**3. The BM microenvironment**

The BM microenvironment plays a pivotal role during MM disease progression by mean neovascularization, bone disease, and activity of inflammatory cells. All the BM microenvironment components surround and support MM plasma cells proliferation, migration, and survival, and are implicated in drug resistance [34, 37].

#### **3.1. Endothelial cells**

BM endothelial cells of patients with MM are altered in shape and characterized by different phenotype (in term of expression of cell adhesion molecules, receptors for cytokines and growth factors together with FVIII-RA, and VE-cadherin) from those of normal resting endothelial cells and shows the capacity to proliferate rapidly and spontaneously enhanced angiogenesis [36–39]. In fact, Vacca et al. [38] demonstrated that the phenotype of MM endothelial cells is characterized by expression of surface receptors such as VEGFR-2 and Tie2/Tek (indicators of active angiogenesis), increased expression of the β3-integrin (that plays a pivotal role in the prevention of apoptosis, adhesion to the ECM, proliferation, migration, and capillarogenesis), expression of endoglin (implicated in the expression of the ligand of the plasma cell CD38 (CD31) enhancing plasma cells interaction with the new-formed blood vessels, favoring plasma cells entry into circulation, and disseminate). The expression of a water transporter, namely aquaporin 1, has been also demonstrated [39]. It enhances vascular permeability, facilitates plasma extravasation, increases interstitial pressure, induces hypoxia, and upregulates hypoxia-inducible factor-1 alpha (HIF-1α) and VEGF [39]. Some MM endothelial cells express the CD133 indicating their derivation from a subset of CD133+ progenitor cells which contribute to the formation of blood neovessels [26, 40, 41]. MM plasma cells recruit BM and circulating CD133+ progenitor cells into the tumor microenvironment by mean the release of a high quantity of VEGF, FGF-2, and IGF [26]. In the BM microenvironment, CD133+ progenitor cells differentiate into MM endothelial cells and complete the formation of the new vessel wall [26].

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].

#### **3.2. Fibroblasts**

**2.3. Vasculogenic mimicry**

100 Update on Multiple Myeloma

**3. The BM microenvironment**

**3.1. Endothelial cells**

wall [26].

survival, and are implicated in drug resistance [34, 37].

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

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

The BM microenvironment plays a pivotal role during MM disease progression by mean neovascularization, bone disease, and activity of inflammatory cells. All the BM microenvironment components surround and support MM plasma cells proliferation, migration, and

BM endothelial cells of patients with MM are altered in shape and characterized by different phenotype (in term of expression of cell adhesion molecules, receptors for cytokines and growth factors together with FVIII-RA, and VE-cadherin) from those of normal resting endothelial cells and shows the capacity to proliferate rapidly and spontaneously enhanced angiogenesis [36–39]. In fact, Vacca et al. [38] demonstrated that the phenotype of MM endothelial cells is characterized by expression of surface receptors such as VEGFR-2 and Tie2/Tek (indicators of active angiogenesis), increased expression of the β3-integrin (that plays a pivotal role in the prevention of apoptosis, adhesion to the ECM, proliferation, migration, and capillarogenesis), expression of endoglin (implicated in the expression of the ligand of the plasma cell CD38 (CD31) enhancing plasma cells interaction with the new-formed blood vessels, favoring plasma cells entry into circulation, and disseminate). The expression of a water transporter, namely aquaporin 1, has been also demonstrated [39]. It enhances vascular permeability, facilitates plasma extravasation, increases interstitial pressure, induces hypoxia, and upregulates hypoxia-inducible factor-1 alpha (HIF-1α) and VEGF [39]. Some MM endothelial cells express the CD133 indicating their derivation from a subset of CD133+ progenitor cells which contribute to the formation of blood neovessels [26, 40, 41]. MM plasma cells recruit BM and circulating CD133+ progenitor cells into the tumor microenvironment by mean the release of a high quantity of VEGF, FGF-2, and IGF [26]. In the BM microenvironment, CD133+ progenitor cells differentiate into MM endothelial cells and complete the formation of the new vessel

capillary-like structures in vitro when stimulated by VEGF and/or FGF-2 [30–36].

formation of the capillary network has been directly demonstrated [35, 36].

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].

#### **3.3. Macrophages**

There are several published data on the association between macrophage infiltration, vascularity, and prognosis in cancer [49–52].

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 phenotypic and functional adaptation but retain their own CD14 and CD68 lineage markers which can be evidenced in the neovessel wall [35]. They display oblong and spindle shape with thin cytoplasmic expansions, some of which are either arranged to form a microvessellike lumen or anastomosed with each other and with those of nearby macrophages to form tubular-like structures [35]. In the BM of patients with active MM, plasma cells secrete VEGF and FGF-2 that bind to VEGFR-1 and FGFR-1, -2 and -3 expressed on monocytes/macrophages surface and induce monocyte migration and infiltration and macrophage to secrete their own VEGF and FGF-2 [17, 35, 49, 52]. These cytokine circuits further promote angiogenesis and vasculogenic mimicry [17].

