Stem Cell Rev DOI 10.1007/s12015-008-9009-1

Targeting Cancer Stem Cells to Modulate Alternative Vascularization Mechanisms Elena Monzani & Caterina AM La Porta

# Humana Press Inc. 2008

Abstract Recently, many papers have shown that tumor vascularization can be explained by angiogenesis, recruitment, cooption, vasculogenic mimicry and by mosaic vessels. In particular, vasculogenic mimicry seems to be different from mosaic blood vessels, where tumor cells form a part of the surface of the vessel while the remaining part is covered by endothelium. In this case, tumor cells in apparent contact with the lumen do not show an endothelial phenotype. More recently, vasculogenic mimicry was proposed to occur in patients with multiple myeloma due to bone marrow macrophages. Herein, all these data are, for the first time, discussed critically in comparison to cancer stem cells—which show high trans-differentiative capacity— and bone-marrow derived stem cells. In fact, the presence of alternative vasculogenic patterns might be due to the presence of stem cell population (cancer stem cells or bone-marrow stem cells). In this connection, the literature is discussed extensively and possible models are proposed. Pharmacological perspectives will also discuss. Keywords Cancer stem cell . Vasculogenic mimicry . Vascularization . Endothelial progenitor cells . Melanoma Recently the cancer stem cell (CSC) hypothesis suggests that neoplastic clones are maintained exclusively by a rare fraction of cells with stem cell proprieties. Like normal stem cells, these rare CSCs possess the extensive proliferative and self-renewal potential necessary to create a new tumor and generate a hierarchy of phenotypically diverse E. Monzani : C. A. La Porta (*) Molecular Oncology Laboratory, Department of Biomolecular Science and Biotechnology, University of Milan, 20133 Milan, Italy e-mail: [email protected]

downstream cells, which are successively more limited in these properties. On the other hand, like as normal tissue stem cells, CSCs should display the ability to undergo a broad range of differentiation events. Recently a stem cell population was found in melanoma biopsy [1] and it was demonstrated to increase during melanoma progression [2]. Considering that melanoma is one of the most aggressive forms of skin cancer and it is strongly resistant to conventional therapeutic agents [3], the molecular biology of CSCs opens interesting new perspectives from the pharmacological point of view [3].

Non Sprouting Angiogenesis and Melanoma Until recently, vascularization of malignant tumors was considered the exclusive result of directed capillary ingrowth. However, recent advances have been made in identifying the processes involved in vascular remodelling. The simplistic model of an invading capillary sprout has been deemed insufficient to describe the entire spectrum of morphogenic and molecular events required to form a neovascular network. The different forms of “non sprouting angiogenesis” are vascular co-option of pre-existing vessels, intussusceptive microvascular growth, postnatal vasculogenesis, glomeruloid angiogenesis and vasculogenic mimicry. In vessel co-option a subset of tumors, organised around few functional vessels, rapidly coopts existing host vessels to form an initially well-vascularized tumor mass that, regressing, leading to a secondarily avascular tumor and massive tumor cell loss [4–7]. This process has now been observed in different tumour types like murine Lewis lung carcinoma, murine ovarian cancer, human melanoma and human Kaposi sarcoma [4–7]. However, the remaining

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tumor is ultimately rescued by robust angiogenesis at the tumor margin [4, 8]. In vascular co-option, therefore, fused vessels together with the newly synthesized connective tissue, is incorporated into the tumor [9]. The expression patterns of Vascular endothelial growth factor (VEGF) and the natural Tie-2 receptor antagonist Ang-2 seem to play complementary and coordinated roles in this process [4, 10]. An additional mode of tumor angiogenesis different from sprouting, is intussusceptive (growth within itself) angiogenenesis (IMG), described approximately 18 years ago. It is a fast process, occurs within hours or even minutes, that does not need proliferation of endothelial cells but the remodelling of these cells by increasing in volume, in complexity (intussusceptive microvascular growth), and becoming thinner; for this region it is more economical from the energetic and metabolic point of view [11]. It is characterized by physiological levels of vascular transpermeability, a condition essential for uncompromized tissue and organ function and it may represent the unique means in the mechanisms of vascular tree formation and vascular remodelling [11]. Recently, IMG growth has been reported during angiogenesis in the mesenteric and peritoneal tissue induced by tumor ascites fluid [12] and in a human colon adenocarcinoma [13]. The fact that IMG angiogenesis does not involve intense cell proliferation, implies that neo-vascularisation by this mechanism would be resistant to cytotoxic treatment. As a consequence, cancerous cells could still cover their nutritional needs and continue to grow [14]. In general, the mechanisms that are involved in the regulation of intussusception are poorly understood but there is good evidence that flow alterations have a major influence in initiating the process of pillar formation [11]. The rapid vascular remodelling by IMG could possibly contribute to intermittent blood flow in tumors. Factors, that are known to be involved in these interactions in sprouting angiogenesis, such as the angiopoietins and their Tie receptors [15], platelet derived growth factor-B [16], monocyte chemotactic protein-1 [17], ephrins and EphB-receptors [18], are candidates for the mediation of IMG [11]. Postnatal angiogenesis is due to circulating bone marrow-derived endothelial progenitor cells (EPCs) that home to sites of physiological and pathological neovascularization and differentiate into endothelial cells. Until 1997, the growth of new blood vessels in adults was considered to exclusively occur through the mechanism of sprouting and intussusceptive angiogenesis. This paradigm of vascular development changed after the discovery, by Asahara et al. of CD34-enriched subpopulation of mononuclear blood cells named EPCs or angioblasts [19], located in a distinct zone of the vascular wall identified to be localized between smooth muscle and adventitial layer of human adult vascular wall [20]. Vascular wall areas

