Neoadjuvant sorafenib treatment of clear cell renal cell carcinoma and release of circulating tumor fragments.

BACKGROUND: Clear cell renal cell carcinoma (ccRCC) is characterized by high constitutive vascular endothelial growth factor A (VEGF-A) production that induces a specific vascular phenotype. We previously reported that this phenotype may allow shedding of multicellular tumor fragments into the circulation, possibly contributing to the development of metastasis. Disruption of this phenotype through inhibition of VEGF signaling may therefore result in reduced shedding of tumor fragments and improved prognosis. To test this hypothesis, we investigated the effect of neoadjuvant sorafenib treatment on tumor cluster shedding. PATIENTS AND METHODS: Patients with renal cancer (n = 10, of which 8 have ccRCC) received sorafenib for 4 weeks before tumor nephrectomy. The resection specimens were perfused, and the perfundate was examined for the presence of tumor clusters. Effects of the treatment on the tumor morphology and overall survival were investigated (follow-up of 2 years) and compared with a carefully matched control group. RESULTS: Neoadjuvant sorafenib treatment induced extensive ischemic tumor necrosis and, as expected, destroyed the characteristic ccRCC vascular phenotype. In contrast to the expectation, vital groups of tumor cells with high proliferation indices were detected in postsurgical renal venous outflow in 75% of the cases. Overall survival of patients receiving neoadjuvant treatment was reduced compared to a control group, matched with regard to prognostic parameters. CONCLUSIONS: These results suggest that neoadjuvant sorafenib therapy for ccRCC does not prevent shedding of tumor fragments. Although this is a nonrandomized study with a small patient group, our results suggest that neoadjuvant treatment may worsen survival through as yet undefined mechanisms.

Circulating tumour tissue fragments in patients with pulmonary metastasis of clear cell renal cell carcinoma.

Tumour metastasis is the result of a complex sequence of events, including migration of tumour cells through stroma, proteolytic degradation of stromal and vessel wall elements, intravasation, transport through the circulation, extravasation and outgrowth at compatible sites in the body (the 'seed and soil' hypothesis). However, the high incidence of metastasis from various tumour types in liver and lung may be explained by a stochastic process as well, based on the anatomical relationship of the primary tumour with the circulation and mechanical entrapment of metastatic tumour cells in capillary beds. We previously reported that constitutive VEGF-A expression in tumour xenografts facilitates this type of metastatic seeding by promoting shedding of multicellular tumour tissue fragments, surrounded by vessel wall elements, into the circulation. After transport through the vena cava, such fragments may be trapped in pulmonary arteries, allowing them to expand to symptomatic lesions. Here we tested whether this process has clinical relevance for clear cell renal cell carcinoma (ccRCC), a prototype tumour in the sense of high constitutive VEGF-A expression. To this end we collected and analysed outflow samples from the renal vein, directly after tumour nephrectomy, in 42 patients diagnosed with ccRCC. Tumour fragments in venous outflow were observed in 33% of ccRCC patients and correlated with the synchronous presence or metachronous development of pulmonary metastases (p < 0.001, Fisher's exact test). In patients with tumours that, in retrospect, were not of the VEGF-A-expressing clear cell type, tumour fragments were never observed in the renal outflow. These data suggest that, in ccRCC, a VEGF-A-induced phenotype promotes a release of tumour cell clusters into the circulation that may contribute to pulmonary metastasis.

Semaphorin 3E expression correlates inversely with Plexin D1 during tumor progression.

Plexin D1 (PLXND1) is broadly expressed on tumor vessels and tumor cells in a number of different human tumor types. Little is known, however, about the potential functional contribution of PLXND1 expression to tumor development. Expression of semaphorin 3E (Sema3E), one of the ligands for PLXND1, has previously been correlated with invasive behavior and metastasis, suggesting that the PLXND1-Sema3E interaction may play a role in tumor progression. Here we investigated PLXND1 and Sema3E expression during tumor progression in cases of melanoma. PLXND1 was not expressed by melanocytic cells in either naevi or melanomas in situ, whereas expression increased with invasion level, according to Clark's criteria. Furthermore, 89% of the metastatic melanomas examined showed membranous PLXND1-staining of tumor cells. Surprisingly, expression of Sema3E was inversely correlated with tumor progression, with no detectable staining in melanoma metastasis. To functionally assess the effects of Sema3E expression on tumor development, we overexpressed Sema3E in a xenograft model of metastatic melanoma. Sema3E expression dramatically decreased metastatic potential. These results show that PLXND1 expression during tumor development is strongly correlated with both invasive behavior and metastasis, but exclude Sema3E as an activating ligand.