in the MM bone marrow and that the inhibition of VEGF produced by plasma and stromal cells and osteopontin produced by osteoclasts, reduce angiogenesis and osteoclastogenic

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Some issues demonstrated that CD38 is expressed by effectors and inhibitory cells, and by both osteoblasts and demonstrating the role of CD38 in bone remodeling, in mice and rabbit models [60] as in human [61]. Horenstein AL. et al. [62] recently shown that the ectoenzymatic network CD73/CD203a is active even in MM bone niche in the alternative production of ADO, which levels correlate with disease aggressiveness and ISS staging of MM patients [61]. Moreover, the role of CD38 in human OC differentiation and as well as the reduction of the area of osteoclast bone resorption in vitro by the anti-CD38 monoclonal antibody daratumumab have been also demonstrated [63]. Overall these findings suggest the possibility of a role of CD38 during osteoclast formation supporting the potential activity of daratumumab on MM bone disease and on the protection of MM plasma cells by stromal cells of the bone niche [60–63].

Hematopoietic stem cells (HSCs) reside in the BM in the endosteum niche and in the vascular niche, where they self-renew and differentiate into mature blood cells [64, 65]. This is a finely controlled-process by mean numerous signals from the bone marrow components [64, 65]. In MM, the BM niches (endosteum and vascular) components play a pivotal role in the regulation of vasculogenesis and angiogenesis [11, 14, 26, 64], and alterations of the signals in niche

In the BM of patients with MM, the expression of the CD133 staminal antigen in some cells of the neovessel wall has been demonstrated [26, 38]. Moreover, a subset of CD34+/CD133+ cells mobilized in the peripheral blood for collection during transplant procedure express VEGFR-2 and are able to differentiate in mature endothelial cells in appropriate culture conditions [28].

Various studies have demonstrated that endothelial progenitor cells (EPCs) can be isolated from patients with MM [40, 66–69] and contribute to the formation of new blood vessels [40]. Moreover, circulating EPCs expressing CD146+, CD105+, and CD34+ are increased in MM

Rigolin et al. [69] have hypothesized a possible origin of EPCs and plasma cells from a common progenitor namely hemangioblast in MM patients. In their work, they demonstrated that EPCs, isolated from MM patients presents the 13q14 deletion and the great part of them are positive for the CD133 [69]. Finally, some evidence indicates a prognostic significance of the

Mesenchymal stem cells (MSCs) are the major component of BM stroma [11, 57, 70–72]. These cells, of unclear origin in MM [71], are potentially able to differentiate into multiple histotypes (i.e. fibroblasts, adipocytes, chondrocytes, and osteoblasts) and in the BM form specialized niches namely "vascular niche" and "osteoblast niche" [57, 70, 71]. MSCs support tumor cell

microenvironment modulate myeloma progression and spread [26, 64].

activity by vascular endothelial cells [11, 57].

**3.6. Hematopoietic stem cells**

**3.7. Endothelial progenitor cells**

**3.8. Mesenchymal stem cells**

patients compared to healthy controls [66, 67].

circulating EPCs also after treatment with new drugs [66, 68].

#### **3.4. Mast cells**

Mast cells recruitment in the tumor bed has been associated with enhanced growth and invasion in solid and hematological malignancies [49, 53–56]. In MM, tumor plasma cells secrete stem cell factor (SCF), FGF-2, VEGF-2, and platelet-derived growth factor (PDGF) that recruit mast cells [14, 52]. The granules of mast cells contain several angiogenic factors: (i) tryptase and chymase that favor the formation of capillary structures via a direct action on endothelial cells and activate latent metalloproteinases and plasminogen activator [53]; (ii) heparin that induces endothelial cell proliferation and migration [54]; (iii) histamine, that has a direct angiogenic effect, induces VEGF production in the granulation tissue [54] and contributes to the hyperpermeability of newly formed microvessels, increasing leakage of plasma proteins and hence deposition of fibrin whose degradation products are angiogenic in vivo [55]; and (iv) TGF-β, TNF-α, IL-8, FGF-2, and VEGF, which are all angiogenic factors [52, 53]. Moreover, in the new vessels wall typical tryptase-positive mast cells connected by a junctional system with the endothelial cells can be evidenced. As macrophage, mast cells keep their lineage marker indicating their adaptation to contribute to vasculogenesis mimicry [33]. In patients with MM BM angiogenesis, evaluated as microvessel area, and mast cells counts are highly correlated [53, 56] and both parameters increase simultaneously in the active phase of disease [56].

#### **3.5. Osteoclasts and osteoblasts**

MM plasma cells that home and expand in the BM causes an unbalanced bone remodeling that induces osteolytic lesions and causes pain, the main symptom of MM [34]. In MM, plasma celldependent alterations of Runx2 and the Wnt pathways induce the differentiation of resident macrophages in osteoclasts and plasma cells themselves can transdifferentiate to functional osteoclasts [57, 58]. Bone disease results from the local production of osteoclast-activating factors (OAF), as well as IL-6, IL-1α or -1β, IL-11, TNF-α, TNF-β, and M-CSF [11]. In particular, the receptor activator of nuclear factor ligand (RANKL), the decoy receptor osteoprotegerin (OPG), its receptor (RANKR), and the chemokine macrophage inflammatory protein-1α (MIP-1α) trigger differentiation and activation signals in osteoclasts precursors, and thus promoting bone resorption [48]. Adhesion molecules, such as β1 integrins, mediate the binding of MM plasma cells to stromal cells and VCAM1 induces overexpression of RANKL in both cell types and suppresses OPG production by stromal cells. Furthermore, plasma cells interfere with the regulation of the bone resorption by the secretion of IL-7 and DKK1, a Wnt inhibitor [59].

