**2. Role of BM microenvironment and angiogenesis in MM progression and prognosis**

MM is a B-cell tumor, characterized by clonal proliferation of malignant plasma cells inside the BM, production of a monoclonal paraprotein, and associated clinical features, including lytic bone lesions, renal insufficiency, hypercalcemia and anemia. It accounts for approxi‐ mately 1% of neoplastic diseases and 13% of hematologic cancers. Albeit significant advan‐ ces have been recently achieved in the treatment of MM, the disease still remains incurable, prompting the development of new therapeutic strategies [8].

MM is thought to evolve from a pre-malignant syndrome known as Monoclonal Gammopathy of Uncertain Significance (MGUS), that progresses to smoldering (asymp‐ tomatic) myeloma and, finally, to symptomatic myeloma. In addition to genetic abnor‐ malities accumulating in MM cells, BM microenvironment actively participates to the pathogenesis and progression of the disease. Indeed, host stromal components pro‐ foundly influence many steps of tumor progression, such as tumor proliferation, inva‐ sion, angiogenesis, metastasis, and even malignant transformation [9]. The BM, where MM cells specifically home, provides a highly specialized microenvironment, which op‐ timally "soils" neoplastic plasma cells, and, in turn, is shaped by the interactions with MM cells [5,6,10].

BM microenvironment consists of a series of cellular components, including hematopoietic cells, immune cells, BM stromal cells (BMSC), osteoclasts, osteoblasts and endothelial cells (EC), all embedded in an extracellular matrix (ECM) (Fig.1).

MM cells specifically localize inside the BM milieu through the CXCR4/CXCL12-SDF1-alpha axis [11] and then interact with ECM and BM cellular components by means of adhesion molecules, including integrins. The complex interplay between MM cells and BM milieu, to‐ gether with the ensuing pathogenetic events, are depicted in Fig. 2 (upper panel).

sult in re-shaping of microenvironment, and architectural changes involve in particular the

The establishment of tight links between MM plasma cells and their microenvironment un‐ derlines the need for appropriate models for studying MM biology and predicting the im‐

In the present paper, we briefly summarize the role of BM microenvironment and, particularly, of MM associated angiogenesis, in MM pathogenesis, progression and prognosis. We then provide an overview of the currently available MM models, in‐ cluding animal models and a new three-dimensional (3D), gel-based, *in vitro* model of human MM microenvironment. Finally, we discuss the potential of RCCSTM bioreactorbased, dynamic 3D model systems (cell and tissue culture) to investigate critical as‐ pects of human MM pathobiology and possible clinical applications. Advantages and limitations of each model, relative to MM investigation and assessment of drug sensi‐

**2. Role of BM microenvironment and angiogenesis in MM progression**

MM is a B-cell tumor, characterized by clonal proliferation of malignant plasma cells inside the BM, production of a monoclonal paraprotein, and associated clinical features, including lytic bone lesions, renal insufficiency, hypercalcemia and anemia. It accounts for approxi‐ mately 1% of neoplastic diseases and 13% of hematologic cancers. Albeit significant advan‐ ces have been recently achieved in the treatment of MM, the disease still remains incurable,

MM is thought to evolve from a pre-malignant syndrome known as Monoclonal Gammopathy of Uncertain Significance (MGUS), that progresses to smoldering (asymp‐ tomatic) myeloma and, finally, to symptomatic myeloma. In addition to genetic abnor‐ malities accumulating in MM cells, BM microenvironment actively participates to the pathogenesis and progression of the disease. Indeed, host stromal components pro‐ foundly influence many steps of tumor progression, such as tumor proliferation, inva‐ sion, angiogenesis, metastasis, and even malignant transformation [9]. The BM, where MM cells specifically home, provides a highly specialized microenvironment, which op‐ timally "soils" neoplastic plasma cells, and, in turn, is shaped by the interactions with

BM microenvironment consists of a series of cellular components, including hematopoietic cells, immune cells, BM stromal cells (BMSC), osteoclasts, osteoblasts and endothelial cells

MM cells specifically localize inside the BM milieu through the CXCR4/CXCL12-SDF1-alpha axis [11] and then interact with ECM and BM cellular components by means of adhesion

prompting the development of new therapeutic strategies [8].

