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56 Recent Advances in Angiogenesis and Antiangiogenesis, 2009 Marco Presta their fate and to study their impact on zebrafish development [23]. In these studies, tumor cells were injected at the blastula stage to explore potential bidirectional interactions between cancer cells and embryonic stem cells. The results indicate that developing zebrafish can be used as a biosensor for tumor-derived signals. However, grafting of tumor cells at this stage, well before vascular development, results in their reprogramming toward a nontumorigenic phenotype, thus hampering any attempt to investigate tumor-driven vascularization. At variance, injection of melanoma cells into the hindbrain ventricle or yolk sac of 48 hpf embryos results in the formation of tumor masses within 4 days [24]. Immunostaining analysis of the grafts reveals the presence of blood vessels within the brain and abdominal lesions, even though the high vascularity of the invaded regions may not allow easy discrimination between developmental and tumor-induced angiogenesis [24]. Fig. (1). Tumor cell xenograft in zebrafish embryo. Tumor cells were injected in the perivitelline space of zebrafish embryo at 48 hpf. After 24 hours, transverse sections of the embryo were stained with DAPI. In A, the graft is marked with an asterisk (nc, notochord). In B, the graft is shown at higher magnification to visualize proliferating tumor cells (arrows). Recently, a novel zebrafish embryo/tumor xenograft angiogenesis assay has been described [25, 26]. The assay is based on the grafting of mammalian tumor cells in the proximity of the developing SIV plexus at 48 hpf (Fig. 1). Pro-angiogenic factors released locally by the tumor graft affect the normal developmental pattern of the SIVs by stimulating the migration and growth of sprouting vessels towards the implant. Onetwo days after tumor cell grafting, whole mount phosphatase alkaline staining allows the macroscopic evaluation of the angiogenic response (Fig. 2A). The use of transgenic zebrafish embryos, in which endothelial cells express GFP under the control of endothelial-specific promoters ([27] and references therein), represents an improvement of the method, allowing the observation and time-lapse recording of newly formed blood vessels in live embryos by epifluorescence microscopy as well as by in vivo confocal microscopy [25, 26] (Fig. 2B). Also, quantum dots may be used as labeling agents of the zebrafish embryo vasculature for long-lasting intravital time-lapse studies [28]. Fig. (2). Tumor angiogenesis in zebrafish embryo. Tumor cells were injected in the perivitelline space of zebrafish embryo at 48 hpf. A) After 24 hours, embryos were stained for alkaline phosphatase activity to visualize the newly formed blood vessels (arrows) converging towards the graft (asterisk). In B, cryosection of a tumor graft (asterisk) in transgenic VEGFR2:G-RCFP zebrafish embryo [11]. Note the GFP-tagged blood vessels within the graft (c, d). Sections were counterstained with DAPI (a) and anti- -tubulin antibodies (b). When compared to other in vivo tumor angiogenesis assays, the zebrafish embryo/tumor xenograft model presents several advantages. i) The model allows the delivery of a very limited number of cells, mimicking the initial stages of tumor angiogenesis and metastasis. ii) Labeled tumor cells (e.g. GFPtransduced or fluorescent dye-loaded cells) can be easily visualized within the embryo. Thus, analysis of the spatial/temporal relationship among tumor cells and newly formed blood vessels may represent an important feature of this model. iii) Several techniques can be applied within the constraints of paraffin or gelatin embedding, including histochemistry and immunohistochemistry. Electron microscopy can also be used in combination with light microscopy. Moreover, reverse transcriptase-polymerase chain reaction analysis with species-specific primers allows the study of gene expression by grafted tumor cells and by the host under different experimental conditions [26].
