Vascular phenotypes in primary non-small cell lung carcinomas and matched brain metastases

BACKGROUND: Anti-angiogenic therapy with bevacizumab (an anti-vascular endothelial growth factor (VEGF) antibody) predominantly targets immature blood vessels. Bevacizumab has shown a survival benefit in non-small cell lung carcinoma (NSCLC) and has recently been demonstrated to be safe in patients with brain metastases. However, it is not known whether bevacizumab is effective against brain metastases or whether metastases are representative of their primary in terms of VEGF expression, hypoxia, proliferation and vascular phenotype. The aim of this study was to evaluate these factors in a series of matched primary NSCLCs and brain metastases. METHODS AND RESULTS: Immunohistochemistry showed strong correlation of carbonic anhydrase 9 expression (a marker of hypoxia) in primary and secondary cancers (P=0.0002). However, the proliferation index, VEGF expression, microvessel density and the proportion of mature vessels were discordant between primary and secondary cancers. The mean proportion of mature vessels was 63.2% higher in the brain metastases than the primary tumours (P=0.004). Moreover, the vascular pattern of the primary tumour was not representative of the metastasis. CONCLUSIONS: Brain metastases have a significantly higher proportion of mature vasculature, suggesting that they may be refractory to anti-VEGF therapy. These findings may have implications for clinical trials and biomarker studies evaluating anti-angiogenic agents in brain metastases

Expression of delta-like ligand 4 (Dll4) and markers of hypoxia in colon cancer

BACKGROUND: Delta-like ligand 4 (Dll4) is a Notch ligand that is upregulated by hypoxia and vascular endothelial growth factor-A (VEGF-A) and is reported to have a role in tumor angiogenesis. Evidence from xenograft studies suggests that inhibiting Dll4-Notch signalling may overcome resistance to anti-VEGF therapy. The aim of this study was to characterise the expression of Dll4 in colon cancer and to assess whether it is associated with markers of hypoxia and prognosis. METHOD: In all, 177 colon cancers were represented in tissue microarrays. Immunohistochemistry was performed using validated antibodies against Dll4, VEGF, hypoxia-inducible factor (HIF)-1alpha, HIF-2alpha, prolyl hydroxylase (PHD)1, PHD2, PHD3 and carbonic anhydrase 9 (CA9). RESULTS: The expression of Dll4 was observed preferentially in the endothelium of 71% (125 out of 175) of colon cancers, but not in the endothelium adjacent to normal mucosa (none out of 107, P<0.0001). The expression of VEGF was significantly associated with HIF-2alpha (P<0.0001) and Dll4 (P=0.010). Only HIF-2alpha had a significant multivariate prognostic effect (hazard ratio 1.61, 95% confidence interval 1.01-2.57). Delta-like ligand 4 was also expressed by neoplastic cells, particularly neoplastic goblet cells. CONCLUSION: Endothelial expression of Dll4 is not a prognostic factor, but is significantly associated with VEGF. Assessing endothelial Dll4 expression may be critical in predicting response to anti-VEGF therapies

Vasohibin inhibits angiogenic sprouting in vitro and supports vascular maturation processes in vivo

<p>Abstract</p> <p>Background</p> <p>The murine homologue of human vasohibin (mVASH1), a putative antiangiogenic protein, was investigated for its effects on <it>in vitro </it>and <it>in vivo </it>angiogenesis.</p> <p>Methods</p> <p>Cell growth and migration were analyzed in murine fibroblasts, smooth muscle cells and endothelial cells. Angiogenic sprouting was studied in human umbilical vein endothelial cells (HUVECs) in the spheroid sprouting assay. <it>In vivo </it>effects on blood vessel formation were investigated in the chorioallantoic membrane (CAM) assay and in the C57BL/6 melanoma xenograft model.</p> <p>Results</p> <p>Purified murine and human VASH1 protein induced apoptosis of murine fibroblasts <it>in vitro</it>, but not of vascular aortic smooth muscle cells (AoSMC) or endothelial cells. Adenoviral overexpression of murine and human VASH1 inhibited capillary sprouting of HUVECs in the spheroid assay. Administration of recombinant murine and human VASH1 inhibited growth of large vessels in the CAM assay and promoted the formation of a dense, fine vascular network. Murine VASH1-overexpressing B16F10 melanomas displayed a reduction in large vessels and vascular area. Moreover, tumors showed more microvessels that stained positive for the mural cell markers α-smooth muscle cell actin (ASMA) and proteoglycan (NG2).</p> <p>Conclusion</p> <p>Our data imply that murine VASH1 causes angiogenic remodelling by inhibiting angiogenic sprouting and large vessel growth, thereby supporting the formation of a vascular bed consisting predominantly of mature microvessels.</p

Identification of serum angiopoietin-2 as a biomarker for clinical outcome of colorectal cancer patients treated with bevacizumab-containing therapy