(EC), all embedded in an extracellular matrix (ECM) (Fig.1).

vascular compartment [7].

40 Multiple Myeloma - A Quick Reflection on the Fast Progress

tivity, are also considered.

**and prognosis**

MM cells [5,6,10].

pact of drugs.

<sup>1:</sup> erithrocytes; 2: megacaryocytes; 3: basophils; 4: adipocytes; 5: osteocytes; 6: B lymphocytes; 7: monocytes; 8: lining osteoblasts; 9: osteoblasts; 10: osteoclasts; 11: hematopoietic stem cells "niche"; 12: T lymphocytes; 13: NK cells; 14: eosinophils; 15: neutrophils; 16: monocytes; 17: stromal cells; 18: mesenchymal stem cells "niche"; 19: dendritic cells; 20: thrombocytes (platelets).

**Figure 1. Bone Marrow microenvironment.** Bone homeostasis is the result of a complex network of stimuli, includ‐ ing hormones, vitamins and physico-mechanical forces. In addition to osteoblats and osteoclasts, which are responsi‐ ble for bone deposition/resorption, BM microenvironment encompasses several cell types, like hematopoietic cells, endothelial cells and mesenchimal cells, all embedded in a complex extra-cellular-matrix (ECM).

Interactions between MM cells and ECM and cellular components (Fig. 2, lower panel) trig‐ ger the release of soluble factors, which, in turn, determine autocrine/paracrine loops of MM survival/proliferation and also promote osteoclastogenesis, defective immune functions and the "angiogenic switch", overall leading to MM cells growth, survival, and resistance to che‐ motherapeutic agents [10]. In particular, adhesion of MM cells to BMSC and to ECM compo‐ nents triggers anti-apoptotic signals and also the release of the pro-survival factor Interleukin (IL)-6. Moreover, MM plasma cells and BM stroma release osteoclast-acivating factors, including IL-1, IL-6, tumor necrosis factor (TNF)-α, RANK-L(Ligand) and Macro‐ phage Inflammatory Protein (MIP)-1α. MM cells have also a unique ability to evade im‐ mune surveillance through several mechanisms, including impairment of cytotoxic activity and induction of dendritic cells dysfunction (Fig. 2).

Angiogenesis, the sprouting of capillaries from existing blood-vessels, is a complex, dynam‐ ic and tightly regulated process, that occurs physiologically during normal growth, wound repair after injury and regeneration [12,13]. Angiogenesis is controlled by the balance be‐ tween positive and negative regulators. In a tumor microenvironment, the exaggerate ex‐ pression of pro-angiogenic cyto-chemokines starts the ''angiogenic switch'', leading to increased micro vessel density (MVD) [14]. The occurrence of an "angiogenic switch", re‐ sponsible for the transition from the avascular "dormant" phase to the vascular phase of ex‐ ponential tumor growth [15,16], has also been proposed for MM. Pro- and anti-angiogenic soluble molecules are produced and released by myeloma cells and components of microen‐ vironment, including MMEC, stromal cells and inflammatory cells [17-19] (Fig.2 A, upper panel). Major angiogenic cytokines are VEGF-A, fibroblast growth factor (FGF) and hepato‐ cyte growth factor (HGF). Both EC in general, and in particular MMEC, and MM cells se‐ crete VEGF and express its receptors, thereby contributing to autocrine/paracrine pathways of tumor growth, survival and angiogenesis [19]. Finally, Angiopoietins (Angs, Ang-1 and-2) are important mediators in vasculature homeostasis and their circulating levels are

Innovative Models to Assess Multiple Myeloma Biology and the Impact of Drugs

http://dx.doi.org/10.5772/54312

43

Overall, BM angiogenesis in MM contributes to disease progression; accordingly, new antimyeloma agents target not only MM cells, but also the microenvironment, and in particular vessels [21]. This notion is exemplified by the proteasome inhibitor Bortezomib (PS-341, Vel‐ cade), which has been approved for treatment of patients with relapsed and refractory MM and more recently used in front-line therapy for the disease. *In vitro*, proteasome inhibition by bor‐ tezomib causes apoptosis in both solid tumor and haematological malignancies, particularly MM [22]. More recently, Bortezomib has also been reported to affect viability of angiogenic EC, as shown in *in vitro* experimental conditions as well in animal models [23,24]. Notably, neither reliable biomarkers measurable *in vivo* nor *ex vivo* models of human BM microenvironment are

currently available to assess the anti-angiogenic effect of drugs in MM patients.

