VEGF and c-Met blockade amplify angiogenesis inhibition in pancreatic islet cancer.

Angiogenesis inhibitors that block VEGF receptor (VEGFR) signaling slow the growth of many types of tumors, but eventually the disease progresses. Multiple strategies are being explored to improve efficacy by concurrent inhibition of other functionally relevant receptor tyrosine kinases (RTK). XL880 (foretinib, GSK1363089) and XL184 (cabozantinib) are small-molecule inhibitors that potently block multiple RTKs, including VEGFR and the receptor of hepatocyte growth factor c-Met, which can drive tumor invasion and metastasis. This study compared the cellular effects of XL880 and XL184 with those of an RTK inhibitor (XL999) that blocks VEGFR but not c-Met. Treatment of RIP-Tag2 mice with XL999 resulted in 43% reduction in vascularity of spontaneous pancreatic islet tumors over 7 days, but treatment with XL880 or XL184 eliminated approximately 80% of the tumor vasculature, reduced pericytes and empty basement membrane sleeves, caused widespread intratumoral hypoxia and tumor cell apoptosis, and slowed regrowth of the tumor vasculature after drug withdrawal. Importantly, XL880 and XL184 also decreased invasiveness of primary tumors and reduced metastasis. Overall, these findings indicate that inhibition of c-Met and functionally related kinases amplifies the effects of VEGFR blockade and leads to rapid, robust, and progressive regression of tumor vasculature, increased intratumoral hypoxia and apoptosis, and reduced tumor invasiveness and metastasis.

Semaphorin 3A is an endogenous angiogenesis inhibitor that blocks tumor growth and normalizes tumor vasculature in transgenic mouse models.

Tumor growth and progression rely upon angiogenesis, which is regulated by pro- and antiangiogenic factors, including members of the semaphorin family. By analyzing 3 different mouse models of multistep carcinogenesis, we show here that during angiogenesis, semaphorin 3A (Sema3A) is expressed in ECs, where it serves as an endogenous inhibitor of angiogenesis that is present in premalignant lesions and lost during tumor progression. Pharmacologic inhibition of endogenous Sema3A during the angiogenic switch, the point when pretumoral lesions initiate an angiogenic phase that persists throughout tumor growth, enhanced angiogenesis and accelerated tumor progression. By contrast, when, during the later stages of carcinogenesis following endogenous Sema3A downmodulation, Sema3A was ectopically reintroduced into islet cell tumors by somatic gene transfer, successive waves of apoptosis ensued, first in ECs and then in tumor cells, resulting in reduced vascular density and branching and inhibition of tumor growth and substantially extended survival. Further, long-term reexpression of Sema3A markedly improved pericyte coverage of tumor blood vessels, something that is thought to be a key property of tumor vessel normalization, and restored tissue normoxia. We conclude, therefore, that Sema3A is an endogenous and effective antiangiogenic agent that stably normalizes the tumor vasculature.

Antiangiogenic therapy decreases integrin expression in normalized tumor blood vessels.

Tumor blood vessels normalized by antiangiogenic therapy may provide improved delivery of chemotherapeutic agents during a window of time but it is unknown how protein expression in tumor vascular endothelial cells changes. We evaluated the distribution of RGD-4C phage, which binds alpha(v)beta(3), alpha(v)beta(5), and alpha(5)beta(1) integrins on tumor blood vessels before and after antiangiogenic therapy. Unlike the control phage, fd-tet, RGD-4C phage homed to vascular endothelial cells in spontaneous tumors in RIP-Tag2 transgenic mice in a dose-dependent fashion. The distribution of phage was similar to alpha(v)beta(3) and alpha(5)beta(1) integrin expression. Blood vessels that survived treatment with AG-013736, a small molecule inhibitor of vascular endothelial growth factor and platelet-derived growth factor receptors, had only 4% as much binding of RGD-4C phage compared with vessels in untreated tumors. Cellular distribution of RGD-4C phage in surviving tumor vessels matched the alpha(5)beta(1) integrin expression. The reduction in integrin expression on tumor vessels after antiangiogenic therapy raises the possibility that integrin-targeted delivery of diagnostics or therapeutics may be compromised. Efficacious delivery of drugs may benefit from identification by in vivo phage display of targeting peptides that bind to tumor blood vessels normalized by antiangiogenic agents.

