Arne Scholz

Interferon-α: Regulatory Effects on Cell Cycle and Angiogenesis

In the current study, we investigated the effects of interferon-α (IFN-α) on proliferation and angiogenesis in neuroendocrine tumor disease. Using a panel of human neuroendocrine tumor cell lines, we confirmed functionally active IFN-α signaling by STAT activation and nuclear translocation as well as transactivation. IFN-α results in anchorage-dependent and -independent growth inhibition due to a delayed progression from S-phase to G2 phase of the cell cycle. This was due to substantial reduction in cellular cyclin B levels resulting in the inhibition of Cdc2 kinase activity. In parallel to growth inhibition, we observed a profound inhibition of VEGF gene transcription by IFN-α in human neuroendocrine tumor cells due to an Sp1/Sp3-dependent inhibition of VEGF promoter activity. Treatment of neuroendocrine tumors with IFN-α in nude mice resulted in growth inhibition and inhibition of angiogenesis. Furthermore, treatment of neuroendocrine tumor patients with IFN-α resulted in decreased VEGF expression as well as tumor angiogenesis in liver metastases. In summary, IFN-α acts via direct antiproliferative effects as well as inhibition of tumor angiogenesis mediated by suppression of VEGF gene expression in neuroendocrine tumor disease.

VEGF-D promotes tumor growth and lymphatic spread in a mouse model of hepatocellular carcinoma.

Lymphatic spread is an important clinical determinant for the prognosis of hepatocellular carcinoma (HCC), but little is known about the control of lymphangiogenesis in HCC. We addressed expression and biological role of the pro‐(lymph), angiogenic protein VEGF‐D in this tumor entity. Using immunohistochemistry and in situ hybridization on specimens of HCC, cirrhotic and normal liver we found abundant expression of VEGF‐D exclusively in the tumor cells. The cognate receptor VEGFR‐3 was detected on blood and lymphatic vessels. By clinicopathological analysis VEGF‐D expression was correlated with pT‐stage of the primary, lymph node metastasis and lymphangiosis carcinomatosa. Three out of 4 human HCC cell lines expressed and secreted VEGF‐D. To approach its biological function, VEGF‐D deficient SKHep‐1 cells were stably transfected with VEGF‐D cDNA and effects on tumor progression were determined in vivo. Compared to mock‐transfected controls, subcutaneous tumors derived from VEGF‐D expressing cells were larger and more frequently metastasized to regional lymph nodes. VEGF‐D expressing tumors exhibited increased microvessel density and increased abundance of peri‐ and intratumoral lymphatics, as assessed by immunostaining for CD31 and for LYVE‐1 and/or podoplanin, respectively. Furthermore, coexpression of the soluble extracellular VEGFR‐3 domain blocked VEGF‐D‐induced tumor growth and lymphatic spread via reduction of angiogenesis and lymphangiogenesis. In the orthotopic approach, VEGF‐D expression resulted in an increased rate of intra‐ and extrahepatic as well as lymph node metastasis. In conclusion, our study suggests that expression of VEGF‐D is involved in growth and lymphatic spread of HCC. Therefore, VEGF‐D might represent a therapeutic target in HCC.

Tumor-associated angiogenesis and lymphangiogenesis correlate with progression of intrahepatic cholangiocarcinoma.

OBJECTIVES:Little is known about the function of tumor-associated neovascularization in the progression of intrahepatic cholangiocarcinoma (IHC). This study was conducted to evaluate the influence of tumor-associated angiogenesis and lymphangiogenesis on progression of IHC.METHODS:We analyzed tissue specimens of IHC (N=114) by immunohistochemistry using the endothelial-specific antibody CD31 and the lymphendothelial-specific antibody D2-40 and subsequently quantified microvessel density (MVD) and lymphatic microvessel density (LVD). To analyze the influence of tumor-associated angiogenesis and lymphangiogenesis on tumor progression, tumors were allocated according to mean MVD and LVD, respectively, into groups of “high” and “low” MVD and LVD, respectively, and various clinicopathological characteristics as well as recurrence and survival data were analyzed.RESULTS:IHC revealed an induction of tumor-associated angiogenesis and lymphangiogenesis. Tumors of “high” MVD displayed more frequently advanced primary tumor stages and multiple tumor nodes. Furthermore, patients with tumors of “high” MVD had an inferior curative resection rate and suffered more frequently from recurrence. A “high” LVD was correlated with increased nodal spread, and patients with “high” LVD tumors more frequently developed recurrence. In the univariate analysis, MVD and LVD revealed significant influence on survival, and MVD was identified as an independent prognostic factor for survival in the multivariate analysis. The 5-year survival of patients with “low” MVD tumors was 42.1%, compared with 2.2% in patients with “high” MVD tumors (P<0.001).CONCLUSIONS:This study suggests a critical function of tumor-associated angiogenesis and lymphangiogenesis for progression of IHC. Therefore, antiangiogenic and antilymphangiogenic approaches may have therapeutic potency in this tumor entity.

