranial Glioma Growth and Negatively RegulatesIn vivoAngiogenesis through a CD36-Dependent Mechanism

Angiogenesis is a critical physiologic process that is appropriated during tumorigenesis. Little is known about how this process is specifically regulated in the brain. Brain angiogenesis inhibitor-1 (BAI1) is a brain-predominant seven-transmembrane protein that contains five antiangiogenic thrombospondin type-1 repeats (TSR). We recently showed that BAI1 is cleaved at a conserved proteolytic cleavage site releasing a soluble, 120 kDa antiangiogenic factor called vasculostatin (Vstat120). Vstat120 has been shown to inhibit in vitro angiogenesis and suppress subcutaneous tumor growth. Here, we examine its effect on the intracranial growth of malignant gliomas and further study its antitumor mechanism. First, we show that expression of Vstat120 strongly suppresses the intracranial growth of malignant gliomas, even in the presence of the strong proangiogenic stimulus mediated by the oncoprotein epidermal growth factor receptor variant III (EGFRvIII). This tumor-suppressive effect is accompanied by a decrease in tumor vascular density, suggesting a potent antiangiogenic effect in the brain. Second, and consistent with this interpretation, we find that treatment with Vstat120 reduces the migration of cultured microvascular endothelial cells in vitro and inhibits corneal angiogenesis in vivo. Third, we show that these antivascular effects critically depend on the presence of the cell surface receptor CD36 on endothelial cells in vitro and in vivo, supporting the role of Vstat120 TSRs in mediating these effects. These results advance the understanding of brain-specific angiogenic regulation, and suggest that Vstat120 has therapeutic potential in the treatment of brain tumors and other intracerebral vasculopathies.

The temporal correlation of dynamic contrast-enhanced magnetic resonance imaging with tumor angiogenesis in a murine glioblastoma model

Objective: Glioblasomta multiforme (GBM) is a WHO grade IV malignant brain tumor with poor prognosis, despite advances in surgical and adjuvant therapy. GBM is characterized by areas of central necrosis and high levels of angiogenesis, during which increased vascular permeability allows for the extravasation of endothelial progenitor cells to support blood vessel and tumor growth. The purpose of this study was to characterize changes in tumor vascular permeability, vascular density and vessel morphology in vivo during angiogenesis.Methods: An orthotropic murine (GL26) glioblastoma model was used in this study. in vivo serial dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) in combination with histologic and molecular genetic analyses was performed to correlate in vivo imaging of vascular development.Results: DCE-MRI revealed a significant change in tumor vessel permeability dependent upon tumor progression and size. Time to max signal intensity displayed a stepwise increase between days 21 and 24 (p<0.05), a critical period before exponential tumor growth during which a significant increase in tumor vascular density and vessel caliber is observed on histology. Furthermore, quantitative real-time PCR revealed a corollary increase in angiogenic signaling molecules before the observed changes on DCE-MRI.Discussion: In vivo changes of orthotopic glioma blood vessel permeability as shown by DCE-MRI correlates with histologic quantification of vascular density and vessel caliber as well as with the molecular expression of angiogenic factors. DCE-MRI is a useful tool for non-invasive in vivo monitoring of angiogenesis in pre-clinical tumor models.

Effect of combined therapy with the antiestrogen agent toremifene and local hyperthermia on breast cancer cells implanted in nude mice

We evaluated the effects of combined treatment with the antiestrogen agent toremifene (TOR) and local hyperthermia (LHT) on the MCF-7 breast cancer cell line. BALB/c mice implanted with MCF-7 cells were divided into six treatment groups: a control group, a TOR30 group (given 30 mg/kg/day), a TOR120 group (given 120 mg/kg/day), an LHT group (43.5 degrees C), a TOR30 + LHT group, and a TOR120 + LHT group. The effects of the treatments on tumor cells, estrogen receptor (ER) expression, and cell cycle kinetics were measured after 21 days. We calculated the apoptotic index and vascular density inside the tumors and evaluated the efficacy of the transmigration of TOR into the tumors. The antitumor effects were significantly greater in both combined therapy groups than in any of the single therapy groups. Estrogen receptor expression was weaker in the combined therapy groups than in the single therapy groups, and there were more G0/G1-phase cells and fewer S-phase cells in both combined therapy groups than in the single therapy groups. The apoptotic index was increased and the tumor vascular density was decreased in the combined therapy groups. We attributed the effects of this combined therapy to the induction of apoptosis, the decrease in vascular density, and the increase and decrease in G0/G1-phase and S-phase cells, respectively, in the tumors.