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.

Cysts of PRKCSH mutated polycystic liver disease patients lack hepatocystin but express Sec63p.

Polycystic liver disease (PCLD) is an inherited disorder caused by mutations in either PRKCSH (hepatocystin) or SEC63 (Sec63p). However, expression patterns of the implicated proteins in diseased and normal liver are unknown. We analyzed subcellular and cellular localization of hepatocystin and Sec63p using cell fractionation, immunofluorescence, and immunohistochemical methods. Expression patterns were assessed in fetal liver, PCLD liver, and normal adult liver. We found hepatocystin and Sec63p expression predominantly in the endoplasmic reticulum. In fetal tissue, there was intense expression of hepatocystin in ductal plate, bile ducts, and hepatocytes. However, Sec63p staining was prominent in early hepatocytes only and weak in bile ducts throughout development. In PCLD tissue, hepatocystin was expressed in hepatocytes, bile ducts, and in cyst epithelium of patients negative for PRKCSH mutation. In contrast, the majority of cysts from PRKCSH mutation carriers did not express hepatocystin. Sec63p expression was observed in all cyst epithelia regardless of mutational state. We conclude that hepatocystin is probably required for development of bile ducts and does not interact with Sec63p. The results support the hypothesis that cyst formation in PCLD results from a cellular recessive mechanism involving loss of hepatocystin. Cystogenesis in SEC63-associated PCLD occurs via a different mechanism.

Magnetic resonance imaging-based detection of glial brain tumors in mice after antiangiogenic treatment.

Proper delineation of gliomas using contrast-enhanced magnetic resonance imaging (CE-MRI) poses a problem in neuro-oncology. The blood brain barrier (BBB) in areas of diffuse-infiltrative growth may be intact, precluding extravasation and subsequent MR-based detection of the contrast agent gadolinium diethylenetriaminepenta-acetic acid (Gd-DTPA). Treatment with antiangiogenic compounds may further complicate tumor detection as such compounds can restore the BBB in angiogenic regions. The increasing number of clinical trials with antiangiogenic compounds for treatment of gliomas calls for the development of alternative imaging modalities. Here we investigated whether CE-MRI using ultrasmall particles of iron oxide (USPIO, Sinerem) as blood pool contrast agent has additional value for detection of glioma in the brain of nude mice. We compared conventional T1-weighted Gd-DTPA-enhanced MRI to T2*-weighted USPIO-enhanced MRI in mice carrying orthotopic U87 glioma, which were either or not treated with the antiangiogenic compound vandetanib (ZD6474, ZACTIMA). In untreated animals, vessel leakage within the tumor and a relatively high tumor blood volume resulted in good MRI visibility with Gd-DTPA- and USPIO-enhanced MRI, respectively. Consistent with previous findings, vandetanib treatment restored the BBB in the tumor vasculature, resulting in loss of tumor detectability in Gd-DTPA MRI. However, due to decreased blood volume, treated tumors could be readily detected in USPIO-enhanced MRI scans. Our findings suggest that Gd-DTPA MRI results in overestimation of the effect of antiangiogenic therapy of glioma and that USPIO-MRI provides an important complementary diagnostic tool to evaluate response to antiangiogenic therapy of these tumors.

Peroxisome proliferator-activated receptor alpha protects against obesity-induced hepatic inflammation.