Pericytes limit tumor cell metastasis.

Previously we observed that neural cell adhesion molecule (NCAM) deficiency in beta tumor cells facilitates metastasis into distant organs and local lymph nodes. Here, we show that NCAM-deficient beta cell tumors grew leaky blood vessels with perturbed pericyte-endothelial cell-cell interactions and deficient perivascular deposition of ECM components. Conversely, tumor cell expression of NCAM in a fibrosarcoma model (T241) improved pericyte recruitment and increased perivascular deposition of ECM molecules. Together, these findings suggest that NCAM may limit tumor cell metastasis by stabilizing the microvessel wall. To directly address whether pericyte dysfunction increases the metastatic potential of solid tumors, we studied beta cell tumorigenesis in primary pericyte-deficient Pdgfb(ret/ret) mice. This resulted in beta tumor cell metastases in distant organs and local lymph nodes, demonstrating a role for pericytes in limiting tumor cell metastasis. These data support a new model for how tumor cells trigger metastasis by perturbing pericyte-endothelial cell-cell interactions.

Effect of inhibition of vascular endothelial growth factor signaling on distribution of extravasated antibodies in tumors.

Antibodies and other macromolecular therapeutics can gain access to tumor cells via leaky tumor vessels. Inhibition of vascular endothelial growth factor (VEGF) signaling can reduce the vascularity of tumors and leakiness of surviving vessels, but little is known about how these changes affect the distribution of antibodies within tumors. We addressed this issue by examining the distribution of extravasated antibodies in islet cell tumors of RIP-Tag2 transgenic mice and implanted Lewis lung carcinomas using fluorescence and confocal microscopic imaging. Extravasated nonspecific immunoglobulin G (IgG) and antibodies to fibrin or E-cadherin accumulated in irregular patchy regions of stroma. Fibrin also accumulated in these regions. Anti-E-cadherin antibody, which targets epitopes on tumor cells of RIP-Tag2 adenomas, was the only antibody to achieve detectable levels within tumor cell clusters at 6 hours after i.v. injection. Treatment for 7 days with AG-013736, a potent inhibitor of VEGF signaling, reduced the tumor vascularity by 86%. The overall area density of extravasated IgG/antibodies decreased after treatment but the change was less than the reduction in vascularity and actually increased when expressed per surviving tumor vessel. Accumulation of anti-E-cadherin antibody in tumor cell clusters was similarly affected. The patchy pattern of antibodies in stroma after treatment qualitatively resembled untreated tumors and surprisingly coincided with sleeves of basement membrane left behind after pruning of tumor vessels. Together, the findings suggest that antibody transport increases from surviving tumor vessels after normalization by inhibition of VEGF signaling. Basement membrane sleeves may facilitate this transport. Antibodies preferentially distribute to tumor stroma but also accumulate on tumor cells if binding sites are accessible.

Quantitative tissue blood flow evaluation of pancreatic tumor: comparison between xenon CT technique and perfusion CT technique based on deconvolution analysis.

PURPOSE: There has been one report that tissue blood flow (TBF) quantification with xenon CT was effective in predicting the therapeutic response to an anticancer drug in pancreatic cancer. The purpose of this study was to evaluate the correlation between the TBF of pancreatic tumors calculated with xenon CT and those with perfusion CT, in order to evaluate whether perfusion CT could replace xenon CT. MATERIALS AND METHODS: Nine patients with pathologically proved pancreatic tumors who underwent both xenon CT and perfusion CT were included. RESULTS: Quantitative TBF of pancreatic tumors measured by perfusion CT ranged from 22.1 to 196.2 ml/min/100 g (mean+/-SD, 52.6+/-54.8 ml/min/100 g). In contrast, those obtained by xenon CT ranged from 10.3 to 173.6 ml/min/100 g (mean+/-SD, 47.4+/-49.4 ml/min/100 g). There was a good linear correlation between xenon CT and perfusion CT (y=0.8537x+2.48, R2=0.895: p<0.05). CONCLUSION: The TBF of pancreatic tumors measured by xenon CT and perfusion CT techniques showed a close linear correlation. We can expect that perfusion CT based on the deconvolution algorithm may replace xenon CT to predict the effect of pancreatic tumor treatment with anticancer drugs.