The presence of tumor-infiltrating FOXP3 + lymphocytes correlates with intratumoral angiogenesis in endometrial cancer

Objectives CD4 +CD25 + regulatory T-cells (Tregs), that express the transcription factor FOXP3, suppress effector T-cell populations and can enable tumour cells to evade the host immune response. In this study, we investigated the numbers of FOXP3 + Tregs in the normal and malignat endometrium and examined potential links with tumor angiogenesis. Methods Paraffin-embedded tissues from 79 patients with stage I endometrial adenocarcinoma and 12 samples from normal endometrium were analyzed using immunohistochemistry for the detection of FOXP3 + lymphocytes. The presence of FOXP3 + lymphocytic infiltration was correlated with the tumor vascular density, the hypoxia inducible factors HIF-1α and HIF-2α, VEGF, estrogen and progesterone receptor expression. Survival analysis was also performed. Results In normal endometrium, FOXP3 was expressed by stroma infiltrating lymphocytes, with a mean number 8 (range 5–11) lymphocytes per ×100 optical field. In tumors, 55/79 (69.6%) cases showed little FOXP3 + lymphocytic infiltration (0–2 per ×100 optical field). In the remaining 24/79 (30.4%) cases that were scored as positive the mean score ranged from 3–8 (median 5). Low numbers of FOXP3 + lymphocytes significantly correlated with tumoral ER negativity and low vascular density. Survival analysis showed no significant impact of FOXP3 lymphocytic infiltration, although there was a trend towards worse prognosis. Conclusions The correlation between the presence of FOXP3 + Tregs and high vessel density in endometrial adenocarcinomas suggests a link between immunity, intratumoral angiogenesis and poor prognosis. However, further studies are required as significantly fewer Tregs were detected in the tumor microenvironment compared to normal endometrium.

Relationship Between Retention of a Vascular Endothelial Growth Factor Receptor 2 (VEGFR2)-Targeted Ultrasonographic Contrast Agent and the Level of VEGFR2 Expression in an In Vivo Breast Cancer Model

The aim of this study was to characterize the relationship between retention of a vascular endothelial growth factor receptor 2 (VEGFR2)-targeted ultrasonographic contrast agent (UCA) and VEGFR2 expression in tumor vasculature of breast cancer. 67NR breast cancer tumors implanted in mice were evaluated in vivo with both VEGFR2-targeted and nontargeted UCAs, and a high-frequency ultrasound system. A bolus of the UCA was injected and allowed to circulate for 4 minutes to allow binding of targeted microbubbles. After that, 2 sets of images before and after a high-power ultrasonic destruction sequence were acquired. The average video intensity of predestruction and postdestruction images was measured and used as a relative measure of retention of the UCA in the tumor. Levels of VEGFR2 expression and tumor vascular density were quantified by immunohistochemical staining and compared with retention of the VEGFR2-targeted UCA. Retention of VEGFR2-targeted microbubbles in tumors was significantly higher than retention of nontargeted microbubbles (mean +/- SD, 47.75+/-9.85 versus 18.5+/-5.46 dB; P< .001). Retention of the VEGFR2-targeted UCA was found to correlate with the level of VEGFR2 expression in the studied tumors (r(2)=0.41). In contrast, retention of the nontargeted UCA was not correlated with the level of VEGFR2 expression (r(2)=0.08). Furthermore, retention of the VEGFR2-targeted UCA was not correlated with the level of tumor vascularity. The magnitude of the molecular ultrasonographic signal from a VEGFR2-targeted UCA retained by tissue correlates with VEGFR2 expression. These results validate the use of molecular ultrasonography for in vivo detection and quantification of VEGFR2 expression in this breast cancer model.