**3. Advantages of models which mimic tumor microenvironment**

Since BM microenvironment is of most importance in supporting myeloma cell growth and survival, experimental models of MM should provide insights into the mechanisms that, at molecular level, regulate the complex interplay between MM cells and biochemical and

Traditional two-dimensional (2D) *in vitro* models (static culture of single cells kept as mono‐ layer on flat, artificial surfaces) still represent the most popular models for *in vitro* studies, even if they present severe limitations, being unable to reproduce the behaviour and physio‐ logical responses of various normal and pathological cell types/tissues. It is now generally accepted that any attempt aimed at the generation of reliable and physiologically relevant *in vitro* tissue analogues, tumors included, should take into account the need of reproducing (or preserving) the specific characteristics of their original microenvironment, which in‐

considered of prognostic significance in MM [20].

**exploiting the third dimension**

physical cues coming from BM ECM and cell components.

**Figure 2. Interactions between MM cellsand BM microenviroment.** Upper panel: schematic representation of MM cells inside BM microenvironment; the soluble factors involved in the major pathogenetic events, including tumor pro‐ liferation/survival, angiogenesis, osteoclastogenesis and defective immune function are depicted. Lower panel illus‐ trates the major growth factor receptors and adhesion molecules used by MM plasma cells to interact with ECM and cellular components of BM microenvironment

Angiogenesis, the sprouting of capillaries from existing blood-vessels, is a complex, dynam‐ ic and tightly regulated process, that occurs physiologically during normal growth, wound repair after injury and regeneration [12,13]. Angiogenesis is controlled by the balance be‐ tween positive and negative regulators. In a tumor microenvironment, the exaggerate ex‐ pression of pro-angiogenic cyto-chemokines starts the ''angiogenic switch'', leading to increased micro vessel density (MVD) [14]. The occurrence of an "angiogenic switch", re‐ sponsible for the transition from the avascular "dormant" phase to the vascular phase of ex‐ ponential tumor growth [15,16], has also been proposed for MM. Pro- and anti-angiogenic soluble molecules are produced and released by myeloma cells and components of microen‐ vironment, including MMEC, stromal cells and inflammatory cells [17-19] (Fig.2 A, upper panel). Major angiogenic cytokines are VEGF-A, fibroblast growth factor (FGF) and hepato‐ cyte growth factor (HGF). Both EC in general, and in particular MMEC, and MM cells se‐ crete VEGF and express its receptors, thereby contributing to autocrine/paracrine pathways of tumor growth, survival and angiogenesis [19]. Finally, Angiopoietins (Angs, Ang-1 and-2) are important mediators in vasculature homeostasis and their circulating levels are considered of prognostic significance in MM [20].

Overall, BM angiogenesis in MM contributes to disease progression; accordingly, new antimyeloma agents target not only MM cells, but also the microenvironment, and in particular vessels [21]. This notion is exemplified by the proteasome inhibitor Bortezomib (PS-341, Vel‐ cade), which has been approved for treatment of patients with relapsed and refractory MM and more recently used in front-line therapy for the disease. *In vitro*, proteasome inhibition by bor‐ tezomib causes apoptosis in both solid tumor and haematological malignancies, particularly MM [22]. More recently, Bortezomib has also been reported to affect viability of angiogenic EC, as shown in *in vitro* experimental conditions as well in animal models [23,24]. Notably, neither reliable biomarkers measurable *in vivo* nor *ex vivo* models of human BM microenvironment are currently available to assess the anti-angiogenic effect of drugs in MM patients.
