Tumor Vascularization

Belgium

Preface

Domenico Ribatti and Francesco Pezzella

The interaction between neoplastic cells and blood vessels, both newly formed or preexisting normal vessels, is one of the fundamental biological events involved in the development and progression of most solid and hematological tumors including the formation of metastases. Tumor angiogenesis is viewed as the consequence of an angiogenic switch, that is, a genetic event that endows the tumor with the ability to recruit blood vessels from the neighboring tissue. The newly formed tumor blood vessels have specific characteristics that allow discrimination from resting blood vessels. They are characterized by rapid proliferation, increased permeability, and disorganized architecture.

Initially thought to be a must for the grow and progression of tumors, the formation of new vessels was regarded as one of the hallmark of cancer. However, it was discovered that tumors can also growth without neoangiogenesis, mainly by co-opting preexisting vessels but also by vascular mimicry.

Since its discovery by Dr. Judah Folkman, tumor angiogenesis has been proposed as a target for novel tumor therapies. However, the success in the clinic of antiangiogenic compounds has been limited in contrast to many preclinical results obtained in animal models. This is in part due to the fact that tumors can be nonangiogenic, in part to several newly discovered mechanisms of resistance due both to the biology of the cancer cells and of the endothelium.

The field has therefore turned out to be more complex than previously thought.

We have attempted to create a book that will be of benefit not only to basic scientists working in this field, but also to clinicians by offering an overview of the field which now include the nonangiogenic tumors and the process vascular co-option.

We express our gratitude to all our colleagues who have contributed to this book covering some still controversial aspects of tumor angiogenesis.

Chapter 1

Sprouting and nonsprouting angiogenesis in tumors

Domenico Ribatti¹ and Francesco Pezzella²,    ¹Department of Basic Medical Sciences, Neurosciences and Sensory Organs, University of Bari Medical School, Bari, Italy,    ²Nuffield Division of Laboratory Science, Radcliffe Department of Medicine, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom

Abstract

The current wisdom is that tumor growth, invasion, and metastasis are angiogenesis dependent. This chapter summarizes the literature data that indicate that some tumors may be vascularized without significant angiogenesis, through intussusceptive microvascular growth, an alternative or additional mechanism of capillary growth whereby the vascular network expands by insertion of newly formed columns of interstitial tissue structures into the vascular lumen called tissue pillars or posts. Glomeruloid vascular proliferation represents another alternative mode of tumor vascular growth. Finally, potential therapeutic implications are also discussed.

Keywords

Angiogenesis; glomeruloid vascular proliferation; intussusceptive microvascular growth; tumor growth

Introduction

In 1971 Judah Folkman first advanced the hypothesis that tumor growth depends on the formation of new blood vessels from the preexisting vascular bed [1]. According to this hypothesis, endothelial cells must be switched from a resting state to a rapid growth phase by a diffusible chemical signal emanating from the tumor cells. New vessels in the body can be produced according to two main mechanisms: vasculogenesis and angiogenesis. Vasculogenesis occurs predominantly in the embryo and is characterized by the differentiation of hemoangioblasts into endothelial vessels. Vasculogenesis has been described in tumors and is discussed in Chapter 6. Angiogenesis is instead defined as a new blood vessel sprouting from a preexisting vessel, that is, capillaries and postcapillary venules [2].

Three types of classic angiogenic have been described: sprouting angiogenesis, the commonest observed in tumors, nonsprouting or intussusceptive microvascular growth (IMG) [3], and glomeruloid vascular proliferation [4]. Tumor growth starts with an avascular phase followed by a vascular phase (Fig. 1.1) [6]. The avascular phase appears to correspond to the histopathological picture presented by a small colony of neoplastic cells that reaches a steady state before it proliferates and becomes rapidly invasive. In this scenario, metabolites and catabolites are transferred by simple diffusion through the surrounding tissue. Dormant tumors have been discovered during autopsies of individuals who died of causes other than cancer [7]. Carcinoma in situ is found in 98% of individuals aged 50–70 years who died of trauma, but is diagnosed in only 0.1% during life.

Figure 1.1 Steps of tumor angiogenesis and growth. Source: Reproduced from Ribatti D, Vacca A. Overview of tumor angiogenesis. In: Figg WD, Folkman J, editors. Angiogenesis: an integrative approach from science to medicine. New York: Springer; 2008, p. 161–8 [5].