It has demonstrated a close link between myeloma cells, osteoclasts, and vascular endothelial cells to form a vicious cycle between bone destruction, angiogenesis, and myeloma expansion in the MM bone marrow and that the inhibition of VEGF produced by plasma and stromal cells and osteopontin produced by osteoclasts, reduce angiogenesis and osteoclastogenic activity by vascular endothelial cells [11, 57].

Some issues demonstrated that CD38 is expressed by effectors and inhibitory cells, and by both osteoblasts and demonstrating the role of CD38 in bone remodeling, in mice and rabbit models [60] as in human [61]. Horenstein AL. et al. [62] recently shown that the ectoenzymatic network CD73/CD203a is active even in MM bone niche in the alternative production of ADO, which levels correlate with disease aggressiveness and ISS staging of MM patients [61]. Moreover, the role of CD38 in human OC differentiation and as well as the reduction of the area of osteoclast bone resorption in vitro by the anti-CD38 monoclonal antibody daratumumab have been also demonstrated [63]. Overall these findings suggest the possibility of a role of CD38 during osteoclast formation supporting the potential activity of daratumumab on MM bone disease and on the protection of MM plasma cells by stromal cells of the bone niche [60–63].

#### **3.6. Hematopoietic stem cells**

phenotypic and functional adaptation but retain their own CD14 and CD68 lineage markers which can be evidenced in the neovessel wall [35]. They display oblong and spindle shape with thin cytoplasmic expansions, some of which are either arranged to form a microvessellike lumen or anastomosed with each other and with those of nearby macrophages to form tubular-like structures [35]. In the BM of patients with active MM, plasma cells secrete VEGF and FGF-2 that bind to VEGFR-1 and FGFR-1, -2 and -3 expressed on monocytes/macrophages surface and induce monocyte migration and infiltration and macrophage to secrete their own VEGF and FGF-2 [17, 35, 49, 52]. These cytokine circuits further promote angiogenesis and

Mast cells recruitment in the tumor bed has been associated with enhanced growth and invasion in solid and hematological malignancies [49, 53–56]. In MM, tumor plasma cells secrete stem cell factor (SCF), FGF-2, VEGF-2, and platelet-derived growth factor (PDGF) that recruit mast cells [14, 52]. The granules of mast cells contain several angiogenic factors: (i) tryptase and chymase that favor the formation of capillary structures via a direct action on endothelial cells and activate latent metalloproteinases and plasminogen activator [53]; (ii) heparin that induces endothelial cell proliferation and migration [54]; (iii) histamine, that has a direct angiogenic effect, induces VEGF production in the granulation tissue [54] and contributes to the hyperpermeability of newly formed microvessels, increasing leakage of plasma proteins and hence deposition of fibrin whose degradation products are angiogenic in vivo [55]; and (iv) TGF-β, TNF-α, IL-8, FGF-2, and VEGF, which are all angiogenic factors [52, 53]. Moreover, in the new vessels wall typical tryptase-positive mast cells connected by a junctional system with the endothelial cells can be evidenced. As macrophage, mast cells keep their lineage marker indicating their adaptation to contribute to vasculogenesis mimicry [33]. In patients with MM BM angiogenesis, evaluated as microvessel area, and mast cells counts are highly correlated [53, 56] and both parameters increase simultaneously in the active phase of disease [56].

MM plasma cells that home and expand in the BM causes an unbalanced bone remodeling that induces osteolytic lesions and causes pain, the main symptom of MM [34]. In MM, plasma celldependent alterations of Runx2 and the Wnt pathways induce the differentiation of resident macrophages in osteoclasts and plasma cells themselves can transdifferentiate to functional osteoclasts [57, 58]. Bone disease results from the local production of osteoclast-activating factors (OAF), as well as IL-6, IL-1α or -1β, IL-11, TNF-α, TNF-β, and M-CSF [11]. In particular, the receptor activator of nuclear factor ligand (RANKL), the decoy receptor osteoprotegerin (OPG), its receptor (RANKR), and the chemokine macrophage inflammatory protein-1α (MIP-1α) trigger differentiation and activation signals in osteoclasts precursors, and thus promoting bone resorption [48]. Adhesion molecules, such as β1 integrins, mediate the binding of MM plasma cells to stromal cells and VCAM1 induces overexpression of RANKL in both cell types and suppresses OPG production by stromal cells. Furthermore, plasma cells interfere with the reg-

ulation of the bone resorption by the secretion of IL-7 and DKK1, a Wnt inhibitor [59].

