Lymphatic vessels assessment in feline mammary tumours

BACKGROUND: The lymphatic vessels play a crucial role in a variety of human cancers since tumour cell lymphatic invasion significantly influences prognosis. It is not known if pre-existing lymphatics are enough for tumour dissemination or de novo development is necessary. VEGFR-3 is an angiogenetic mediator for both lymphatic and blood vessels during embryonic development, and only for lymphatics after birth. VEGF is a mediator of both vasculogenesis and angiogenesis, regulates the growth of lymphatics in various experimental models, and is produced in many solid tumours. CD44 mediates hyaluronic acid (HA)-dependent cell adhesion: besides promoting invasion, this interaction also supports neoangiogenesis that indirectly stimulates tumour cell proliferation. The expression of VEGF-C (Vascular Endothelial Growth Factor – C), its receptor VEGFR-3 and CD44, were studied on feline mammary samples to assess the importance of lymphangiogenesis and lymphangiotrophism in neoplasia. METHODS: Samples were taken from six normal mammary glands (NMG), ten benign (BT) and 32 malignant (MT) tumours. Immunohistochemical laminin/VEGFR-3 double stain, VEGF-C and CD44 stains were applied to 4 μm-thick sections, and their expression evaluated in intratumoral/extratumoral and intramammary/extramammary fields. RESULTS: All groups revealed a higher number of lymphatics in the extratumoral/extramammary areas. VEGF-C expression in the epithelium paralleled the number of positive vessels in the NMG, BT and MT, whereas VEGF-C higher expression was noted in the intratumoral fields only in infiltrating MT. CD44 score was lower in extratumoral than intratumoral fields in tumours and showed a significant increase in extramammary/extratumoral fields from NMG to MT. Pearson test showed a significant and inversely proportional correlation between CD44 expression and the number of lymphatic vessels with VEGFR-3 in malignant infiltrating tumours. CONCLUSION: The number of both VEGFR-3 positive and negative lymphatics in the extratumoral and extramammary stroma was significantly higher than intratumoral and intramammary fields respectively in the NMG, BT and MT. This suggests a scant biological importance of intratumoral lymphatics while their higher number is due to the concentration of existing vessels following compression of the extratumoral stroma in spite of a non demonstrable increase from NMG to MT. The tumour model employed provided no evidence of lymphangiogenesis, and metastasis in the regional lymph node develops following the spread through the pre-existing lymphatic network

Targeting lymphangiogenesis to prevent tumour metastasis

Recent studies involving animal models of cancer and clinicopathological analyses of human tumours suggest that the growth of lymphatic vessels (lymphangiogenesis) in or nearby tumours is associated with the metastatic spread of cancer. The best validated molecular signalling system for tumour lymphangiogenesis involves the secreted proteins vascular endothelial growth factor-C (VEGF-C) and VEGF-D that induce growth of lymphatic vessels via activation of VEGF receptor-3 (VEGFR-3) localised on the surface of lymphatic endothelial cells. In this review, we discuss the evidence supporting a role for this signalling system in the spread of cancer and potential approaches for blocking this system to prevent tumour metastasis

Elevated level of inhibin-α subunit is pro-tumourigenic and pro-metastatic and associated with extracapsular spread in advanced prostate cancer

The biological function of inhibin-α subunit (INHα) in prostate cancer (PCa) is currently unclear. A recent study associated elevated levels of INHα in PCa patients with a higher risk of recurrence. This prompted us to use clinical specimens and functional studies to investigate the pro-tumourigenic and pro-metastatic function of INHα. We conducted a cross-sectional study to determine a link between INHα expression and a number of clinicopathological parameters including Gleason score, surgical margin, extracapsular spread, lymph node status and vascular endothelial growth factor receptor-3 expression, which are well-established prognostic factors of PCa. In addition, using two human PCa cell lines (LNCaP and PC3) representing androgen-dependent and -independent PCa respectively, we investigated the biological function of elevated levels of INHα in advanced cancer. Elevated expression of INHα in primary PCa tissues showed a higher risk of PCa patients being positive for clinicopathological parameters outlined above. Over-expressing INHα in LNCaP and PC3 cells demonstrated two different and cell-type-specific responses. INHα-positive LNCaP demonstrated reduced tumour growth whereas INHα-positive PC3 cells demonstrated increased tumour growth and metastasis through the process of lymphangiogenesis. This study is the first to demonstrate a pro-tumourigenic and pro-metastatic function for INHα associated with androgen-independent stage of metastatic prostate disease. Our results also suggest that INHα expression in the primary prostate tumour can be used as a predictive factor for prognosis of PCa