When tumors start to growth beyond the critical size of 2 mm at their site of origin they can ether undergo the angiogenic switch and move to induce new vessel growth or can keep growing remaining nonangiogenic by exploiting the host’s preexisting vessels [8]. Sometime, as it has been show in the rat brain after an initial nonangiogenic phase, in which the tumor grows but only by exploiting preexisting vessels, a late angiogenic switch can occur [9].

In this chapter, we will discuss the angiogenic switch and the angiogenic processes while the nonangiogenic tumors will be described subsequently in Chapter 2.

The angiogenic switch

The mechanism of the switch was formally described by Hanahan, who developed transgenic mice in which the large T oncogene is hybridized to the insulin promoter [10]. In this model for β-islet cell tumorigenesis (RIP-Tag model), all islet cells in a transgenic mouse line express the large T antigen at birth. By 12 weeks, 75% of islets have progressed to small foci of proliferating cells, but only 4% are angiogenic and their number is closely correlated with the incidence of tumor formation [10]. The switch depends on increased production of one or more positive regulators of angiogenesis, such as vascular endothelial growth factor (VEGF), fibroblast growth factor-2 (FGF-2), interleukin-8 (IL-8), placental growth factor (PlGF), transforming growth factor-β (TGF-ß), platelet-derived growth factor (PDGF), pleiotrophins, and others, which cannot be held in check by the levels of antiangiogenic factors [6]. These can be exported from tumor cells, mobilized from the extracellullar matrix, or released from host cells recruited to the tumor. The switch clearly involves more than a simple upregulation of angiogenic activity and has thus been regarded as the result of the net balance between positive and negative regulators.

Sprouting tumor angiogenesis

In absence of pathological stimuli, vessels and their endothelial cells are quiescent and they remain so mostly because of a homeostatic mechanism relying on the Angiopoietin (Ang) family and their receptors. The first description of sprouting angiogenesis in tumor growth was reported by Ausprunk and Folkman [11]. They described the following stages: (1) the basement membrane is locally degraded on the side of the dilated peritumoral postcapillary venule situated closed to the angiogenic stimulus; (2) interendothelial contacts are weakened and endothelial cells migrate into the connective tissue; (3) a solid cord of endothelial cells form; and (4) lumen formation occurs proximal to the migrating front, contiguous tubular sprouts anastomose to form functionally capillary loops, parallel with the synthesis of the new basement membrane, and the recruitment of pericytes. In Ref. [12], Paku and Paweletz integrated the schema by Ausprunk and Folkman by means of ultrastructural observations, as follows: (1) a structural alteration of the basement membrane occurs, characterized by the loss of electron density over the entire circumference of the dilated mother vessel, followed by a partial degradation of the basement membrane at places where endothelial cell processes are projecting into the connective tissue; (2) endothelial cells migrate arranged in parallel, maintaining their basal-luminal polarity, forming a slit-like lumen and sealed by intact interendothelial junctions; (3) basement membrane is deposited continuously by the polarized endothelial cell while only the tip of the growing capillary bud is devoid of basement membrane; and (4) proliferating pericyte migrate along the basement membrane of the capillary bud, resulting in a complete coverage of the new vessel.

Initiation of sprouting require a growth signal usually an increased level of one of the VEGFs, as the basement membrane is degraded and the endothelial cells start to detach, the vessels becomes leaky. As the endothelial cells proliferate, they migrate in the surrounding stroma and they arrange themselves into a stalk. As the length of the cell row increases, the newly formed endothelium differentiates into tip and stalk cells bearing different morphologies and functional properties (Fig. 1.2). Endothelial tip cells primarily migrate but proliferate only minimally, in contrast to the proliferating endothelial stalk cells [14]. Tip cells have been compared to axonal growth cones during neurite outgrowth. The phenotypic specialization of endothelial cells as tip or stalk cells is very transient and reversible, depending on the balance between proangiogenic factors, such as VEGF and Jagged-1 (JAG-1), and suppressors of endothelial cell proliferation, such as delta-like ligand 4 (Dll-4)-Notch activity [15,16]. Endothelial cells are exposed to VEGF-A which leads to an activation of VEGF receptor-2 (VEGFR-2) and Dll4 in exposed cells. Lateral inhibition between activated cells mediated by Delta-Notch signaling enhances relative differences between neighboring cells and triggers the formation of a single tip cell [13]. The stalk cells induce deposit of basement membrane and recruit pericytes while becoming quiescent, thus stabilizing the newly formed vessel.