BACKGROUND: The combination of chemotherapy with the vascular endothelial growth factor (VEGF) antibody bevacizumab is a standard of care in advanced colorectal cancer (CRC). However, biomarkers predicting outcome of bevacizumab-containing treatment are lacking. As angiopoietin-2 (Ang-2) is a key regulator of vascular remodelling in concert with VEGF, we investigated its role as a biomarker in metastatic CRC. METHODS: Serum Ang-2 levels were measured in 33 healthy volunteers and 90 patients with CRC. Of these, 34 had metastatic disease and received bevacizumab-containing therapy. To determine the tissue of origin of Ang-2, quantitative real-time PCR was performed on microdissected cryosections of human CRC and in a murine xenograft model of CRC using species-specific amplification. RESULTS: Ang-2 originated from the stromal compartment of CRC tissues. Serum Ang-2 levels were significantly elevated in patients with metastatic CRC compared with healthy controls. Amongst patients receiving bevacizumab-containing treatment, low pre-therapeutic serum Ang-2 levels were associated with a significant better response rate (82 vs 31%; P<0.01), a prolonged median progression-free survival (14.1 vs 8.5 months; P<0.01) and a reduction of 91% in the hazard of death (P<0.05). CONCLUSION: Serum Ang-2 is a candidate biomarker for outcome of patients with metastatic CRC treated with bevacizumab-containing therapy, and it should be further validated to customise combined chemotherapeutic and anti-angiogenic treatment. British Journal of Cancer (2010) 103, 1407-1414. doi: 10.1038/sj.bjc.6605925 www.bjcancer.com Published online 5 October 2010 (C) 2010 Cancer Research U

Ascl2 Knockdown Results in Tumor Growth Arrest by miRNA-302b-Related Inhibition of Colon Cancer Progenitor Cells

Background: Achaete scute-like 2 (Ascl2), a basic helix-loop-helix (bHLH) transcription factor, controls the fate of intestinal stem cells. However, the role of Ascl2 in colon cancer progenitor cells remains unknown. The cell line HT-29 (47.5–95 % of CD133 + population) and LS174T (0.45 % of CD133 + population) were chosen for functional evaluation of Ascl2 in colon cancer progenitor cells after gene knockdown by RNA interference. Methodology/Principal Findings: Immunohistochemistry demonstrated that Ascl2 was significantly increased in colorectal adenocarcinomas. Downregulation of Ascl2 using RNA interference in cultured colonic adenocarcinoma HT-29 and LS174T cells reduced cellular proliferation, colony-forming ability, invasion and migration in vitro, and resulted in the growth arrest of tumor xenografts in vivo. The Ascl2 protein level in CD133 + HT-29 cells was significantly higher than in CD133 2 HT-29 cells. Ascl2 blockade via shRNA interference in HT-29 cells (shRNA-Ascl2/HT-29 cells) resulted in 26.2 % of cells staining CD133 + compared with 54.7 % in control shRNA-Ctr/HT-29 cells. The levels of ‘stemness ’ associated genes, such as CD133, Sox2, Oct4, Lgr5, Bmi1, and C-myc, were significantly decreased in shRNA-Ascl2/HT-29 and shRNA-Ascl2/LS174T cells in vitro as well as in the corresponding tumor xenograft (CD133 was not performed in shRNA-Ascl2/LS174T cells). The shRNA-Ascl2/ HT-29 cells had inhibited abilities to form tumorspheres compared with control. The microRNA (miRNAs) microarrays, identified 26 up-regulated miRNAs and 58 down-regulated miRNAs in shRNA-Ascl2/HT-29 cells. Expression levels of let-7b

Retrospective exploratory analysis of VEGF polymorphisms in the prediction of benefit from first-line FOLFIRI plus bevacizumab in metastatic colorectal cancer

<p>Abstract</p> <p>Background</p> <p>Molecular predictors of bevacizumab efficacy in colorectal cancer have not been identified yet. Specific <it>VEGF </it>polymorphisms may affect gene transcription and therefore indirectly influence the efficacy of bevacizumab.</p> <p>Methods</p> <p>Genomic DNA of 111 consecutive metastatic colorectal cancer patients treated with first-line FOLFIRI plus bevacizumab was obtained from blood samples. <it>VEGF </it>-2578 C/A, -1498 C/T, + 405 C/G, + 936 C/T polymorphisms were analyzed by means of PCR-RFLP. DNA samples from 107 patients treated with FOLFIRI alone served as historical control group. The relation of <it>VEGF </it>polymorphisms with PFS, evaluated through Kaplan-Meier method and log-rank test, was the primary end-point. An interaction test with a Cox model has been performed in order to demonstrate the heterogeneity of the effect of <it>VEGF </it>-1498 C/T polymorphism between bevacizumab-and control group.</p> <p>Results</p> <p>In the bevacizumab-group median PFS and OS of patients carrying <it>VEGF </it>-1498 C/C, C/T and T/T allelic variants were, respectively, 12.8, 10.5, 7.5 months (p = 0.0046, log-rank test) and 27.3, 20.5, 18.6 months (p = 0.038, log-rank test). <it>VEGF </it>-1498 T/T genotype was associated with shorter PFS (HR = 2.13, [1.41-5.10], p = 0.0027). In the control group no significant association of <it>VEGF </it>-1498 C/T allelic variants and PFS or OS was found. Interaction between <it>VEGF </it>-1498 C/T variants and treatment effect suggested that the relation of <it>VEGF </it>-1498 T/T genotype with shorter PFS was caused by the effect of bevacizumab (p = 0.011). Other investigated polymorphisms did not affect the outcome.</p> <p>Conclusions</p> <p>These data suggest a possible role for <it>VEGF </it>-1498 C/T variants in predicting the efficacy of bevacizumab in the up-front treatment of metastatic colorectal cancer patients. A molecular tool for selecting subjects candidate to benefit from the anti-VEGF could be important for clinical practice. The retrospective and exploratory design of the present study, coupled with the non-randomized nature of the comparison between treated and untreated patients, imply that these results should be considered as hypothesis generators. A prospective validating trial is currently ongoing.</p