Recently it has become evident that obesity is associated with low-grade chronic inflammation. The transcription factor peroxisome proliferator-activated receptor alpha (PPARalpha) has been shown to have a strong antiinflammatory action in liver. However, the role of PPARalpha in obesity-induced inflammation is much less clear. Therefore, the aim of our study was to determine whether PPARalpha plays a role in obesity-induced hepatic inflammation. To induce obesity, wild-type sv129 and PPARalpha(-/-) mice were exposed to a chronic high-fat diet (HFD), using a low-fat diet (LFD) as control. In wild-type mice, HFD significantly increased the hepatic and adipose expression of numerous genes involved in inflammation. Importantly, this effect was amplified in PPARalpha(-/-) mice, suggesting an antiinflammatory role of PPARalpha in liver and adipose tissue. Further analysis identified specific chemokines and macrophage markers, including monocyte chemotactic protein 1 and F4/80(+), that were elevated in liver and adipose tissue of PPARalpha(-/-) mice, indicating increased inflammatory cell recruitment in the knockout animals. When all groups of mice were analyzed together, a significant correlation between hepatic triglycerides and expression of inflammatory markers was observed. Many inflammatory genes that were up-regulated in PPARalpha(-/-) livers by HFD were down-regulated by treatment with the PPARalpha ligand Wy-14643 under normal nonsteatotic conditions, either in vivo or in vitro, suggesting an antiinflammatory effect of PPARalpha that is independent of reduction in liver triglycerides. In conclusion, our results suggest that PPARalpha protects against obesity-induced chronic inflammation in liver by reducing hepatic steatosis, by direct down-regulation of inflammatory genes, and by attenuating inflammation in adipose tissue.

Angiostatin generating capacity and anti-tumour effects of D-penicillamine and plasminogen activators.

BACKGROUND: Upregulation of endogenous angiostatin levels may constitute a novel anti-angiogenic, and therefore anti-tumor therapy. In vitro, angiostatin generation is a two-step process, starting with the conversion of plasminogen to plasmin by plasminogen activators (PAs). Next, plasmin excises angiostatin from other plasmin molecules, a process requiring a donor of a free sulfhydryl group. In previous studies, it has been demonstrated that administration of PA in combination with the free sulfhydryl donor (FSD) agents captopril or N-acetyl cysteine, resulted in angiostatin generation, and anti-angiogenic and anti-tumour activity in murine models. METHODS: In this study we have investigated the angiostatin generating capacities of several FSDs. D-penicillamine proved to be most efficient in supporting the conversion of plasminogen to angiostatin in vitro. Next, from the optimal concentrations of tPA and D-penicillamine in vitro, equivalent dosages were administered to healthy Balb/c mice to explore upregulation of circulating angiostatin levels. Finally, anti-tumor effects of treatment with tPA and D-penicillamine were determined in a human melanoma xenograft model. RESULTS: Surprisingly, we found that despite the superior angiostatin generating capacity of D-penicillamine in vitro, both in vivo angiostatin generation and anti-tumour effects of tPA/D-penicillamine treatment were impaired compared to our previous studies with tPA and captopril. CONCLUSION: Our results indicate that selecting the most appropriate free sulfhydryl donor for anti-angiogenic therapy in a (pre)clinical setting should be performed by in vivo rather than by in vitro studies. We conclude that D-penicillamine is not suitable for this type of therapy.

Cerebral amyloid angiopathy with severe secondary vascular pathology: a histopathological study.

Cerebral amyloid angiopathy (CAA) is a common neuropathological finding and is characterized by deposition of fibrillar amyloid in cortical and leptomeningeal vessels. In this study we describe the macroscopic and microscopic neuropathological findings of 5 patients with severe CAA-associated secondary vascular changes, including smooth muscle cell degeneration, hyalinization, 'double-barreling' phenomenon, macrophage infiltration, and aneurysmal dilatation of the vessel wall. In 3 of the 5 patients these vascular changes were associated with multiple small hemorrhages, whereas in 2 patients areas of ischemic necrosis were observed. However, none of these patients suffered from large (lobar) hemorrhagic accidents. Nevertheless, severe CAA, particularly when associated with secondary vascular pathology, may lead to vascular dementia-like ischemic changes. Hence, the distinction between patients with severe CAA and secondary vascular abnormalities from those suffering from vascular dementia can be difficult. We speculate that CAA, particularly when associated with secondary vascular pathology, although not resulting in large hemorrhages, may contribute to cognitive decline. The functional impact of CAA and CAA-related secondary vascular changes on cognitive performance warrants further exploration.

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.

Histological analysis of defective colonic healing as a result of angiostatin treatment.