It has demonstrated a close link between myeloma cells, osteoclasts, and vascular endothelial cells to form a vicious cycle between bone destruction, angiogenesis, and myeloma expansion

vasculogenic mimicry [17].

**3.5. Osteoclasts and osteoblasts**

**3.4. Mast cells**

102 Update on Multiple Myeloma

Hematopoietic stem cells (HSCs) reside in the BM in the endosteum niche and in the vascular niche, where they self-renew and differentiate into mature blood cells [64, 65]. This is a finely controlled-process by mean numerous signals from the bone marrow components [64, 65]. In MM, the BM niches (endosteum and vascular) components play a pivotal role in the regulation of vasculogenesis and angiogenesis [11, 14, 26, 64], and alterations of the signals in niche microenvironment modulate myeloma progression and spread [26, 64].

In the BM of patients with MM, the expression of the CD133 staminal antigen in some cells of the neovessel wall has been demonstrated [26, 38]. Moreover, a subset of CD34+/CD133+ cells mobilized in the peripheral blood for collection during transplant procedure express VEGFR-2 and are able to differentiate in mature endothelial cells in appropriate culture conditions [28].

#### **3.7. Endothelial progenitor cells**

Various studies have demonstrated that endothelial progenitor cells (EPCs) can be isolated from patients with MM [40, 66–69] and contribute to the formation of new blood vessels [40]. Moreover, circulating EPCs expressing CD146+, CD105+, and CD34+ are increased in MM patients compared to healthy controls [66, 67].

Rigolin et al. [69] have hypothesized a possible origin of EPCs and plasma cells from a common progenitor namely hemangioblast in MM patients. In their work, they demonstrated that EPCs, isolated from MM patients presents the 13q14 deletion and the great part of them are positive for the CD133 [69]. Finally, some evidence indicates a prognostic significance of the circulating EPCs also after treatment with new drugs [66, 68].

#### **3.8. Mesenchymal stem cells**

Mesenchymal stem cells (MSCs) are the major component of BM stroma [11, 57, 70–72]. These cells, of unclear origin in MM [71], are potentially able to differentiate into multiple histotypes (i.e. fibroblasts, adipocytes, chondrocytes, and osteoblasts) and in the BM form specialized niches namely "vascular niche" and "osteoblast niche" [57, 70, 71]. MSCs support tumor cell growth, metastasis, survival, bone marrow colonization, and evasion of the immune system [72]. MSCs can migrate toward primary tumors and metastatic sites, implying that these cells might modulate tumor growth and metastasis. In the BM of patients with MM, functional abnormalities of MSCs and complex interaction with MM plasma cells have been demonstrated indicating that they play a critical role in MM development and disease outcome [70, 71]. In fact, MSCs can induce bortezomib-resistance in MM plasma cell by increasing Bcl2 expression and enhance NF-κB activity via cell-cell contact [73, 74]. Moreover, MSCs are able to modulate engraftment of HSC, to suppress T- and B-lymphocyte activation and proliferation, and to affect dendritic cell maturation [71]. Finally, since MSCs represents the osteoblasts progenitors, in the BM of MM patients, MSCs play a critical role in the pathophysiology of myeloma bone disease [75]. They present reduced osteogenic potential and promoting osteoclasts formation and activity by increasing RANKL to OPG expression, augmenting secretion of activin A, uncoupling ephrinB2-EphB4 signaling, and augmenting Wnt5a production [75].

in vitro and in vivo [92, 93]. Finally, FGF-2 triggers paracrine MM-stromal cell interactions in an IL-6/FGF-2 paracrine loop [92, 95] and syndecan-1 (CD138), a low-affinity receptor of FGF-2, is also expressed by MM cells [96]. The high expression/activation of the FGF2 signaling in active MM also overcomes the inhibitory effect of the Pentraxin 3 (PTX3) [97, 98], a soluble pattern recognition receptor that binds with high affinity and selectivity to FGF2 inhibiting its pro-angiogenic activity, autocrine loops usually activated to self-limit physiologic angiogen-

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HGF has been identified in human MM cell lines and in freshly isolated plasma cells from patients with MM [99, 100]. Serum levels of this factor are higher in newly diagnosed MM patients and decline after induction therapy in the responding patients. Ferrucci et al. demonstrated the co-expression of HGF and c-MET in MM endothelial cells, suggesting autocrine stimulation [99]. Moreover, BM stromal cells produce HGF, paracrine stimulation of MM cells within the BM microenvironment can also take place [99, 100]. The inhibition of this pathway causes reduction of spontaneous and plasma cell-induced angiogenesis in MM endothelial

The Ang-1/Ang-2 expression in MM patient serum and BM samples correlates with the BM microvascular density [102–106]. It has been demonstrated that Ang-1, as well as Ang-2 expression, is upregulated in MM cell lines and in plasma cells obtained from MM patients [103, 105] and that the angiopoietin receptor Tie-2 is upregulated in the BM endothelial cells in the presence of MM cells [104]. Moreover, anti-Tie-2 antibodies blocked the in vitro angiogenic activity of MM cells [104]. Higher levels of Ang-1 and Ang-2 have been detected in MM patients as compared to controls [102] and their ratio may represent an independent prognos-

