Iris A. Schulkens

Expression, Regulation and Function of Human Metallothioneins in Endothelial Cells

Metallothioneins (MTs) are small cysteine-rich proteins which are involved in e.g. metal homeostasis, metal detoxification and protection against oxidative stress. In addition, several MTs have been shown to regulate expression of proangiogenic growth factors like vascular endothelial growth factor. Detailed information about the expression and regulation of specific MT isoforms in endothelial cells (EC) is limited. We therefore performed extensive mRNA expression profiling of all known human MTs in EC. We found that the basal endothelial expression is restricted to MT1E, MT1X, MT2A, and MT3. Physiological activation of EC by exposure to serum increased the expression of MT1E and MT2A and induced the expression of MT1M. Furthermore, exposure to zinc or copper induced the expression of most MT1 isoforms, while hypoxia specifically increased the expression of MT1E, MT1M, MT1X, and MT3. Finally, knockdown of the dominant MT isoform in EC, i.e. MT2A, resulted in decreased proliferation and sprouting as well as in increased migration of human umbilical vein EC. Together, these findings provide a link between MTs and angiogenesis.

Galectin expression profiling identifies galectin-1 and Galectin-9Δ5 as prognostic factors in stage I/II non-small cell lung cancer.

Approximately 30–40% of the patients with early stage non-small cell lung cancer (NSCLC) will present with recurrent disease within two years of resection. Here, we performed extensive galectin expression profiling in a retrospective study using frozen and paraffin embedded tumor tissues from 87 stage I/II NSCLC patients. Our data show that galectin mRNA expression in NSCLC is confined to galectin-1, -3, -4, -7, -8, and -9. Next to stage, univariable Cox regression analysis identified galectin-1, galectin-9FL and galectin-9Δ5 as possible prognostic markers. Kaplan-Meier survival estimates revealed that overall survival was significantly shorter in patients that express galectin-1 above median levels, i.e., 23.0 (2.9–43.1) vs. 59.9 (47.7–72.1) months (p = 0.020) as well as in patients that express galectin-9Δ5 or galectin-9FL below the median, resp. 59.9 (41.9–75.9) vs. 32.8 (8.7–56.9) months (p = 0.014) or 23.2 (−0.4–46.8) vs. 58.9 (42.9–74.9) months (p = 0.042). All three galectins were also prognostic for disease free survival. Multivariable Cox regression analysis showed that for OS, the most significant prognostic model included stage, age, gal-1 and gal-9Δ5 while the model for DFS included stage, age and gal-9Δ5. In conclusion, the current study confirms the prognostic value of galectin-1 and identifies galectin-9Δ5 as novel potential prognostic markers in early stage NSCLC. These findings could help to identify early stage NSCLC patients that might benefit most from adjuvant chemotherapy.

Low dose angiostatic treatment counteracts radiotherapy-induced tumor perfusion and enhances the anti-tumor effect.

The extent of tumor oxygenation is an important factor contributing to the efficacy of radiation therapy (RTx). Interestingly, several preclinical studies have shown benefit of combining RTx with drugs that inhibit tumor blood vessel growth, i.e. angiostatic therapy. Recent findings show that proper scheduling of both treatment modalities allows dose reduction of angiostatic drugs without affecting therapeutic efficacy. We found that whilst low dose sunitinib (20 mg/kg/day) did not affect the growth of xenograft HT29 colon carcinoma tumors in nude mice, the combination with either single dose RTx (1x 5Gy) or fractionated RTx (5x 2Gy/week, up to 3 weeks) substantially hampered tumor growth compared to either RTx treatment alone. To better understand the interaction between RTx and low dose angiostatic therapy, we explored the effects of RTx on tumor angiogenesis and tissue perfusion. DCE-MRI analyses revealed that fractionated RTx resulted in enhanced perfusion after two weeks of treatment. This mainly occurred in the center of the tumor and was accompanied by increased tissue viability and decreased hypoxia. These effects were accompanied by increased expression of the pro-angiogenic growth factors VEGF and PlGF. DCE-MRI and contrast enhanced ultrasonography showed that the increase in perfusion and tissue viability was counteracted by low-dose sunitinib. Overall, these data give insight in the dynamics of tumor perfusion during conventional 2 Gy fractionated RTx and provide a rationale to combine low dose angiostatic drugs with RTx both in the palliative as well as in the curative setting.