A model for co-expression pattern analysis of genes implicated in angiogenesis and tumour cell invasion in cervical cancer

To date, numerous genes have been identified which are involved in both tumour neovascularisation (angiogenesis) and tumour cell invasion, and most of them are also expressed to some extent under normal physiological conditions. However, little is known about how these genes co-express in these settings. This study was undertaken to quantitate mRNA levels in normal and malignant cervical tissues of nine selected genes (VEGF121, VEGF165, VEGF189, VEGF-C, eIF-4E, b-FGF, TSP-2, MMP-2 and MMP-9) implicated in the above processes using real-time quantitative RT–PCR. In addition, the Spearman's rank correlation was used to determine their co-expression patterns. The transcript levels for the different VEGF-A splice variants (VEGF121, VEGF165, VEGF189) were at least 10-fold higher in the cancer cases, with the highest levels in the primary tumours demonstrating lympho-vascular space involvement. The lymphangiogenic factor VEGF-C and MMP-9 were upregulated 130- and 80-fold respectively in cervical cancers. The highest levels of VEGF-C mRNA were found in the lymph-node positive group. The transcript levels for b-FGF were similar in normal cervical tissue and early-stage cervical cancer, however, higher levels were found in the cervical cancers with advanced stage disease. Comparing gene transcript levels between recurrent and non-recurrent cervical cancer patients revealed significant differences (P=0.038) in transcript levels for the angiogenesis inhibitor TSP-2, with the highest levels in non-recurrent cases. Co-expression pattern analysis in normal cervical tissue revealed highly significant co-expressions (P<0.0001) between TSP-2 and most other genes analysed (VEGF121, VEGF165, VEGF-C, b-FGF and MMP-2). In cervical cancer, TSP-2 appears only to be highly co-expressed with MMP-2 (P<0.0001). In contrast to normal cervical tissue, we found a highly significant co-expression (P<0.0001) between MMP-9 and VEGF189 in cervical cancer. The combined application of real-time quantitative RT–PCR and Spearman's rank correlation identifies gene transcripts which are simultaneously co-expressed. Our results revealed a significant co-expression between the angiogenesis inhibitor TSP-2 and most other genes analysed in normal cervical tissue. In cervical cancer, we found a strong upregulation of VEGF-C and MMP-9 mRNA, with a highly significant co-expression between MMP-9 and VEGF189

Expression and Functional Roles of Angiopoietin-2 in Skeletal Muscles

Angiopoietin-1 (ANGPT1) and angiopoietin-2 (ANGPT2) are angiogenesis factors that modulate endothelial cell differentiation, survival and stability. Recent studies have suggested that skeletal muscle precursor cells constitutively express ANGPT1 and adhere to recombinant ANGPT1 and ANGPT2 proteins. It remains unclear whether or not they also express ANGPT2, or if ANGPT2 regulates the myogenesis program of muscle precursors. In this study, ANGPT2 regulatory factors and the effects of ANGPT2 on proliferation, migration, differentiation and survival were identified in cultured primary skeletal myoblasts. The cellular networks involved in the actions of ANGPT2 on skeletal muscle cells were also analyzed.Primary skeletal myoblasts were isolated from human and mouse muscles. Skeletal myoblast survival, proliferation, migration and differentiation were measured in-vitro in response to recombinant ANGPT2 protein and to enhanced ANGPT2 expression delivered with adenoviruses. Real-time PCR and ELISA measurements revealed the presence of constitutive ANGPT2 expression in these cells. This expression increased significantly during myoblast differentiation into myotubes. In human myoblasts, ANGPT2 expression was induced by H(2)O(2), but not by TNFα, IL1β or IL6. ANGPT2 significantly enhanced myoblast differentiation and survival, but had no influence on proliferation or migration. ANGPT2-induced survival was mediated through activation of the ERK1/2 and PI-3 kinase/AKT pathways. Microarray analysis revealed that ANGPT2 upregulates genes involved in the regulation of cell survival, protein synthesis, glucose uptake and free fatty oxidation.Skeletal muscle precursors constitutively express ANGPT2 and this expression is upregulated during differentiation into myotubes. Reactive oxygen species exert a strong stimulatory influence on muscle ANGPT2 expression while pro-inflammatory cytokines do not. ANGPT2 promotes skeletal myoblast survival and differentiation. These results suggest that muscle-derived ANGPT2 production may play a positive role in skeletal muscle fiber repair