Osteopontin (OPN) contributes to angiogenesis in MM [107–109]. Its expression correlates with BM microvascular density, and OPN-immunodepleted conditioned media from myeloma cells fail to induce a pro-angiogenic effect [107, 108] and an anti-OPN antibody block myeloma-induced angiogenesis [107]. Moreover, OPN may represent a useful serum

Matrix Metalloproteinase-2 and -9 (MMP-2 and MMP-9) secretion is increased in patients with active MM versus non-active MM or MGUS [92, 110, 111] and usually, the MMP-2 expression is stronger [92, 110]. MM cell lines and freshly isolated BM plasma cells of MM patients produce MMP-9 [112], and MMP secretion of MM cells is triggered by BM stromal or

PDGF-Receptor Beta (PDGF-Rbeta) is expressed in plasma cells of MM patients [111, 113], and PDGF-BB/PDGF-Rbeta kinase axis promotes MM tumor growth by activating ERK-1/2 and AKT [113, 114]. Dasatinib, an orally bioactive TK-inhibitor significantly delays MM tumor

Airoldi et al. [115] demonstrated that IL-12 receptor B2 (IL-12Rbeta2) is downregulated in MM plasma cells and IL-12 reduces their pro-angiogenic activity by downregulation of a wide panel of angiogenic factors, including FGF-2, VEGF, Ang-2, and IL-6 and upregulation of some inhibitors of angiogenesis, including CXCL-4, interferon alpha and gamma (IFN-α

marker of bone disease and BM angiogenic extent in myeloma patients [109].

growth acting as an inhibitor of PDGF-Rbeta kinase activation [113].

and IFN-γ), and tissue inhibitor of metalloproteinase-2 (TIMP-2).

esis in a normal subject or MGUS patient [97].

cells in vitro and in vivo [99–101].

tic factor in these patients [106].

endothelial cells [92, 112].

#### **3.9. Adipocytes**

The cancer-associated adipocytes (also namely peritumoral, intratumoral, or tumor-infiltrating adipocytes) influence tumor biology also by promoting angiogenesis [76–81]. A great number of signaling factors contributing to angiogenesis in both adipose tissue and tumors: VEGF, Ang-1 and -2, leptin, adiponectin, TNF-α, FGF, TGF-β, HGF, IL-6, and IL-8 [77–79, 81]. The VEGF/VEGFR system is the main mediator of angiogenic activity in adipose tissue [77]. In particular, adipocytes produce VEGF, Ang-2, and HGF [77, 81]. In MM, the hypoxic environment of BM favors the production of angiogenic factors by adipocytes, particularly VEGF, and decreases adipogenic differentiation increasing adipose-derived stem cell proliferation and migration [82, 83], supporting aberrant microvessel growth and neovascularization, and MM plasma cell proliferation [82, 83]. Paracrine and autocrine signaling of VEGFA between BM adipocytes and MM cells have been also demonstrating [77, 80, 81].

#### **3.10. Soluble factors and transduction pathways**

The progression from in situ to invasive and metastatic solid tumors are accompanied and enhanced by the switch from the perivascular to the vascular phase [84, 85]. The same process has been demonstrated in MM in which active disease represent the 'vascular phase' of plasma cell tumors, and non-active disease (remission or plateau phase), smoldering MM and MGUS their 'perivascular phase' [22, 26, 43].

VEGF is the main angiogenic cytokine secreted in the BM of patients with MM [86–88]. VEGF carries out its activity through the MEK-1/ERK pathway by the interaction with his receptors (VEGFR1–3) [86]. In the BM of patients with MM paracrine loops between endothelial cells and plasma cells [89] and autocrine loops on the same endothelial cells have been demonstrated [90]. Moreover, plasma cell-derived VEGF stimulates IL-6 and VEGF secretion in BM stromal cells, whereas stromal cells-derived IL-6 promotes proliferation, survival, and VEGF production in plasma cells, activating a loop between both growth factors [91].

Levels of FGF isoforms are significantly higher in the serum and plasma cell lysates of patients with active MM compared with non-active MM and MGUS patients [92–94]. Moreover, FGF-2 inhibition suppresses the angiogenic potential of plasma cells from patients with active MM in vitro and in vivo [92, 93]. Finally, FGF-2 triggers paracrine MM-stromal cell interactions in an IL-6/FGF-2 paracrine loop [92, 95] and syndecan-1 (CD138), a low-affinity receptor of FGF-2, is also expressed by MM cells [96]. The high expression/activation of the FGF2 signaling in active MM also overcomes the inhibitory effect of the Pentraxin 3 (PTX3) [97, 98], a soluble pattern recognition receptor that binds with high affinity and selectivity to FGF2 inhibiting its pro-angiogenic activity, autocrine loops usually activated to self-limit physiologic angiogenesis in a normal subject or MGUS patient [97].