Rapid access of antibodies to alpha5beta1 integrin overexpressed on the luminal surface of tumor blood vessels.

Integrin alpha(5)beta(1) is overexpressed on endothelial cells of tumor vessels and is uniformly and rapidly accessible to antibodies in the bloodstream. Here, we determined whether antibodies rapidly gain access to integrin overexpressed on the abluminal (basolateral) surface of endothelial cells through vascular leakiness or whether the rapid accessibility results instead because the integrin is overexpressed on the luminal (apical) surface of endothelial cells due to loss of cell polarity. Using tumors in RIP-Tag2 transgenic mice as a model, we first compared the binding pattern of intravascular anti-alpha(5)beta(1) integrin antibody with the leakage pattern of nonspecific IgG. The distributions did not match: anti-alpha(5)beta(1) integrin antibody uniformly labeled the tumor vasculature, but IgG was located in patchy sites of leakage. We next injected an antibody to fibrinogen/fibrin, which resulted in patchy labeling of tumors that matched the leakage of IgG and the overall distribution of fibrin in tumors. Similarly, injected antibodies to the basement membrane protein fibronectin, a ligand of alpha(5)beta(1) integrin, or type IV collagen produced patchy sites of leakage instead of uniform labeling of vascular basement membrane. Differences in the kinetics of labeling, which for alpha(5)beta(1) integrin antibody was near maximal by 10 minutes but for the other antibodies gradually increased over 6 hours, indicated differences in accessibility of their respective targets. Isosurface rendering of confocal microscopic images was consistent with antibody binding to alpha(5)beta(1) integrin on the luminal surface of endothelial cells. Together, these findings indicate that the rapid accessibility of alpha(5)beta(1) integrin in RIP-Tag2 tumors results from overexpression of the integrin on the luminal surface of tumor vessels.

Microvascular density and hypoxia-inducible factor pathway in pancreatic endocrine tumours: negative correlation of microvascular density and VEGF expression with tumour progression.

Tumour-associated angiogenesis is partly regulated by the hypoxia-inducible factor (HIF) pathway. Endocrine tumours are highly vascularised and the molecular mechanisms of their angiogenesis are not fully delineated. The aim of this study is to evaluate angiogenesis and expression of HIF-related molecules in a series of patients with pancreatic endocrine tumours (PETs). The expression of vascular endothelial growth factor (VEGF), HIF-1alpha, HIF-2alpha and carbonic anhydrase 9 (CA9) was examined by immunohistochemistry in 45 patients with PETs and compared to microvascular density (MVD), endothelial proliferation, tumour stage and survival. Microvascular density was very high in PETs and associated with a low endothelial index of proliferation. Microvascular density was significantly higher in benign PETs than in PETs of uncertain prognosis, well-differentiated and poorly differentiated carcinomas (mean values: 535, 436, 252 and 45 vessels mm(-2), respectively, P < 0.0001). Well-differentiated tumours had high cytoplasmic VEGF and HIF-1alpha expression. Poorly differentiated carcinomas were associated with nuclear HIF-1alpha and membranous CA9 expression. Low MVD (P = 0.0001) and membranous CA9 expression (P = 0.0004) were associated with a poorer survival. Contrary to other types of cancer, PETs are highly vascularised, but poorly angiogenic tumours. As they progress, VEGF expression is lost and MVD significantly decreases. The regulation of HIF signalling appears to be specific in pancreatic endocrine tumours.

Soluble Eph A receptors inhibit tumor angiogenesis and progression in vivo.