Recent Advances in Angiogenesis and Antiangiogenesis, 2009, 59-66 59 CHAPTER 7 The Contribution of Circulating Endothelial Cells to Tumor Angiogenesis Francesco Bertolini 1 , Patrizia Mancuso 1 , Paola Braidotti 2 , Yuval Shaked 3 and Robert S. Kerbel 4 . 1 2 European Institute of Oncology, and University of Milan, Department of Medicine, Surgery and Dentistry and San Paolo Hospital, Milan, Italy. 3 Technion- Israel Institute of Technology, Rappaport Faculty of Medicine, Department of Molecular Pharmacology, Haifa, Israel, 4 Sunnybrook Health Sciences Centre, Molecular and Cellular Biology, Department of Medical Biophysics, University of Toronto, Toronto, Ontario M4N 3M5, Canada. Address correspondence to: Dr Francesco Bertolini, European Institute of Oncology, via Ripamonti 435, 20141 Milan, Italy; Tel: 02-57489-535; Fax 02-574-89-537; Email: francesco.bertolini@ieo.it 1. INTRODUCTION Abstract: Immunohistochemistry, flow cytometry and cell culture procedures have demonstrated the presence of circulating endothelial cells (CECs) and circulating endothelial progenitors (CEPs) in the blood of vertebrates. CECs and CEPs are currently being investigated in a variety of diseases as markers of vascular turnover or damage and, also in the case of CEPs, vasculogenesis. CEPs appear to have a “catalytic” role in different steps of cancer progression and recurrence after therapy, and there are preclinical and clinical data suggesting that CEC enumeration might be useful to select and predict clinical response in patients who are candidates for anti-angiogenic treatments. In some types of cancer, CECs and CEPs might be one of the possible hidden identities of cancer stem cells. The definition of CEC and CEP phenotype and the standardization of CEC and CEP enumeration strategies are highly desirable goals in order to exploit these cells as reliable biomarkers in oncology clinical trials. By means of microscopy, cells with a possible endothelial morphology were found to circulate in the blood in the early ‘70s [1]. In the ‘90s, immunohistochemistry (IHC) studies confirmed the endothelial nature of these cells, and their enumeration by means of positive enrichment, IHC or flow cytometry (FC) indicated that levels of circulating endothelial cells (CECs) are increased in a very wide spectrum of disorders encompassing vascular, autoimmune, infectious and ischemic diseases [2-8]. Over the past ten years, increased CEC counts were observed in some cancer patients [9-21], and these cells were studied as surrogate biomarkers of angiogenesis and anti-angiogenic drug activity in preclinical models and medical oncology [22-26]. These studies also indicated that the endothelial phenotype was expressed by cells displaying a wide variety of different features [6-8]. Some CECs had a phenotype compatible with terminally differentiated endothelial cells (EC), in some cases being apoptotic or necrotic and thus most likely derived from the turnover of vessel walls. Some other cells expressed progenitor-associated antigens in addition to endothelial antigens, and were considered candidates as “circulating endothelial progenitors” (CEPs). This chapter will describe some of the most recent findings about CECs and CEPs in cancer, along with novel Domenico Ribatti (Ed.) All rights reserved - © 2009 Bentham Science Publishers Ltd. (and possibly provocative) hypotheses emerging from these studies. 2. TOWARDS A MOLECULAR, ULTRA- STRUCTURAL AND PHENOTYPIC DEFINITION OF CECs AND CEPs The two most frequently used CEC enumeration strategies, IHC and FC, have provided in many cases different CEC frequencies in health and disease [6-8]. According to IHC enumeration, CECs are large cells present in a frequency of 10-100/mL in healthy subjects [6-7]. According to FC, events with an EC phenotype show in most cases small dimensions and are counted with a frequency of 100-10,000/mL [8-11, 16-18]. Antigenic promiscuity between CECs and platelets has prompted the development and validation of new FC enumeration procedures where DNA staining reagents have allowed the count and the sorting of platelet-depleted, DNA-containing cells with an EC phenotype (CD45-,CD31+CD146+; Mancuso et al, in press). Studies which have used transmission electron microscopy (TEM), confirmed that these sorted CECs are of endothelial nature by virtue of the presence of EC-specific Weibel-Palade bodies (Fig. 1) and of RNA transcripts for the ECspecific gene VE-cadherin. Transmission electron microscopy (TEM) studies also offered an explanation of the controversies about CEC frequency in the
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Page 4 and 5: FOREWORD It has been known for a ve
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Page 8 and 9: v Vito Pistoia Head, Laboratory of
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Page 16 and 17: The Role of Mesenchymal Stem Cells
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