growth, metastasis, survival, bone marrow colonization, and evasion of the immune system [72]. MSCs can migrate toward primary tumors and metastatic sites, implying that these cells might modulate tumor growth and metastasis. In the BM of patients with MM, functional abnormalities of MSCs and complex interaction with MM plasma cells have been demonstrated indicating that they play a critical role in MM development and disease outcome [70, 71]. In fact, MSCs can induce bortezomib-resistance in MM plasma cell by increasing Bcl2 expression and enhance NF-κB activity via cell-cell contact [73, 74]. Moreover, MSCs are able to modulate engraftment of HSC, to suppress T- and B-lymphocyte activation and proliferation, and to affect dendritic cell maturation [71]. Finally, since MSCs represents the osteoblasts progenitors, in the BM of MM patients, MSCs play a critical role in the pathophysiology of myeloma bone disease [75]. They present reduced osteogenic potential and promoting osteoclasts formation and activity by increasing RANKL to OPG expression, augmenting secretion of activin A, uncoupling ephrinB2-EphB4 signaling, and augmenting Wnt5a production [75].

The cancer-associated adipocytes (also namely peritumoral, intratumoral, or tumor-infiltrating adipocytes) influence tumor biology also by promoting angiogenesis [76–81]. A great number of signaling factors contributing to angiogenesis in both adipose tissue and tumors: VEGF, Ang-1 and -2, leptin, adiponectin, TNF-α, FGF, TGF-β, HGF, IL-6, and IL-8 [77–79, 81]. The VEGF/VEGFR system is the main mediator of angiogenic activity in adipose tissue [77]. In particular, adipocytes produce VEGF, Ang-2, and HGF [77, 81]. In MM, the hypoxic environment of BM favors the production of angiogenic factors by adipocytes, particularly VEGF, and decreases adipogenic differentiation increasing adipose-derived stem cell proliferation and migration [82, 83], supporting aberrant microvessel growth and neovascularization, and MM plasma cell proliferation [82, 83]. Paracrine and autocrine signaling of VEGFA between

The progression from in situ to invasive and metastatic solid tumors are accompanied and enhanced by the switch from the perivascular to the vascular phase [84, 85]. The same process has been demonstrated in MM in which active disease represent the 'vascular phase' of plasma cell tumors, and non-active disease (remission or plateau phase), smoldering MM and

VEGF is the main angiogenic cytokine secreted in the BM of patients with MM [86–88]. VEGF carries out its activity through the MEK-1/ERK pathway by the interaction with his receptors (VEGFR1–3) [86]. In the BM of patients with MM paracrine loops between endothelial cells and plasma cells [89] and autocrine loops on the same endothelial cells have been demonstrated [90]. Moreover, plasma cell-derived VEGF stimulates IL-6 and VEGF secretion in BM stromal cells, whereas stromal cells-derived IL-6 promotes proliferation, survival, and VEGF

Levels of FGF isoforms are significantly higher in the serum and plasma cell lysates of patients with active MM compared with non-active MM and MGUS patients [92–94]. Moreover, FGF-2 inhibition suppresses the angiogenic potential of plasma cells from patients with active MM

production in plasma cells, activating a loop between both growth factors [91].

BM adipocytes and MM cells have been also demonstrating [77, 80, 81].

**3.10. Soluble factors and transduction pathways**

MGUS their 'perivascular phase' [22, 26, 43].

**3.9. Adipocytes**

104 Update on Multiple Myeloma

HGF has been identified in human MM cell lines and in freshly isolated plasma cells from patients with MM [99, 100]. Serum levels of this factor are higher in newly diagnosed MM patients and decline after induction therapy in the responding patients. Ferrucci et al. demonstrated the co-expression of HGF and c-MET in MM endothelial cells, suggesting autocrine stimulation [99]. Moreover, BM stromal cells produce HGF, paracrine stimulation of MM cells within the BM microenvironment can also take place [99, 100]. The inhibition of this pathway causes reduction of spontaneous and plasma cell-induced angiogenesis in MM endothelial cells in vitro and in vivo [99–101].

The Ang-1/Ang-2 expression in MM patient serum and BM samples correlates with the BM microvascular density [102–106]. It has been demonstrated that Ang-1, as well as Ang-2 expression, is upregulated in MM cell lines and in plasma cells obtained from MM patients [103, 105] and that the angiopoietin receptor Tie-2 is upregulated in the BM endothelial cells in the presence of MM cells [104]. Moreover, anti-Tie-2 antibodies blocked the in vitro angiogenic activity of MM cells [104]. Higher levels of Ang-1 and Ang-2 have been detected in MM patients as compared to controls [102] and their ratio may represent an independent prognostic factor in these patients [106].

Osteopontin (OPN) contributes to angiogenesis in MM [107–109]. Its expression correlates with BM microvascular density, and OPN-immunodepleted conditioned media from myeloma cells fail to induce a pro-angiogenic effect [107, 108] and an anti-OPN antibody block myeloma-induced angiogenesis [107]. Moreover, OPN may represent a useful serum marker of bone disease and BM angiogenic extent in myeloma patients [109].

Matrix Metalloproteinase-2 and -9 (MMP-2 and MMP-9) secretion is increased in patients with active MM versus non-active MM or MGUS [92, 110, 111] and usually, the MMP-2 expression is stronger [92, 110]. MM cell lines and freshly isolated BM plasma cells of MM patients produce MMP-9 [112], and MMP secretion of MM cells is triggered by BM stromal or endothelial cells [92, 112].