Correlations between immune response and vascularization qRT-PCR gene expression clusters in squamous cervical cancer

BackgroundThe tumour microenvironment comprises a network of immune response and vascularization factors. From this network, we identified immunological and vascularization gene expression clusters and the correlations between the clusters. We subsequently determined which factors were correlated with patient survival in cervical carcinoma.MethodsThe expression of 42 genes was investigated in 52 fresh frozen squamous cervical cancer samples by qRT-PCR. Weighted gene co-expression network analysis and mixed-model analyses were performed to identify gene expression clusters. Correlations and survival analyses were further studied at expression cluster and single gene level.ResultsWe identified four immune response clusters: ‘T cells’ (CD3E/CD8A/TBX21/IFNG/FOXP3/IDO1), ‘Macrophages’ (CD4/CD14/CD163), ‘Th2’ (IL4/IL5/IL13/IL12) and ‘Inflammation’ (IL6/IL1B/IL8/IL23/IL10/ARG1) and two vascularization clusters: ‘Angiogenesis’ (VEGFA/FLT1/ANGPT2/ PGF/ICAM1) and ‘Vessel maturation’ (PECAM1/VCAM1/ANGPT1/SELE/KDR/LGALS9). The ‘T cells’ module was correlated with all modules except for ‘Inflammation’, while ‘Inflammation’ was most significantly correlated with ‘Angiogenesis’ (p < 0.001). High expression of the ‘T cells’ cluster was correlated with earlier TNM stage (p = 0.007). High CD3E expression was correlated with improved disease-specific survival (p = 0.022), while high VEGFA expression was correlated with poor disease-specific survival (p = 0.032). Independent predictors of poor disease-specific survival were IL6 (hazard ratio = 2.3, p = 0.011) and a high IL6/IL17 ratio combined with low IL5 expression (hazard ratio = 4.2, p = 0.010).Conclusions‘Inflammation’ marker IL6, especially in combination with low levels of IL5 and IL17, was correlated with poor survival. This suggests that IL6 promotes tumour growth, which may be suppressed by a Th17 and Th2 response. Measuring IL6, IL5 and IL17 expression may improve the accuracy of predicting prognosis in cervical cancer.

A key role for galectin‐1 in sprouting angiogenesis revealed by novel rationally designed antibodies

Galectins are carbohydrate binding proteins that function in many key cellular processes. We have previously demonstrated that galectins are essential for tumor angiogenesis and their expression is associated with disease progression. Targeting galectins is therefore a potential anti‐angiogenic and anti‐cancer strategy. Here, we used a rational approach to generate antibodies against a specific member of this conserved protein family, i.e. galectin‐1. We characterized two novel mouse monoclonal antibodies that specifically react with galectin‐1 in human, mouse and chicken. We demonstrate that these antibodies are excellent tools to study galectin‐1 expression and function in a broad array of biological systems. In a potential diagnostic application, radiolabeled antibodies showed specific targeting of galectin‐1 positive tumors. In a therapeutic setting, the antibodies inhibited sprouting angiogenesis in vitro and in vivo, underscoring the key function of galectin‐1 in this process.

Examination of the Role of Galectins During In Vivo Angiogenesis Using the Chick Chorioallantoic Membrane Assay

Angiogenesis is a complex multi-process involving various activities of endothelial cells. These activities are influenced in vivo by environmental conditions like interactions with other cell types and the microenvironment. Galectins play a role in several of these interactions and are therefore required for proper execution of in vivo angiogenesis. In this chapter we describe a method to study galectins and galectin inhibitors during physiologic and pathophysiologic angiogenesis in vivo using the chicken chorioallantoic membrane (CAM) assay.