Tumor-Associated Lymphangiogenesis Correlates with Lymph Node Metastases and Prognosis in Hilar Cholangiocarcinoma

BackgroundTumor-associated lymphangiogenesis has been shown to promote nodal spread and is of prognostic significance in some tumor entities. Currently, nothing is known about the impact of lymphangiogenesis on progression and prognosis in hilar cholangiocarcinoma.MethodsWe analyzed tissue specimens of normal liver and hilar cholangiocarcinoma (nxa0=xa060) by immunohistochemistry using the lymphendothelial-specific antibody D2-40 and subsequently quantified lymphatic microvessel density (LVD). The LVD was correlated with clinicopathological characteristics and recurrence pattern of the tumors as well as patients’ survival.ResultsIn contrast to the low abundance of lymphatic vessels in nontransformed liver tissue, we found an induction of lymphangiogenesis in hilar cholangiocarcinoma. Tumors with a high LVD (34 out of 60) had a significant higher incidence of lymph node involvement (pxa0<xa00.001), perivascular (pxa0=xa00.017), and perineural (pxa0=xa00.033) lymphangiosis and local recurrence (pxa0<xa00.001). Furthermore, a high LVD was identified to be a significant overall (three-year: 24.4% versus 90.5%; five-year: 7.0% versus 76.4%; pxa0<xa00.001) and disease-free (three-year: 8.3% versus 76.6%; five-year: 5.9% versus 61.4%; pxa0<xa00.001) survival disadvantage, with LVD representing an independent prognostic factor for survival (pxa0<xa00.001) in the multivariate analysis.ConclusionsLymphangiogenesis is associated with increased frequency of tumor cells in lymphatics and lymph nodes in hilar cholangiocarcinoma. The prognostic importance of tumor-associated lymphangiogenesis was reflected by LVD serving as an independent prognostic factor. In addition, lymphangiogenesis may represent a potential target in the development of new therapeutic approaches in hilar cholangiocarcinoma.

Microvessel density correlates with lymph node metastases and prognosis in hilar cholangiocarcinoma.

BackgroundNeovascularization was shown to be critically involved in the progression of multiple cancers, and treatment approaches targeting tumor-associated neovascularization provide convincing results in recent years in some tumor entities. However, little is known about the tumor-associated neovascularization in hilar cholangiocarcinoma. The present study was conducted to analyze tumor-associated neovascularization in hilar cholangiocarcinoma and to determine its influence on tumor growth, metastasis, recurrence, and prognosis.MethodsWe analyzed tissue specimens of hilar cholangiocarcinoma (n = 60) by immunohistochemistry using the endothelial-specific antibody CD31 and subsequently quantified the microvessel density (MVD). The MVD was correlated with clinicopathological characteristics and recurrence pattern of the tumors as well as survival of patients.ResultsHilar cholangiocarcinoma revealed a high degree of vascularization, with a calculated mean MVD of 28.1 ± 14.5 vessels. Tumors with a high MVD had a significant higher incidence of lymph node involvement (P = 0.009) and local recurrence (P < 0.001). Furthermore, a high MVD was identified to be a significant overall survival disadvantage (3-year, 28% vs. 93%; 5-year, 8% vs. 78%; P < 0.001) as well as disease-free survival disadvantage (3-year, 7% vs. 88%, 5-year, 7% vs. 72%; P < 0.001), with MVD representing an independent prognostic factor for survival.ConclusionsNeovascularization is associated with nodal spread as well as local recurrence and serves as an independent prognostic factor for survival after curative resection of hilar cholangiocarcinoma. Therefore, tumor-associated neovascularization seems to be critically involved in the progression of this tumor entity. In addition, neovascularization may represent a potential target in he development of new therapeutic approaches in hilar cholangiocarcinoma.