Figure 1.2 The functional specialization of endothelial cells during the sprouting process. VEGF-A induces the formation and extension of filopodia as well as the expression of Dll4 protein in the tip cells. Tip cells express high levels of Dll-4, PDGF-B, UNC5b, VEGFR-2, and VEGFR-3, and have low levels of Notch signaling activity. During both mouse and zebrafish angiogenesis, VEGFR-3 is most strongly expressed in the leading tip cell and is downregulated by Notch signaling in the stalk cell. Stalk cells produce fewer filopodia, are more proliferative, form tubes and branches, and form a vascular lumen. They also establish junctions with neighboring cells and synthesize basement membrane components. Stalk cells have high levels of Notch signaling activity and elevated expression of JAG-1. Nrarp is a downstream of Notch that counteracts Notch signaling and is expressed in stalk cells at branch points. Adherens junction formation is associated with the inhibition of endothelial cell migration in monolayers. This process is mediated by VE-cadherin. Another cell adhesion molecule expressed in endothelial cells is the N-cadherin. VE-cadherin is strictly required for the polarization of endothelial cells. Endothelial cell polarization starts with the delivery of deadhesive apical glycoproteins to the cell–cell contact via exocytosis. Deadhesive molecules include CD34-sialomucins, such as CD34 and PODXL. Source: Reproduced from Ribatti D, Crivellato E. Sprouting angiogenesis: a reappraisal. Dev Biol 2012;372:157–65 [13].

During the transition from active sprouting to quiescence endothelial cells, tip cell adopts a phalanx phenotype (so-called as the endothelial cells form an ordered monolayer reminiscent of the military phalanx formation of the ancient Greek soldiers) that acquires a lumen, nonproliferating, and immobile cells, which promotes vessel integrity and stabilizes the vasculature through increased cell adhesion and dampened response to VEGF (Fig. 1.2) [17].

Lumen formation, essential to allow a flow of blood through the new vessel, involves a complex molecular mechanism composed of endothelial cell repulsion at the cell–cell contacts within the endothelial cell cords, junctional rearrangement, and endothelial cell shape change [18]. After the vascular lumen has been established, blood initiates to flow through the newly formed vessel. As angiogenesis progress, some of newly formed vessels may become redundant and regresses: this process is called remodeling.

Remodeling involves the regression of some of the newly formed vessels as well as changes in the diameter of vessel lumens and vascular wall thickening. Remodeling determines the formation of large and small vessels, the establishment of directional flow, the association with mural cells (pericytes and smooth muscle cells), and the adjustment of vascular density to meet the nutritional requirements of the surrounding tissue. Pruning involves the removal of excess of endothelial cells which form redundant channels and occurs physiologically during embryonic development and during corpus luteum regression, pathologically in intratumor angiogenesis and upon therapeutic VEGF manipulation [19]. In fact, in both preclinical and clinical settings, anti-VEGF drugs induce remodeling of tumor blood vessels leading to a more normalized vasculature [20]. As a consequence of the structural and functional normalization of tumor blood vessels, blood flow is increased and cytotoxic drugs can more easily be delivered to the tumor.

Pruning and remodeling of the vascular network may be stimulated by tissue-derived signaling molecules and blood flow conditions (e.g., wall-shear stress and pressure). The stabilization of the newly formed vessel and the maintenance of the existing vasculature are late events in the angiogenic process. Pericyte adhesion to native capillaries and endothelial cell wrapping by surrounding pericytes are basic events in blood vessel stabilization and maturation (Fig. 1.3) [21]. Pericytes provide structural support for the capillary wall, acting as a scaffold along which endothelial cells migrate during sprouting. Reduced pericyte coverage is associated with metastasis, and overexpression of PDGF-B increases pericyte coverage and inhibits tumor growth.

Figure 1.3 Schematic drawing that illustrates the paracrine interactions occurring between pericyte precursor cells and endothelial cells in PDGF-mediated angiogenesis. Endothelial cells secrete PDGF-B that causes pericyte precursor cell proliferation and migration through activation of PDGFR-β. Pericytes surround and cover early endothelial tubes. By contrast, endothelial cells in vascular sprouts release VEGF, which in turn mediates suppression of PDGFR-β signaling through the induction of VEGFR-2/PDGFR-β complexes. This pathway abrogates pericyte coverage of endothelial sprouts leading to vascular instability and regression. Source: Reproduced from Ribatti D, Nico B, Crivellato, E. The role of pericytes in angiogenesis. Int J Dev Biol