Myeloid Cells Contribute to Tumor Lymphangiogenesis

The formation of new blood vessels (angiogenesis) and lymphatic vessels (lymphangiogenesis) promotes tumor outgrowth and metastasis. Previously, it has been demonstrated that bone marrow-derived cells (BMDC) can contribute to tumor angiogenesis. However, the role of BMDC in lymphangiogenesis has largely remained elusive. Here, we demonstrate by bone marrow transplantation/reconstitution and genetic lineage-tracing experiments that BMDC integrate into tumor-associated lymphatic vessels in the Rip1Tag2 mouse model of insulinoma and in the TRAMP-C1 prostate cancer transplantation model, and that the integrated BMDC originate from the myelomonocytic lineage. Conversely, pharmacological depletion of tumor-associated macrophages reduces lymphangiogenesis. No cell fusion events are detected by genetic tracing experiments. Rather, the phenotypical conversion of myeloid cells into lymphatic endothelial cells and their integration into lymphatic structures is recapitulated in two in vitro tube formation assays and is dependent on fibroblast growth factor-mediated signaling. Together, the results reveal that myeloid cells can contribute to tumor-associated lymphatic vessels, thus extending the findings on the previously reported role of hematopoietic cells in lymphatic vessel formation

A quantitative analysis of lymphatic vessels in human breast cancer, based on LYVE-1 immunoreactivity

This study was undertaken to determine the highly sensitive method for detecting tumour lymphatic vessels in all the fields of each slide (LV), lymphatic microvessel density (LMVD) and lymphatic vessel invasion (LVI) and to compare them with other prognostic parameters using immunohistochemical staining with polyclonal (PCAB) and monoclonal antibodies (MCAB) to the lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1), and the pan-endothelial marker factorVIII in a series of 67 human breast cancers. In all LYVE-1-stained sections, LV (some of which contained red blood cells) were frequently found localised in extralobular stroma, dermis, connective tissue stroma and adjacent to artery and vein, but were rare within the intralobular stroma or the tumour body (3/67 cases) or areas of widespread invasion. In contrast small blood vessels were observed in intra- and extralobular stroma in the factor VIII-stained sections. Quantitation of vessel numbers revealed that LYVE-1/PCAB detected a significantly larger number of LV than either H&E or LYVE-1/MCAB (P<0.0001). LYVE-1/PCAB detected LVI in 25/67 cases (37.3%) and their presence was significantly associated with both lymph node metastasis (χ2=4.698, P=0.0248) and unfavourable overall survival (OS) (P=0.0453), while not relapse- free survival (RFS) (P=0.2948). LMVD had no influence for RFS and OS (P=0.4879, P=0.1463, respectively). Our study demonstrates that immunohistochemistry with LYVE-1/PCAB is a highly sensitive method for detecting tumour LV/LVI in breast cancer and LVI is a useful prognostic indicator for lymphatic tumour dissemination

Hypoxia-Induced Retinal Angiogenesis in Zebrafish as a Model to Study Retinopathy

Mechanistic understanding and defining novel therapeutic targets of diabetic retinopathy and age-related macular degeneration (AMD) have been hampered by a lack of appropriate adult animal models. Here we describe a simple and highly reproducible adult fli-EGFP transgenic zebrafish model to study retinal angiogenesis. The retinal vasculature in the adult zebrafish is highly organized and hypoxia-induced neovascularization occurs in a predictable area of capillary plexuses. New retinal vessels and vascular sprouts can be accurately measured and quantified. Orally active anti-VEGF agents including sunitinib and ZM323881 effectively block hypoxia-induced retinal neovascularization. Intriguingly, blockage of the Notch signaling pathway by the inhibitor DAPT under hypoxia, results in a high density of arterial sprouting in all optical arteries. The Notch suppression-induced arterial sprouting is dependent on tissue hypoxia. However, in the presence of DAPT substantial endothelial tip cell formation was detected only in optic capillary plexuses under normoxia. These findings suggest that hypoxia shifts the vascular targets of Notch inhibitors. Our findings for the first time show a clinically relevant retinal angiogenesis model in adult zebrafish, which might serve as a platform for studying mechanisms of retinal angiogenesis, for defining novel therapeutic targets, and for screening of novel antiangiogenic drugs