**2. Neovessels formation in multiple myeloma**

Neovessels in the BM of patients with active MM appear thin, tortuous, and arborized and are highly permeable showing fenestrae, vesicles, transcellular holes, widened intercellular junctions, and a discontinuous basement membrane [16]. These alterations are consequent to the rapid neovascularization induced by tumor plasma cells by mean of three different processes: (i) angiogenesis, (ii) vasculogenesis, and (iii) vasculogenic mimicry [17].

#### **2.1. Angiogenesis**

**1. Introduction**

98 Update on Multiple Myeloma

and treatment response [3, 4].

and ECM components [5–9].

of MM [11–15].

In the past decades, myeloma research has been focalized on the malignant cell leading to the identification of various genes (i.e., oncogenes and tumor suppressor genes) and of signaling pathways by which the identified genes themselves control survival and proliferation of cancer cells [1–4]. More recently, newly developed technologies have enabled us to investigate cancer cells at the genomic level. Such gene profiling studies are providing insight into the pathogenesis and risk stratification of plasma cell diseases, and help to predict both prognosis

Cancer cells interact with all cells composing the microenvironment and with components of extracellular matrix (ECM) [5, 6]. These interactions play the most important role in the epigenetic control of the malignant phenotype, as in primary sites as in the metastatic ones [6, 7]. Moreover, interactions between host cells in the niche microenvironment and ECM represent an intense area of research [5–9]. The aim of these studies is the better understanding of the pathophysiological events in the tumor process, including malignant cells, surrounding cells,

Multiple myeloma (MM) is a malignancy of plasma cells that home to and expand in the bone marrow (BM) [9]. MM is characterized by a high genomic heterogeneity but, generally, it shows the same histological features, [8–10]. The interactions between MM plasma cells and BM microenvironment (stromal cells, hematopoietic cells, ahnd ECM) represent near genetic modifications an important factor for disease progression [11–14]. Pathophysiological interactions of myeloma cells with the components of BM microenvironment are pivotal during the progression-associated bone disease and neovascularization [13]. These interactions are mediated by autocrine and paracrine loops that regulate multiple signaling pathways and influence many fundamental biological aspects of the malignant phenotype (i.e., apoptosis,

Neovascularization is the formation of new vessels from existing ones (angiogenesis) or from endothelial precursors (vasculogenesis) and represents one of the principal biological process controlled by the interactions between plasma cells and BM microenvironment. It is a constant hallmark of disease progression [11–15]. Angiogenesis is controlled by several angiogenic cytokines [14, 15]. The major of these are vascular endothelial growth factor (VEGF), fibroblast growth factor-2 (FGF-2), and hepatocyte growth factor (HGF) directly secreted not

The observation of an increased BM angiogenesis in MM, an overexpression of angiogenic cytokines, and their correlation with disease activity, overall survival and the development of new antiangiogenic compounds, led to consider angiogenesis as a new target in the treatment

Neovessels in the BM of patients with active MM appear thin, tortuous, and arborized and are highly permeable showing fenestrae, vesicles, transcellular holes, widened intercellular

survival, proliferation, invasion, bone damage, and angiogenesis) [12–14].

only by the tumor plasma cells but also by stromal cells [14, 15].

**2. Neovessels formation in multiple myeloma**

In 1994, Vacca and colleagues [16] demonstrated for the first time that BM microvascular density was significantly increased in MM compared to monoclonal gammopathy of undetermined significance (MGUS) and moreover in active (diagnosis, relapse, and leukemic phase) versus non-active (complete/objective response and plateau) MM. The authors first hypothesized that progression from MGUS to MM is accompanied by an increase in BM microvascular density. Subsequent studies by other groups confirmed the observation of increased angiogenesis in active MM compared to healthy individuals or MGUS patients [17–20].

Angiogenesis is the sprouting of new blood vessels from pre-existing ones and is finely regulated [17, 18]. Angiogenesis is essential for tumor growth, invasion, and metastasis starting from the balanced early avascular phase of cancer up to being uncontrolled and unlimited in time during the vascular phase [6, 17, 20]. The angiogenic switch from the avascular to the vascular phase is controlled by the many oncogenes, among which c-myc, c-fos, c-jun, and ets-1 have been recognized [20, 21]. They are activated in tumor plasma cells as a consequence of immunoglobulin translocations and genetic instability [20, 21], and induce the angiogenic phenotype in MM plasma cells [21]. MM plasma cells become CD45-negative and begin to produce VEGF [22]. The same angiogenic switch represents a crucial event for the progression from asymptomatic to symptomatic MM [23]. So, angiogenesis represents an important process in MM progression as well as an important prognostic factor [17, 19, 20].