The Eph family of receptor tyrosine kinases and their ligands, known as ephrins, play a crucial role in vascular development during embryogenesis. The function of these molecules in adult angiogenesis has not been well characterized. Here, we report that blocking Eph A class receptor activation inhibits angiogenesis in two independent tumor types, the RIP-Tag transgenic model of angiogenesis-dependent pancreatic islet cell carcinoma and the 4T1 model of metastatic mammary adenocarcinoma. Ephrin-A1 ligand was expressed in both tumor and endothelial cells, and EphA2 receptor was localized primarily in tumor-associated vascular endothelial cells. Soluble EphA2-Fc or EphA3-Fc receptors inhibited tumor angiogenesis in cutaneous window assays, and tumor growth in vivo. EphA2-Fc or EphA3-Fc treatment resulted in decreased tumor vascular density, tumor volume, and cell proliferation, but increased cell apoptosis. However, EphA2-Fc had no direct effect on tumor cell growth or apoptosis in culture, yet inhibited migration of endothelial cells in response to tumor cells, suggesting that the soluble receptor inhibited blood vessel recruitment by the tumor. These data provide the first functional evidence for Eph A class receptor regulation of pathogenic angiogenesis induced by tumors and support the function of A class Eph receptors in tumor progression.

[Color Doppler signal enhancement with SH/TH-508 in pancreatic tumors].

In this report, we showed the efficacy of a new contrast agent (SH/TA-508, Schering AG, Germany) for color Doppler imaging of the pancreatic tumors. In pancreatic ductal cancer, no enhancement of the lesion was observed, but vascular invasion by cancer became to be easily evaluated. On the other hand, hypervascular tumors such as islet cell tumor and cystadenocarcinoma, were increased in color Doppler signals of vessels by SH/TA-508. We concluded that SH/TA-508 was useful for evaluating the vascular invasion by pancreatic cancer as well as vascularity of hypervascular mass and solid component of cystic neoplasma.

Helical CT for the preoperative localization of islet cell tumors of the pancreas: value of arterial and parenchymal phase images.

OBJECTIVE: In two-phase helical CT, organs are scanned in both the arterial and the parenchymal phase after IV injection of contrast material. The purpose of our study was to evaluate the usefulness of two-phase helical CT for the preoperative localization of pancreatic islet cell tumors. MATERIALS AND METHODS: Ten patients with 11 surgically proven islet cell tumors were included in the study. Helical CT scans of the pancreas were obtained with 5-mm collimation and overlapping image reconstruction. Images were obtained in the arterial and parenchymal phases after contrast material injection. A test bolus was used for tailored bolus timing. Images were analyzed by two observers. If a tumor was detected, its density relative to the surrounding parenchyma was scored by consensus, using a five-point scale. RESULTS: Nine of 11 tumors could be located using two-phase helical CT (sensitivity, 82%), including one 4-mm gastrinoma. Two lesions smaller than 5 mm could not be visualized. Two tumors were easier to detect (n = 1) or could only be detected on parenchymal-phase images (n = 1). Tumor conspicuity was better on arterial-phase images in two patients. CONCLUSION: Our results show that obtaining CT scans in both the arterial and the parenchymal phase leads to improved detection of pancreatic islet cell tumors.

Embolization in the palliation of complications of inoperable primary pancreatic neoplasms.

Primary pancreatic neoplasm typically presents at an advanced stage where surgical management may not be feasible. These patients are often symptomatic due to biliary obstruction but problems may also include gastrointestinal bleeding and endocrinological complications. We describe two cases illustrating the use of palliative embolization in the control of biochemical and haemorrhagic complications of primary pancreatic neoplasm. In one case, massive gastrointestinal bleeding from an inoperable primary pancreatic carcinoma was controlled by two embolization procedures to produce devascularization of the primary lesion. In a second case, life-threatening hypercalcaemia was thought to be due to secretion of a parathormone-like material from an inoperable islet cell tumour. There was no evidence of liver metastases and the pancreatic mass was embolized, following which serum calcium was reduced to near normal levels with considerable clinical improvement. We conclude that there is a role for embolization of inoperable primary pancreatic neoplasm in the palliation of biochemical or haemorrhagic complications of these tumours.