Tumor-Associated Lymphangiogenesis Correlates with Prognosis after Resection of Human Hepatocellular Carcinoma

BackgroundExperimental results from animal models as well as studies of human cancers indicate a critical role for tumor-associated lymphangiogenesis in tumor progression. However, its significance in hepatocellular carcinoma (HCC) is not well established.MethodsWe analyzed tissue specimens from healthy liver (nxa0=xa036), cirrhotic liver (nxa0=xa024), and HCC (nxa0=xa060) by immunohistochemistry, using antibody D2-40 specific for lymphendothelia. We subsequently quantified lymphatic microvessel density (LVD). The LVD was correlated with clinicopathological characteristics of the tumors as well as survival and disease-free survival of the patients.ResultsIn contrast to healthy as well as cirrhotic liver, lymphangiogenesis was induced in HCC. Lymphatic vessels were detected in the intratumoral septa as well as within the bulk of tumor cells. Tumors with high LVD (24 of 60) had developed significantly more frequently in cirrhotic livers (Pxa0=xa00.001) and were more frequently restricted to one liver lobe (Pxa0=xa00.04). Univariate analysis revealed high LVD as a marker for reduced survival and disease-free survival disadvantage (medianxa0>60 vs. 21xa0months, Pxa0=xa00.018, and 19 vs. 8xa0months, Pxa0=xa00.047, respectively). In multivariate analysis, LVD showed a trend toward association with reduced survival (Pxa0=xa00.059) and represented an independent prognostic factor for disease-free survival (Pxa0=xa00.017).ConclusionsTumor-associated lymphangiogenesis is involved in neovascularization of hepatocellular carcinoma. Quantitative analysis of LVD demonstrated a significant influence of lymphangiogenesis on survival and established LVD as an independent predictor of disease-free survival. Quantification of LVD may be helpful in identifying patients with a high risk of tumor recurrence.

An Orthotopic Model of Pancreatic Somatostatin Receptor (SSTR)-Positive Tumors Allows Bimodal Imaging Studies Using 3T MRI and Animal PET-Based Molecular Imaging of SSTR Expression

Somatostatin receptor (SSTR) scintigraphy is currently used as one standard imaging modality in neuroendocrine tumors (NETs). However, future optimization of NET imaging may be achieved with positron emission tomography based methods utilizing more sensitive and specific tracers in combination with computed tomography or magnetic resonance imaging. Here we established an orthotopic mouse model that reflects relevant aspects of human pancreatic NETs such as SSTR expression, dense vascularization and metastatic disease. This model was then utilized to test the feasibility of combined magnetic resonance imaging and animal positron emission tomography. Orthotopic implantation of amphicrine, SSTR-positive pancreatic AR42J cells resulted in rapidly growing tumors, with concomitant metastatic spread into abdominal lymph nodes and peritoneal cavity. Primary tumors as well as their metastases expressed the neuroendocrine markers chromogranin A and synaptophysin. For imaging experiments, the SSTR ligands 68Ga-DOTATOC or 68Ga-DOTANOC were injected intravenously, and animals were subsequently examined in an animal positron emission tomography scanner and a clinical 3T (tesla) magnetic resonance imager. All animals showed radionuclide accumulation in the primary tumor. Definite anatomical correlation was achieved using digital image fusion of the positron emission tomography and magnetic resonance imaging data. 68Ga-DOTANOC strongly accumulated in the tumor tissue (mean 6.6-fold vs. control tissues) when compared to 68Ga-DOTATOC, which showed a higher renal clearance. In good agreement with the biodistribution data, the kidney-to-tumor ratio was higher for 68Ga-DOTATOC (2.43-fold vs. 1.75-fold). Consequently, 68Ga-DOTANOC achieved better signal enhancement in the primary tumor and allowed for detection of metastatic lesions. In summary, we established a novel orthotopic pancreatic SSTR-positive tumor model and used this model to provide proof of principle for the diagnostic combination of SSTR-based molecular imaging and magnetic resonance imaging. Specifically, the animal model allowed the comparative evaluation of 68Ga-DOTANOC and 68Ga-DOTATOC, with 68Ga-DOTANOC providing better tumor-specific accumulation and renal activity. We conclude that this animal model will be of innovative value for further investigation in the imaging of NETs.