PDGF-Receptor Beta (PDGF-Rbeta) is expressed in plasma cells of MM patients [111, 113], and PDGF-BB/PDGF-Rbeta kinase axis promotes MM tumor growth by activating ERK-1/2 and AKT [113, 114]. Dasatinib, an orally bioactive TK-inhibitor significantly delays MM tumor growth acting as an inhibitor of PDGF-Rbeta kinase activation [113].

Airoldi et al. [115] demonstrated that IL-12 receptor B2 (IL-12Rbeta2) is downregulated in MM plasma cells and IL-12 reduces their pro-angiogenic activity by downregulation of a wide panel of angiogenic factors, including FGF-2, VEGF, Ang-2, and IL-6 and upregulation of some inhibitors of angiogenesis, including CXCL-4, interferon alpha and gamma (IFN-α and IFN-γ), and tissue inhibitor of metalloproteinase-2 (TIMP-2).

IL-27 exert strong antitumor activities against MM cells from patients by binding with its specific IL-27 receptor [116, 117] inhibiting the angiogenic potential of MM plasma cells. In animals injected with the U266 MM cell line, the expression of the genes encoding the chemokines CCL-2, CXCL-3, CXCL-5, and CXCL-6 is significantly downregulated by IL-27 treatment [116, 117].

defined the biological role of EphA3 in MM angiogenesis and their preliminary data indicate

Angiogenesis and Antiangiogenesis in Multiple Myeloma

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

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Focal adhesion kinase (FAK) is a tyrosine kinase that localizes at focal adhesion sites of endothelial cell to the ECM [135–137]. It mediates signaling starting from integrin, is upregulated in many cancer types, controlling tumor aggressiveness, and metastasis [135], and is implicated in endothelial cell survival, proliferation, and migration [136, 137]. Integrin/FAK-mediated signaling cooperate with other growth factor receptor signaling (i.e. FGFR signaling) to pro-

Various growth factor receptors induced an increase in DNA synthesis in MM endothelial cells by mean PI3K/Akt-MEK/ERK pathway inducing angiogenesis [17, 86, 138]. The role of this pathway in promoting angiogenesis is mainly related to the phosphorylation of eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1), S6-kinase (S6K), and MAP kinase interacting kinase mediated by ERK [139, 140]. This process leads to an increased rate of mRNA translation into HIF-1α protein in an oxygen-independent way [139, 140]. ERK is also able to activate the transcription of HIF-1α by the co-activator CBP/p300 that increases

MicroRNAs are small endogenous non-coding RNAs (21–25 nucleotides) involved in regulating normal physiological processes as well as cancer pathogenesis [141–143]. Particularly, some miRNA have been implicated in tumor angiogenesis individuate as potential therapeutic targets/ therapy [143–145]. Evidence suggests that MM cells promote angiogenic activity via HIF-1α, a key transcription factor of hypoxia, leading to the overproduction of angiogenic cytokines [91, 104]. Moreover, communication between plasma cells, stromal cells, and endothelial cells is mediated also by mean the exosomes, small endosome-derived vesicles, containing a wide range of functional proteins, mRNA, and miRNA [146]. In BM of MM, miR-135b has been involved as the principal pro-angiogenic miRNA by targeting factor-inhibiting HIF-1 [147], whereas miR-199a-5p, which directly targets HIF1-α, miR-15a, and miR-16, and VEGF, have been demonstrated to be strong inhibitors of MM-induced angiogenesis [148, 149]. Overall, published data indicate that circulating microRNAs in exosomes and microvesicles can be useful biomarkers of angiogenesis, and synthetic miRNAs may be potential new antiangiogenic

The combination of biological drugs in the actual therapeutic strategies of MM have improved the outcome of MM patients because of their activity on microenvironment [17, 151–153].

Bortezomib, a potent, highly selective, and reversible proteasome inhibitor targeting the 26S proteasome complex [154, 155] act on key cellular processes, such as cell cycle progression, inflammation, immune surveillance, growth arrest, and apoptosis [154]. Bortezomib acts by mean the modulation of NF-κB transcription factor, which mediates the expression and secretion of cytokines, chemokines, cell adhesion molecules involved also in anti-apoptosis and cellular

that EphA3 could represent an angiogenic target in patients with MM [134].

mote angiogenesis in MM [138].

therapeutics tools in MM [150].

**4.1. Proteasome inhibitors**

**4. Antiangiogenesis in multiple myeloma**

HIF-1α/p300 complex formation [139, 140].

Another important paracrine loop between MM endothelial cells and plasma cells involves CXC-chemokines and their cognate receptors have been evidenced in the BM of MM patients [118, 119]. In fact, BM endothelial cells express and secrete high amounts of the CXCchemokines CXCL8/IL-8, CXCL11/interferon-inducible T-cell alpha chemoattractant (I-TAC), CXCL12/stromal cell-derived factor (SDF)-1α, and CCL2/monocyte chemotactic protein (MPC)-1 [118] that mediate the interactions between plasma cells and stromal cells interacting with the respective chemokine receptors (CXCR and CCR) [118, 120].