Antiangiogenic therapy is a highly promising new strategy in the treatment of cancer. One of the first angiogenesis inhibitors described was angiostatin, a 38-kDa internal proteolytically generated fragment of plasminogen. In a previous study we found that angiostatin affected physiological angiogenesis as well as tumor angiogenesis. It impaired healing when administered during repair of experimental colonic anastomoses, as reflected by a decrease in mechanical strength. On histology, we observed a decrease in factor VIII-stained vessel amount and volume in angiostatin-treated colonic anastomoses. The exact working mechanism of angiostatin has not been elucidated. Based on the available studies on proposed working mechanisms of angiostatin, we have attempted to address histological differences in physiological angiogenesis between the tissues of colonic anastomoses of mice with impaired healing and control mice. After angiostatin treatment there was more inflammatory tissue as a result of impaired healing. Furthermore, we found fewer vessels in the granulation tissue after angiostatin treatment. However, especially with respect to extracellular matrix (ECM), endothelial cell apoptosis, proliferation, or neutrophil influx, no gross differences were discerned 1 week following surgery, using histology and immunohistochemistry techniques.

Differential effects of vascular endothelial growth factor A isoforms in a mouse brain metastasis model of human melanoma.

We reported previously that vascular endothelial growth factor isoform A (VEGF-A) expression by Mel57 human melanoma cells led to tumor progression in a murine brain metastasis model in an angiogenesis-independent fashion by dilation of co-opted, pre-existing vessels and concomitant enhanced blood supply (B. Kusters et al., Cancer Res., 62: 341-345, 2002). Here, we compare the activities of the 121, 165, and 189 VEGF-A isoforms in this model by transfecting Mel57 cells with the respective cDNAs and by injecting the resulting stably transfected cell lines in the internal carotid arteries of nude mice (n = 10 for each isoform). Although the three isoforms had similar potency to induce endothelial cell proliferation, VEGF(121) expression did not result in sprouting angiogenesis but rather led to extensive vasodilation and increased permeability of pre-existing, predominantly peritumoral vessels. Sometimes, proliferating endothelial cells accumulated in vessel lumina, giving these a microvascular, glomeruloid, proliferation-like appearance. Expression of VEGF(165) or VEGF(189) was associated with induction of an intratumoral neovascular bed. In VEGF(165)-expressing tumors, daughter endothelial cells were distributed among newly formed vessels that were extensively dilated. This also occurred in VEGF(189) tumors, but there, vasodilation was less pronounced. Using contrast-enhanced magnetic resonance imaging, the different vascular phenotypes were visualized on characteristic radiological images. VEGF(165) expression was the most unfavorable of the three. Mice carrying VEGF(165) tumors became moribund earlier than those carrying VEGF(121)-expressing tumors (16 +/- 4 days versus 22 +/- 3 days). Our data demonstrate that VEGF-A isoforms differ in angiogenic properties that can be visualized by contrast-enhanced magnetic resonance imaging.

Accumulation of heparan sulfate proteoglycans in cerebellar senile plaques.

Alzheimer's disease (AD) brains are characterized by the presence of senile plaques (SPs), which primarily consist of amyloid beta protein (Abeta). Besides Abeta, several other proteins with the ability to modulate amyloid fibril formation accumulate in SPs, e.g. heparan sulfate proteoglycans (HSPGs). Cerebellar SPs are predominantly of the diffuse type, whereas fibrillar SPs are rarely observed. Furthermore, because of the spatial separation of non-fibrillar and fibrillar SPs in the cerebellum, this brain region provides a model for the study of the association of Abeta-associated factors with various stages of SP formation. In the present study, we performed an immunohistochemical analysis to investigate the expression of the HSPG species agrin, perlecan, glypican-1 and the syndecans 1-3 as well as glycosaminoglycan side-chains in cerebellar SPs. We demonstrated that agrin and glypican-1 were expressed in both non-fibrillar and fibrillar cerebellar SPs, whereas the syndecans were only associated with fibrillar cerebellar SPs. Perlecan expression was absent in all cerebellar SPs. Since fibrillar and non-fibrillar SPs may develop independently in the cerebellum, it is likely that agrin, glypican-1 as well as heparan sulfate glycosaminoglycans may contribute to the formation of both cerebellar plaque types, whereas syndecan only seems to play a role in the generation of cerebellar fibrillar plaques.