#### **2.2. Vasculogenesis**

Vasculogenesis is responsible for the primary development of the vascular system during embryogenesis and is fundamental for the formation of the yolk sac vasculature, of the heart, and of the dorsal aortae [24]. It derives from the differentiation of endothelial progenitors, namely angioblasts, deriving from mesoderm and aggregate into a primitive capillary plexus [24]. Important evidence suggests that vasculogenesis contributes to neovascularization in the bone marrow of MM patients [25–27]. In fact, putative endothelial progenitor cells have been isolated from peripheral blood and several studies have suggested that angioblasts contribute to the formation of tumor neovessels [25, 26]. It has been demonstrated that when CD34+ VEGFR-2+ cells isolated from peripheral blood of MM patients were cultured on fibronectincoated plates and exposed to angiogenic cytokines, they acquire a typical spindle-shaped morphology and express endothelial cell markers (CD34, CD31, Flk-1, Tie-2, and E-selectins) [26]. Moreover, in the BM of MM patients, but not of MGUS patients, some endothelial cells of neovessel wall express on their surface the typical endothelial cell markers: factor VIII-related antigen (FVIII-RA), vascular endothelial-cadherin (VE-cadherin), VEGFR-2, and TIE/Tek, as well as the CD133 staminal antigen whose expression was found in the microvascular wall together with FVIII-RA or VE-cadherin in some active MM patients [26].

#### **2.3. Vasculogenic mimicry**

The phenomenon called "vasculogenesis mimicry" represent a model of neovascularization in aggressive solid and hematologic tumors, owing to the specific capacity of malignant cells and other non-endothelial cells to form vessel-like networks [27–33]. This phenomenon can be an escape mechanism for antiangiogenic drugs that are now incorporated into standard clinical practice [29]. Also, inflammatory cells (i.e. macrophages and mast cells) participate in this process [30–33] because they can generate endothelial progenitor and can produce functional capillary-like structures in vitro when stimulated by VEGF and/or FGF-2 [30–36].

MM endothelial cells are functionally different from MGUS endothelial cells, are characterized by an overangiogenic phenotype, and resemble transformed cells because of they downregulate or upregulate some genes like tumor cells [41]. These changes are influenced by the MM microenvironmental and/or plasma cells factors (such as hypoxia, inflammation, expression of multiple cytokines, growth factors, etc.) that render endothelial cells unstable and heterogeneous, with progressive characteristics comparable with a cancer cell. In addition, those factors may have genetic causes and consequences (i.e., increased expression of oncogenes and loss of tumor suppressor genes) [41]. This reciprocal interrelationship and heterogeneity may translate into a site- and stage-specific changes in the regulation of BM-microvessel density and angiogenesis dependence, and ultimately to changes in the proliferation and antiapoptotic potential of MM tumor cells, even in the same patient [17]. Moreover, the overangiogenic activity of MM endothelial cells is linked to a well-defined protein expression [42]. This proteomic signature renders MM endothelial cells very similarly to transformed (such as tumor) cells than normal endothelial cells, confirming the results obtained in the studies at the genomic level [41].

Angiogenesis and Antiangiogenesis in Multiple Myeloma

http://dx.doi.org/10.5772/intechopen.76931

101

The stromal microenvironment is characterized by a modified extracellular matrix, enhanced angiogenesis, and cells with an activated phenotype, including fibroblasts referred to as 'activated myofibroblasts' or 'cancer-associated fibroblasts' (CAFs) [6, 43–48]. In the poorly vascularized hypoxic or necrotic areas of tumors, they accumulate numerous tumor-associated fibroblasts [43, 44]. They respond to experimental hypoxia by producing high amounts of VEGF-2, FGF-2, tumor necrosis factor alpha (TNF-α), urokinase and matrix metalloproteinases and synthesizing inducible nitric oxide synthase, which increases blood flow and promotes angiogenesis [45]. In breast, prostate, and pancreatic carcinomas, the number of CAFs is associated with an increased malignancy grade, tumor progression, and poor prognosis [46]. CAFs are heterogeneous [45] and display phenotypes similar to those of myofibroblasts derived from quiescent fibroblasts that have undergone activation during tissue remodeling in wound healing, fibrosis [47]. CAFs can arise from resident fibroblasts, BM-derived progenitor cells and cells undergoing the endothelial-mesenchymal transition (EndMT) or mesenchymal transition (MT) [47] in the BM of MM patients, an important interplay between CAFs and plasma cells during MM initiation and progression has been demonstrated [48]. Plasma cells induce and maintain the CAF-activated phenotype, which, in turn, supports tumor progression by promoting extracellular matrix remodeling, cell proliferation, apoptosis resistance, and angiogenesis [48]. Moreover, CAFs play a key role in the bortezomib resistance of MM cells. The protective effect is not related to cell-to-cell interactions but to the ability of bortezomib to trigger bortezomib-resistant CAFs to release in the BM microenvironment several cytokine/growth factors with antiapoptotic effects, such as IGF-1, IL-6 IL-8, and exosomes [48].

There are several published data on the association between macrophage infiltration, vascu-

In patients with active MM, macrophages contribute to building neovessels through vasculogenic mimicry [35]. Under a synergistic stimulation by VEGF/FGF-2, they undergo a

**3.2. Fibroblasts**

**3.3. Macrophages**

larity, and prognosis in cancer [49–52].

Scavelli et al. demonstrated that when exposed to VEGF and FGF-2, macrophages isolated from BM of myeloma patients develop phenotypic and biologic properties similar to those of endothelial cells, and exhibit numerous cytoplasmic extroversions arranged in tube-like structures [35]. Finally, in BM biopsies of MM, the participation of inflammatory cells in the formation of the capillary network has been directly demonstrated [35, 36].