Immunohistochemical Evaluation of Giant Cell Tumors of the Jaws Using CD34 Density Analysis

To compare CD34 expression in both aggressive and nonaggressive giant cell lesions of the jaws and identify any associations between tumor vascular density and biologic behavior. This was a retrospective study of subjects treated for giant cell lesions of the jaws at Massachusetts General Hospital from 1992 to 2006. The primary predictor variable was tumor classification (aggressive or nonaggressive); tumors were considered aggressive if they were greater than 5 cm in size, recurred after treatment, or exhibited 3 of the following: presence of root resorption, tooth displacement, or cortical bone thinning or perforation. Secondary predictor variables, recorded for each patient, were demographic, anatomic, and clinical measures. The outcome variable was the average CD34 staining density of histologic specimens quantified in 2 different areas. Descriptive and bivariate statistics were computed to identify predictors associated with vascular density. The study sample was composed of 32 subjects with a mean age of 24.4 +/- 19.77 years (range: 2-83); 23 subjects (71.8%) were female. Of the tumors included, 11 (34.4%) were located in the maxilla, 21 (65.6%) in the mandible. Twenty-six tumors (81.2%) were classified as aggressive; the remainder (18.8%) were nonaggressive. There were no statistically significant differences between subjects with aggressive versus nonaggressive tumors with regard to age, gender, or location. Subjects with aggressive tumors had a significantly higher CD34 staining density (P = .02). None of the secondary predictors was associated with vascular density. Vascular density of giant cell tumors of the jaws is significantly increased in aggressive tumors.

Tissue Inhibitor of Metalloproteinase-3 via Oncolytic Herpesvirus Inhibits Tumor Growth and Vascular Progenitors

Malignant solid tumors remain a significant clinical challenge, necessitating innovative therapeutic approaches. Oncolytic viral therapy is a nonmutagenic, biological anticancer therapeutic shown to be effective against human cancer in early studies. Because matrix metalloproteinases (MMP) play important roles in the pathogenesis and progression of cancer, we sought to determine if "arming" an oncolytic herpes simplex virus (oHSV) with an MMP-antagonizing transgene would increase virus-mediated antitumor efficacy. We generated oHSVs that express human tissue inhibitor of metalloproteinases 3 (TIMP3) or firefly luciferase and designated them rQT3 and rQLuc, respectively. We evaluated the antitumor efficacy of these viruses against neuroblastoma and malignant peripheral nerve sheath tumor (MPNST) xenografts. Relative to rQLuc, rQT3-infected primary human MPNST and neuroblastoma cells exhibited equivalent virus replication but increased cytotoxicity and reduced MMP activity. In vivo, rQT3-treated tumors showed delayed tumor growth, increased peak levels of infectious virus, immature collagen extracellular matrix, and reduced tumor vascular density. Remarkably, rQT3 treatment reduced circulating endothelial progenitors, suggesting virus-mediated antivasculogenesis. We conclude that rQT3 enhanced antitumor efficacy through multiple mechanisms, including direct cytotoxicity, elevated virus titer, and reduced tumor neovascularization. These findings support the further development of combined TIMP-3 and oncolytic virotherapy for cancer.

Tumor Necrosis Factor α Mediates Homogeneous Distribution of Liposomes in Murine Melanoma that Contributes to a Better Tumor Response

Successful treatment of solid tumors with chemotherapeutics requires that adequate levels reach the tumor cells. Tumor vascular normalization has been proposed to enhance drug delivery and improve tumor response to chemotherapy. Differently, augmenting leakage of the tumor-associated vasculature, and as such enhance vascular abnormality, may improve tumor response as well. In the present study, we show that addition of low-dose tumor necrosis factor alpha (TNF) to systemic injections with pegylated long circulating liposomes augmented the tumor accumulation of these liposomes 5- to 6-fold, which strongly correlated with enhanced tumor response. Using intravital microscopy, we could study the liposomal distribution inside the tumor in more detail. Especially 100 nm liposomes effectively extravasate in the surrounding tumor tissue in the presence of TNF and this occurred without any effect on tumor vascular density, branching, and diameter. Next to that, we observed in living animals that tumor cells take up the liposomes intact, followed by intracellular degradation. To our knowledge, this is an unprecedented observation. Taken together, TNF renders more tumor vessels permeable, leading to a more homogeneous distribution of the liposomes throughout the tumor, which is crucial for an optimal tumor response. We conclude that delivery of nanoparticulate drug formulations to solid tumor benefits from augmenting the vascular leakage through vascular manipulation with vasoactive drugs like TNF.