[Clinical role of ultrasound and color Doppler ultrasound in diagnosing pancreatic endocrine tumors].

US, EUS, color Doppler US, and color Doppler EUS were performed for five cases of pancreatic endocrine tumors. US was able to detect four cases of five tumors (80%), but could not sufficiently evaluate the internal echo. Color Doppler US was able to reveal the blood flows inside in only one case (25%). EUS was able to detect clearly all five cases (100%), and color Doppler EUS was able to reveal a significant amount of the internal blood flow in all cases. Color Doppler EUS was reflected in hypervascular findings on angiogram and proliferating vascular findings on histology. Therefore, ultrasound, especially EUS was useful for diagnosing the location of the tumor and for evaluating the internal echo, whereas color Doppler EUS was useful for evaluating the vascularity of the tumors.

Induction of angiogenesis during the transition from hyperplasia to neoplasia.

It is now well established that unrestricted growth of tumours is dependent upon angiogenesis. Previous studies on tumour growth, however, have not revealed when or how the transition to an angiogenic state occurs during early tumour development. The advent of transgenic mice carrying oncogenes that reproducibly elicit tumours of specific cell types is providing a new format for studying multi-step tumorigenesis. In one of these models, transgenic mice expressing an oncogene in the beta-cells of the pancreatic islets heritably recapitulate a progression from normality to hyperplasia to neoplasia. We report here that angiogenic activity first appears in a subset of hyperplastic islets before the onset of tumour formation. A novel in vitro assay confirms that hyperplasia per se does not obligate angiogenesis. Rather, a few hyperplastic islets become angiogenic in vitro at a time when such islets are neovascularized in vivo and at a frequency that correlates closely with subsequent tumour incidence. These findings suggest that induction of angiogenesis is an important step in carcinogenesis.

Islet cell tumors metastatic to the liver: effective palliation by sequential hepatic artery embolization.

The value of sequential percutaneous hepatic artery embolization with polyvinyl alcohol particles was examined in 22 patients with islet cell carcinoma metastatic to the liver. Nine patients had gastrinoma, 2 had glucagonoma, and 11 had no discernible hormonal secretions or syndromes. Ninety-seven embolizations were done with a median number of 4 (range, 1 to 12) per patient. The interval between embolizations ranged from 1 to 8 months. Twelve of twenty evaluable patients had a partial remission, frequently associated with subjective improvement and decrease in hormone levels. The projected median survival of all 22 patients from the initiation of embolization is 33.7 months (range, 1 to 72). Nausea, vomiting, fever, and abdominal pain occurred with each embolization and subsided usually by day 10 (range, 3 to 35). Sequential hepatic artery occlusion is an effective method for prolonged palliation in this selected group of patients.

Localization of neuroendocrine tumors of the pancreas.

The diagnosis of neuroendocrine tumors of the pancreas is based on clinical, biochemical, and ultrastructural data. The different radiological modalities are employed only for localization, except in the case of nonfunctioning neoplasms, for which a differential diagnosis from adenocarcinoma can be suggested based on the radiographic images. Generally, the initial evaluation should consist of the noninvasive studies and should be complemented by angiography and, in cases of functioning neoplasms, with pancreatic venous sampling if the preliminary findings are inconclusive. However, the localizing work-up should be terminated when the information obtained is sufficient to determine the therapy that will follow. If the ultrasonic or computed tomographic findings preclude surgical resection, further work-up is obviously unnecessary.

Nonfunctioning islet cell carcinoma presenting bleeding gastric varices and splenomegaly.

This is a report of a 63-year-old Japanese woman with a nonfunctioning islet cell carcinoma of the pancreas presenting bleeding gastric varices and splenomegaly. These manifestations are extremely rare in patients with nonfunctioning islet cell tumor. The tumor originated in the tail of the pancreas and grew mainly within the spleen. The gastric varices due to increased blood flow to the tumor and arteriovenous fistulas within the tumor were confirmed by angiography and operation. The tumor was resected and she is in a good health for 14-months after the operation.