HIF-1α has been demonstrated to be stabilized in MM plasma cells, in hypoxic as in normoxic conditions [82, 83, 119, 121–123]. The constitutive stabilization of HIF-1α in myeloma cells is associated with the oncogenic c-Myc activity, suggesting that a common signaling pathway is active in MM plasma cells [122]. Among target genes controlled by HIF-1α, the genes coding for the pro-angiogenic cytokines VEGF, IL-8, and OPN have been evidenced, and HIF-1α silencing significantly suppresses the pro-angiogenic properties of MM cells reducing their secretion [87]. Moreover, MM endothelial cells from relapsed/refractory MM patients, but not those of newly diagnosed or non-active MM patients, showed a stabilization and activation of the HIF-1α protein in normoxic conditions [124]. This stabilization is induced by ROS and correlated with the expression of HIF-1α pro-angiogenic targets [124]. The inhibition of HIF-1α in MM plasma cells [123] as well as in endothelial cells [124] impaired the MM plasma cells/stromal cells communication, the angiogenesis-related functions, and revert bortezomib- and lenalidomide-resistance [123, 124]. It may also have prognostic significance because patients with MM endothelial cells expressing the stabilized HIF-1α protein had shorter overall survival [124].

The mammalian target of rapamycin (mTOR) is an intracellular serine/threonine kinase that mediates intracellular metabolism, cell survival, and actin rearrangement. mTOR is made of two independent complexes, mTORC1, involved in protein synthesis and autophagy inhibition, and mTORC2, involved in progression promotion, survival, actin reorganization, and drug resistance [125–127]. In MM endothelial, a significantly higher activation of mTORC2 have been demonstrated. Its inhibition induces a reduction of the angiogenic abilities of MM endothelial cells, suggesting a major role of mTORC2 in the "angiogenic switch" and indicates that mTORC2 might be a new antiangiogenic target in MM [127].

In MM endothelial, cell-to-cell contact-dependent homotypic activation of Notch pathway has been shown [128, 129]. MM plasma cells cocultured with MM endothelial cells trigger Jagged1/2-mediated Notch activation enhancing endothelial angiogenic activity. Moreover, halting Notch axis reduces angiogenesis *in vitro* and *in vivo* suggesting Notch pathway as a novel therapeutic target in MM [129].

The ephrins (Efn) and their receptors (Eph), a large family of receptor tyrosine kinases, are involved in several biological processes including cancer growth, progression, and angiogenesis [130–133]. Caivano et al. [134] recently demonstrated that EphA3 is highly overexpressed in MM endothelial cells and its expression correlates with disease progression. They have also defined the biological role of EphA3 in MM angiogenesis and their preliminary data indicate that EphA3 could represent an angiogenic target in patients with MM [134].

Focal adhesion kinase (FAK) is a tyrosine kinase that localizes at focal adhesion sites of endothelial cell to the ECM [135–137]. It mediates signaling starting from integrin, is upregulated in many cancer types, controlling tumor aggressiveness, and metastasis [135], and is implicated in endothelial cell survival, proliferation, and migration [136, 137]. Integrin/FAK-mediated signaling cooperate with other growth factor receptor signaling (i.e. FGFR signaling) to promote angiogenesis in MM [138].

Various growth factor receptors induced an increase in DNA synthesis in MM endothelial cells by mean PI3K/Akt-MEK/ERK pathway inducing angiogenesis [17, 86, 138]. The role of this pathway in promoting angiogenesis is mainly related to the phosphorylation of eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1), S6-kinase (S6K), and MAP kinase interacting kinase mediated by ERK [139, 140]. This process leads to an increased rate of mRNA translation into HIF-1α protein in an oxygen-independent way [139, 140]. ERK is also able to activate the transcription of HIF-1α by the co-activator CBP/p300 that increases HIF-1α/p300 complex formation [139, 140].

MicroRNAs are small endogenous non-coding RNAs (21–25 nucleotides) involved in regulating normal physiological processes as well as cancer pathogenesis [141–143]. Particularly, some miRNA have been implicated in tumor angiogenesis individuate as potential therapeutic targets/ therapy [143–145]. Evidence suggests that MM cells promote angiogenic activity via HIF-1α, a key transcription factor of hypoxia, leading to the overproduction of angiogenic cytokines [91, 104]. Moreover, communication between plasma cells, stromal cells, and endothelial cells is mediated also by mean the exosomes, small endosome-derived vesicles, containing a wide range of functional proteins, mRNA, and miRNA [146]. In BM of MM, miR-135b has been involved as the principal pro-angiogenic miRNA by targeting factor-inhibiting HIF-1 [147], whereas miR-199a-5p, which directly targets HIF1-α, miR-15a, and miR-16, and VEGF, have been demonstrated to be strong inhibitors of MM-induced angiogenesis [148, 149]. Overall, published data indicate that circulating microRNAs in exosomes and microvesicles can be useful biomarkers of angiogenesis, and synthetic miRNAs may be potential new antiangiogenic therapeutics tools in MM [150].