Outcome prediction for renal cell carcinoma: evaluation of prognostic factors for tumours divided according to histological subtype

A wide variety of parameters have been investigated for their prognostic significance in mixed series of renal cell carcinoma (RCC). The classification of RCC into separate types with differing morphology, genotype and probable clinical outcome has led to a re-evaluation of many prognostic parameters with studies confined to a single RCC morphotype. Tumour stage remains the most important predictor of RCC outcome and recent investigations have focused upon tumour diameter and the prognostic significance of stromal, vascular and lymphatic invasion within the renal sinus. In large tumour series, morphotype has been correlated with patient survival, with clear cell RCC being associated with a less favourable outcome than chromophobe RCC and to a lesser extent papillary RCC, for organ confined tumours. The prognostic significance of nuclear grading remains controversial. Fuhrman grading has been shown to have prognostic utility for clear cell RCC in some series. Recent studies have shown that for papillary RCC, grading should be based upon nucleolar size and that Fuhrman grading is inappropriate for chromophobe RCC. Proliferative indices based upon a variety of markers have been correlated with outcome for clear cell RCC (Ki-67, AgNORs, p21(waf1/cip1) and p27(Kip1)) and papillary RCC (Ki-67, AgNORs), although in some series prognostic significance was lost on multivariate analysis. The presence of tumour necrosis has been shown to predict survival for clear cell and chromophobe RCC, and in clear cell RCC quantification of tumour vascular density has been correlated with outcome. Several molecular markers have been investigated for prognostic significance, mostly in clear cell RCC. Although some of these markers have been shown to be significantly associated with survival, these findings remain to be confirmed in large scale follow-up studies.

Ferumoxtran-10 enhancement in orthotopic xenograft models of human brain tumors: an indirect marker of tumor proliferation?

Ferumoxtran-10 belongs to the Ultra Small Particles of Iron Oxide (USPIO) class of contrast agents and induces delayed tumor enhancement in brain tumors, reflecting the trapping of iron oxide particles by the macrophages and activated microglia. The aim of the study was to compare Ferumoxtran-10 contrast enhancement in four human high-grade glioma xenograft models (TCG2, TCG3, TCG4, and U87) with different growing profiles. Fragments of human malignant glioma were orthotopically xenografted into the brain of four groups of nude mice. All mice underwent a MRI examination 24 h after intravenous administration of Ferumoxtran-10 (axial T1 SE weighted MR images). The contrast enhancement observed in the different tumor types was measured and was correlated to in vivo tumor growth and to histological parameters, such as proliferative tumor cell fraction, apoptosis, vascular density, and Perls' staining score. A good relationship was observed: (a) between tumor-to-background contrast and proliferative index, (b) between tumor-to-background contrast and tumor growth, and (c) between tumor-to-background contrast and Perls' staining score. The registered MR enhancement contrasts were not influenced by apoptotic index and by vascular density in these experimental xenografts. Tumor contrast enhancement 24 h after intravenous Ferumoxtran-10 administration seems to be well correlated to tumor proliferative index and tumor growth and could be used as an indirect marker of tumor proliferation.

A comparison of the vascular density of VEGF expression with microvascular density determined with CD34 and CD31 staining and conventional prognostic markers in renal cell carcinoma

The aim of this study was to compare the vascular density of Vascular endothelial growth factor (VEGF) expression with microvascular density determined by CD34 and CD31 with conventional prognostic parameters. The study involved 50 renal cell carcinoma (RCC) cases. VEGF, CD34, and CD31 were stained by immunohistochemistry, and then preparations were evaluated by two pathologists under light microscopy. The whole tumor area was scrutinized in all the sections. In the evaluation of VEGF, due to the lack of homogenous staining within the tumor, two parameters, distribution and intensity of expression, were evaluated semiquantitively. In the evaluation of microvascular density with CD34 and CD31 staining, three hot areas with the highest density were determined. In x200 magnification of these areas, on a single plane, the quantity of vascular structures with lumens was determined. Intensity of VEGF Expression was higher in papillary type carcinoma of kidney parenchyma (P = 0.014) and it was significantly correlated with tumor stage (P = 0.013), survival time (P = 0.01), and tumor size (P = 0.035). Distribution of VEGF expression was also higher in papillary RCC (P = 0.055) and it was significantly correlated with tumor stage (P = 0.043) and tumor size (P = 0.039). Vascular density determined with CD34 staining was higher in conventional RCC (P < 0.05); in addition, it was significantly correlated with distribution and intensity of VEGF expression (P < 0.05) and tumor stage (P < 0.05). Vascular density determined with CD31 staining was not significantly correlated with tumor type, tumor stage, nuclear grade, and survival time. Intensity and distribution of VEGF were higher in papillary RCC. Both parameters were significantly correlated with tumor size, stage, and vascular density determined with CD34 staining. Intensity of VEGF was also significantly correlated with capsule invasion. Vascular density determined with CD34 staining, however, was higher in conventional RCC, and it was correlated with tumor size and stage.