Different angioregulatory activity of monovalent galectin-9 isoforms

Galectin-9 consists of two peptide-linked carbohydrate recognition domains (CRDs), but alternative splicing and proteolytic processing can give rise to multiple galectin-9 isoforms. Some of these consist of a single CRD and can exert different functions in cell biology. Here, we explored the role of these galectin-9 isoforms in endothelial cell function and angiogenesis. For this, we compared the effects of the two separate CRDs (Gal-9N and Gal-9C) with the tandem repeat galectin-9M on endothelial cell proliferation, migration, sprouting and tube formation in vitro as well as on angiogenesis in vivo using the chicken chorioallantoic membrane (CAM) assay. Galectin-9 isoforms significantly affected proliferation in quiescent endothelial cells and migration in activated endothelial cells. Interestingly, both monovalent gal-9 CRDs displayed opposite effects compared to gal-9M on proliferation and migration. Sprouting was significantly inhibited by gal-9C, while all isoforms appeared to stimulate tube formation. Angiogenesis in vivo was hampered by all three isoforms with predominant effects on vessel length. In general, the isoforms induced only subtle concentration-dependent effects in vitro as well as in vivo. Collectively, the effects of different galectin-9 isoforms in endothelial cell biology depend on the cellular activation status. While opposing effects can be observed on a cellular level in vitro, all galectin-9 isoforms hamper angiogenesis in vivo. This warrants further investigation of the regulatory mechanisms that underlie the diverging roles of galectin-9 isoforms in endothelial cell biology since this could provide therapeutic opportunities.

Examination of the Role of Galectins and Galectin Inhibitors in Endothelial Cell Biology

The growth of new blood vessels is a key event in many (patho)physiological processes, including embryogenesis, wound healing, inflammatory diseases, and cancer. Neovascularization requires different, well-coordinated actions of endothelial cells, i.e., the cells lining the luminal side of all blood vessels. Galectins are involved in several of these activities. In this chapter we describe methods to study galectins and galectin inhibition in three key functions of endothelial cells during angiogenesis, i.e., endothelial cell migration, endothelial cell sprouting, and endothelial cell network formation.

A high IL6/IL17 ratio combined with low IL5 expression is correlated with poor survival in squamous cervical cancer

Cervical cancer is the second leading cause of cancer death in young women worldwide. The aim of the current study was to identify which combination of immunological and vascular factors in the tumor microenvironment of cervical carcinoma is most significant for survival. Using qRTPCR, 42 markers were investigated in frozen squamous cervical cancer samples (n = 52). Weighted gene coexpression network analysis and mixed-model analyses were performed to identify gene expression clusters and study their correlations. Selected individual factors were further investigated by immunohistochemistry. We identified a ‘T cells’ (CD3E/CD8A/TBX21/IFNG/FOXP3/ IDO1), ‘Macrophages’ (CD4/CD14/CD163), ‘Th2’ (IL4/ IL5/IL13/IL12), ‘Inflammation’ (IL6/IL1B/IL8/IL23/IL10/ ARG1), ‘Angiogenesis’ (VEGFA/FLT1/ANGPT2/ PGF/ ICAM1) and ‘Vessel maturation’ (PECAM1/VCAM1/ ANGPT1/SELE/KDR/LGALS9) cluster. The ‘T cells’ cluster significantly correlated with early TNM stage (p = 0.007). High expression of ‘ Tc ells’ marker CD3E correlated with improved disease-specific survival (p = 0.022). High expression of ‘Angiogenesis’ marker VEGFA correlated with poor survival (p = 0.032). High expression of ‘Inflammation’ marker IL6 was an independent predictor of poor survival (hazard ratio = 2.3, p = 0.011). A high IL6/ IL17 ratio combined with low IL5 expression gave a hazard ratio of 4.2 (p = 0.010). Using immunohistochemistry, we have shown that IL-6 is correlated with poor survival, and determined that IL-17 was predominantly expressed by neutrophils (66%), mast cells (23%) and innate lymphoid cells (8%). Remarkably, Th17 cells were only a minor IL-17 expressing population (4%). A similar distribution was found in head and neck, ovarian, endometrial, prostate, breast, lung and colon carcinoma. A high number of IL-17 expressing cells was an independent prognostic factor for poor survival in early stage disease (p = 0.016, n = 160). A high number of Th17 cells was an independent prognostic factor for improved disease-specific survival (p = 0.026), suggesting Th17 cells are part of an anti-tumor immune response. IL6 independently predicted poor survival in cervical cancer. IL17 expressed by Th17 cells could counteract the tumor promoting effects of IL6, even more so combined with a Th2 response characterized by IL5. Since RNA levels, in contrast to protein levels, are extremely low in activated neutrophils, the IL17 RNA levels measured most likely represented Th17 cells. Th17 cells, despite being a minor IL-17 expressing subpopulation, were also associated with improved survival in squamous cervical cancer. Total IL-17 expressing cells, primarily representing neutrophils, rather associated with poor survival in early stage disease. Measuring IL6, especially in combination with IL17 and IL5 expression, may improve the accuracy of predicting prognosis and support the development of anti-IL-6 combined with anti-VEGF-A therapy in cervical cancer.