containing EPCs are found in large and middle sized arteries and veins of different organs [20]. These data suggest the existence of a ‘vasculogenic zone’ in the wall of adult human blood vessels not directly exposed to shear stress, which may serve as niche for progenitor cells for postnatal vasculogenesis, contributing to tumor vascularization and local immune response [20]. These cells express several endothelial specific markers like CD31, VEGF receptor (R)-2, Tie-2 [21] and CD14 [22] and have a role in maintenance of vascular integrity (postnatal vasculogenesis) [19]. On the other hand, bone marrow-derived circulating EPCs or angioblasts have the capacity to proliferate, migrate and differentiate into endothelial lineage cells and can contribute to tumor angiogenesis and growth of certain tumours [23]. Their discovery led to the new concept that vasculogenesis and angiogenesis may occur simultaneously in the postnatal life because these cells are able to differentiate when needed into vascular endothelium, through a mechanism recapitulating embryonic vasculogenesis [21, 24]. In particular, recruitment of EPCs from the bone marrow into the blood system, triggered by the increased availability of angiogenic growth factors or chemokines produced by tumours, such as VEGF and angiopoietin [25, 26], induce their incorporation into the sites of active neovascularization during tissue ischemia, vascular trauma or tumor growth and then promote differentiation in endothelial cells (ECs) [24, 27, 28]. EC progenitors have been identified in the blood vessels of mouse colon tumors and in xenografts of lymphoma, Lewis lung carcinoma, Ewing’s sarcoma, and breast cancer [24, 28–30]. Hypoxia, that can results from the tumour growth, can also mobilize EPCs from the bone marrow in the same way [23]. Some studies have demonstrated an impaired role of EPCs in angiogenesis after specific interventions. In a study by Capillo et al. [31], endostatin was described as a potent inhibitor of mobilization and clonogenic potential of EPCs. Similarly, simultaneous inhibition of VEGFR-2 and VEGFR-1 demonstrated an effective inhibition of mobilization and incorporation of EPCs in tumour vasculature [28]. Glomeruloid microvascular proliferations (GMPs) are focal proliferative buddings of endothelial cells resembling a renal glomerulus [32, 33], that was recently found in 13– 23% of various human tumours (melanoma, breast-, endometrial- and prostate cancer), and this vascular signature was significantly associated with an impaired prognosis [34]. The function of these structural units is not well understood. Formation of GMPs has been considered a dysregulated angiogenic response, due to imbalanced expression of angiogenic stimulators and inhibitors, but little is known about its detailed pathogenesis [35]. Presence of GMPs was associated with increased expression in tumour endothelium of the VEGF-A, receptors KDR, FLT-1 and neuropilin-1, as well as VEGF-D protein [36].

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Finally vasculogenic mimicry was first described by the unique ability of aggressive melanoma cells to express an endothelial phenotype and to form vessel-like networks in three dimensional cultures, “mimicking” the pattern of embryonic vascular networks and recapitulating the patterned networks seen in patients with aggressive tumors correlated with poor prognosis (Fig. 1) [37]. In fact, the word ‘‘vasculogenic’’ was selected to indicate the generation of the pathway de novo and ‘‘mimicry’’ was used because the tumour uses cell pathways for transporting

a tumor

fluid in tissues that were clearly not blood vessels. Additional studies have reported vasculogenic mimicry in several other tumor types, including breast, prostate, ovarian, chorio-, lung carcinomas, synovial-, rhabdomyosarcoma, Ewing sarcomas and paeochromocytoma [38]. Interestingly, global gene analysis of aggressive and poorly aggressive human cutaneous and uveal melanoma cell lines unexpectedly revealed the ability of aggressive tumor cells to express genes associated with multiple cellular phenotypes and stem cells including endothelial, epithelial, fibroblast, and several other cell types [38–40]. These findings were interpreted considering that aggressive melanoma cells might revert to an undifferentiated, embryonic-like phenotype [37]. In this connection, aggressive melanoma cells express endothelium-associated genes and form extracellular matrix rich vasculogenic like networks in three dimensional cultures [41–49]. Microarray analysis indicated a genetic reversion of aggressive melanoma cells to an undifferentiated embryonic-like phenotype [38]. Endothelium associated genes such as VE-cadherin, Ephrin A2 and tissue factor pathway inhibitors, CD34, tyrosine kinase receptor 1, neuropilin 1, E-selectin and endoglin (CD105) had a more than twofold increased expression in