Inhibitory Effects of Castration in an Orthotopic Model of Androgen-Independent Prostate Cancer Can Be Mimicked and Enhanced by Angiogenesis Inhibition

Today, the most important treatment of advanced prostate cancer is castration; unfortunately, however, the long-term effect of this therapy is insufficient. Recent studies suggest that castration-induced prostate involution could be caused by primary effects in the prostate vasculature; therefore, we examined if antivascular treatments could mimic the effects of castration. Androgen-independent AT-1 prostate cancer cells were grown inside the ventral prostate in adult rats. Tumor-bearing animals were treated with an inhibitor of vascular endothelial growth factor receptor 2 and epidermal growth factor receptor signaling, N-(4-bromo-2-fluorophenyl)-6-methoxy-7-[(1-methylpiperidin-4-yl)methoxy]quinazolin-4-amine (ZD6474, AstraZeneca, Södertälje, Sweden), and short-term effects (after 3 days) were compared with those induced by castration. Castration caused decreased vascular density in the normal tissue surrounding the tumor and consequently increased tumor hypoxia and apoptosis, and moderately decreased tumor growth. ZD6474 treatment resulted in decreased tumor vascular density accompanied by increased tumor hypoxia, apoptosis, and decreased tumor growth, suggesting that castration and antiangiogenic therapy work through similar mechanisms. Interestingly, castration or ZD6474 alone worked by reducing vascular density in the surrounding normal tissue and ZD6474 also in the tumor. Combined treatment with castration + ZD6474 was more effective than castration and ZD6474 alone in inducing tumor hypoxia, apoptosis, necrosis, and decreasing tumor vascular density. These findings show that a drug that targets the vasculature in the tumor and in the surrounding ventral prostate lobe could mimic and even enhance the effects of castration. Our present findings thus suggest that castration + ZD6474 could be a particularly effective way to treat prostate tumors.

Vascular Endothelial Growth Factor Tyrosine Kinase Inhibitor AZD2171 and Fractionated Radiotherapy in Mouse Models of Lung Cancer

The vascular endothelial growth factor receptor (VEGFR) tyrosine kinases are being explored as targets for antiangiogenic cancer therapy. Radiotherapy also inhibits tumor growth and affects vasculature. We investigated the combination of the potent VEGFR tyrosine kinase inhibitor AZD2171 and ionizing radiation in cell culture and mouse models of lung cancer. We show that ionizing radiation induces expression of phosphorylated VEGFR-2 (Flk-1) in endothelial cells and that this phosphorylation is inhibited by AZD2171. Human umbilical vascular endothelial cells become more sensitive to radiation after treatment with AZD2171 as determined by clonogenic assay. Matrigel assay showed a decrease in in vitro endothelial tubule formation with AZD2171/radiation combination treatment. When similar combination was applied to the H460 lung cancer xenograft model in nude mice, loss of radiation-induced phosphorylated Flk-1 was observed in the combination treatment group, which also showed a large decrease in tumor vascular density by staining of the von Willebrand factor. H460 tumor growth delay was enhanced in the combination treatment group compared with the groups treated with AZD2171 or radiation alone. Additionally, after therapy, Ki67 index showed >4-fold reduction of tumor proliferation in the combination therapy group, which also showed increased intratumoral apoptotic index by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining. In conclusion, AZD2171 sensitizes lung tumor xenografts to radiation and inhibits angiogenesis both in vitro and in vivo. When used as a radiation enhancer, AZD2171 has the potential to improve tumor growth delay by inhibiting tumor proliferation and promoting apoptosis. Clinical trials are needed to determine the potential of this combination therapy in patients with locally advanced lung cancer.