Blood

Other types of cells

b

EPC VEGF

tumor angiogenesis

Fig. 1 Blood vessels form through angiogenesis when sprouts grow out from existing vessels. However, alternative vascularization mechanisms occur in tumors such as vasculogenic mimicry. Recent evidences demonstrate that endothelial precursor cells (EPC) or macrophage cells can contribute to build neovessels (a). Factors such as VEGF produced by tumors may result in mobilization of these cells in the peripheral circulation and enhance their recruitment into the tumor vasculature (b). In red endothelial cells, in blue other types of cells (i.e., tumor cell, macrophages, EPC)

Table 1 Summary of angiogenic/vasculogeic factors as well as adhesion and stem cell markers expressed by human melanoma cancer stem cells [1] Gene

Expressed (+)

VEGF Flt-1 Flk-1 Nrp-1 Nrp-2 Ang-1 Ang-2 Tie-1 Tie-2 Ephrine A2 CD31 Endogline Notch 4 JAG1 JAG2 DLL1 DLL3 DLL4 Sox18 Semaphorine 3A Semaphorine 3F E-cadherin N-cadherin P-cadherin

+ − + + + + + − + + + + + − − − − − − + − − + −

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vasculogenic mimicry positive cells, while genes related to a melanocytic phenotype, like Melan-A, microphthalmia associated transcription factor and tyrosinase, were more than 20-fold downregulated [8]. Another possibility is that the endothelial cell lining is replaced by tumour cells, resulting in the so-called ‘‘mosaic vessels’’, where both endothelial and tumour cells contribute to the formation of the vascular tube [50]. Mosaic blood vessels constitute 4% of the total surface area of tumor microcirculation. Some researchers believe that they are not related to vasculogenic mimicry (VM) channels and are a mosaicism of endothelial and tumor cells [50]. In contrast, Zhang and co-workers proposed a three-stage phenomenon among VM channels, mosaic blood vessels, and endothelium-dependent blood vessels, according to which all three patterns participate in tumor blood supply [51].

Vasculogenic Mimicry and Cancer Stem Cells: New Therapeutic Perspectives The transdifferentiative capacity of normal stem cells is a common properties of CSCs. Regarding to melanoma CSCs

like as all the other CSCs, they can transdifferentiate into different phenotype [1], they express neurogenic, angiogenic, vasculogenic and also lymphatic markers [1] (Table 1) and they are able to organize pseudovascular network in a physiological assay such as aorta ring [52] (Fig. 2). Thereby, we suggest that CSC subpopulation inside the tumor is able to organize vasculogenic mimicry or a mosaic network in dependence of the environmental condition. In this connection, a transdifferentiative capacity was demonstrated for bone marrow macrophages [53] and for dendritic cells [54], recently. Thereby we believe that, at least for melanoma, VM or mosaic vessel is due to the transdifferentiative capacity of CSCs subpopulation. The evidence of such a subpopulation opens of course new perspectives for the treatment of melanoma. In particular since the expression of proangiogenic/lymphatic factors might confer additional transdifferentiative capacity and a more aggressive phenotype, the use of anti-angiogenic drugs in combination with drugs acting on specific target of CSCs subpopulation, open interesting new therapeutic perspectives. To this aim, many efforts need to be done to better understand the biology of CSCs and to identify specific targets expressed by this subpopulation.

M M Aorta

Aorta

M

Aorta Fig. 2 CSC melanoma cells (WM115) was able to organize a pseudovascular network in rat aorta ring model (vasculogenic mimicry). Rat aorta ring and WM115 spheroids (M) were cocultivated in the presence of VEGF (20 ng/ml) in eight multi-wells collagen-coated. After 8 days, the cells were fixed, treated with 0.5% TRITON X-100 and stained with anti-VE cadherin (1:10) overnight at

M Aorta 4°C to detect human melanoma cells (red) or with fluorescein Griffonia (Bandeiraea) simplicifonia lactin 1, isolectin B4 (Vector) for 30 min at room temperature to detect rat endothelium (green). Nuclei were counterstained with DAPI (1:100) for 30 min. Arrowheads, rat aorta vessels; arrows, human melanoma vessels

Stem Cell Rev Acknowledgment We thank Riccardo Bazzotti for the relevant contribution to the vasculogenic mimicry picture. 18.

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