**Abstract**

Multiple Myeloma (MM) develops almost exclusively within the Bone Marrow (BM), highlighting the critical role of the microenvironment in conditioning disease progression and resistance to drugs. Indeed, while the therapeutic armamentarium for MM has significantly improved over the past 20 years, the disease remains ultimately incurable. This failure may depend on the high phenotypic and genetic heterogeneity of MM, but also on the paucity and inadequacy of two-dimensional (2D) conventional preclinical models in reproducing MM within the BM. In the present paper, we provide a brief updated overview on MM BM microenvironment. We then discuss newly developed preclinical models mimicking MM/microenvironment interactions, including three-dimensional (3D), gel-based, *in vitro* models and a novel *ex vivo* system of isolated tumor and stromal cells cultured in bioreactor. Potential applications of each model, relative to investigation of MM pathogenic mechanisms and prediction of the best drug/combination for each individual patient will be also evaluated.

**Keywords:** multiple myeloma, tumor microenvironment, 2D/3D culture models, 3D culture in bioreactor, drug testing, personalized therapy

## **1. Introduction**

Multiple Myeloma (MM) is a B-cell tumor characterized by clonal proliferation of malignant plasma cells (PC) inside the bone marrow (BM), production of a monoclonal paraprotein and associated clinical features, including hypercalcemia, renal failure, anemia and lytic bone lesions (CRAB features) [1, 2].

MM is the second most common hematological malignancy and is responsible for approximately 20% of deaths from hematological tumors. Despite significant advances in therapy over the past two decades, the disease remains incurable, and more than 90% of MM patients eventually become refractory to therapy and relapse [1, 2].

MM develops along an evolutionary process, leading a normal PC to the premalignant state of monoclonal gammopathy of uncertain significance (MGUS), an intermediate asymptomatic but more advanced pre-malignant state referred to as smoldering MM (SMM) and finally to symptomatic MM [3–6]. This process is driven by the accumulation of cytogenetic modifications in PC. Indeed, while MM is still considered a single disease entity, it should be viewed as a collection of several different cytogenetically distinct PC tumors [7]. Cytogenetic abnormalities encompass translocations involving the immunoglobulin heavy chain (IgH) gene locus on chromosome 14q32 and hyperdiploidy (particularly trisomies), as initiating events [8]. IgH translocations include t(4;14), t(6;14), t(11;14), t(14;16) and t(14;20) translocations, which place the oncogenes Multiple Myeloma SET domain (MMSET)/ fibroblast growth factor receptor 3 (FGFR3), cyclin D3 (CCND3), CCND1, MAF, and MAFB, respectively, under the control of the strong enhancers of the Ig loci. This in turn leads to over-expression of cyclin D protein family members, ultimately driving G1/S checkpoint dysregulation [9, 10]. Hyperdiploidy, which is associated with the gain of the odd numbered chromosomes, including chromosome 3, 5, 7, 9, 11, 15, 19 and 21, also affects this checkpoint, implicating cyclin D dysregulation as an early and unifying oncogenic event in MM [9]. Subsequent studies demonstrated that other cytogenetic changes termed secondary cytogenetic abnormalities, including gain(1q), del(1p), del(17p), del(13), RAS mutations and secondary translocations involving MYC, arise along the disease course of MM, exacerbating the cell cycle dysregulation and driving further proliferation and disease progression [10, 11]. Patients carrying del(17p), t(4;14), t(14;16), t(14;20), gain(1q), or p53 mutation, particularly when in combination (double-hit and triple-hit myeloma), are considered affected by high-risk MM [11], and represent an area of unmet clinical need [8].

In addition to genetic abnormalities, a characteristic feature of myeloma cells is the requirement for an intimate relationship with the BM microenvironment, where plasma cells are nurtured in specialized niches that maintain their longterm survival. Indeed, BM components deeply influence many steps of tumor progression, such as MM proliferation and invasion, angiogenesis and drug resistance [12, 13].

The BM, where MM cells specifically home mainly through the CXCR4/CXCL12- SDF1α axis [14], provides a highly specialized microenvironment, which optimally "soils" neoplastic PC, and, in turn, is shaped by the interactions with the tumor [15, 16]. The BM microenvironment comprises two major compartments, *i.e.*, the cellular and the non-cellular compartment. The latter includes the extracellular matrix (ECM), consisting of collagen I to XI, fibronectin, glycoproteins, matrix proteoglycans and glycosaminoglycans, as well as the liquid milieu (cytokines, chemokines and growth factors). The cellular compartment consists of a series of components, including BM stromal cells (BMSC), hematopoietic cells, osteoclasts, osteoblasts, endothelial cells (EC), adipocytes and immune cells. Inside the BM milieu MM cells realize a complex interplay involving both cellular and ECM components through the engagement of adhesion molecules and the release of soluble factors, including cytokines, growth factors and exosomes [12]. Exosomes are extracellular membranous vesicles known to facilitate the transfer of biologically active molecules, including proteins and nucleic acids (particularly microRNAs -miRNAs), from the original producing cell to the target cell [17]. Exosomes are released by almost all cell types and, depending on their cargo, can induce target cell activation, proliferation/differentiation or death, thus playing a key role in the regulation of physiological as well as pathologic processes, including malignant transformation [17]. In MM, exosomes have been recently shown to reprogram the BM microenvironment, creating a niche for tumor PC and favoring their expansion and the onset of pharmacological resistance [18–20].

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*3D Models of Surrogate Multiple Myeloma Bone Marrow Microenvironments: Insights…*

Another key feature of the BM microenvironment is hypoxia. In the BM, oxygen (O2) tensions fluctuate throughout two specialized niches, the hypoxic endosteal niche and the oxygenated vascular niche, mapping areas with controlled, physiological O2 gradients, instrumental to hematopoietic stem cells homeostasis [21]. BM homing is a common feature of hematological malignancies, that in proximity of hypoxic niches escape drug-inflicted apoptosis and acquire a drug-resistant phenotype. This is particularly true for MM that develops almost exclusively in the BM, where myeloma cells accumulation and the abnormal vasculature contribute to aggravate hypoxia. BM samples from MM patients as well as circulating MM cells are reported to have a hypoxic phenotype [22] and a strong stabilized expression of the hypoxia master regulator hypoxia-inducible factor (HIF)-1α protein [23]. Notably, HIF-1α suppression in myeloma cells blocks tumor growth *in vivo* and interferes negatively with angiogenesis and bone destruction [24]. In addition to conventional cell contact-dependent and -independent signaling pathways, hypoxia promotes MM survival and drug resistance through alternative mechanisms. Hypoxia is indeed a major regulator of exosomal content and angiogenesis in MM settings [25]. Moreover, hypoxia shifts the metabolic profile of MM cells toward elevated glycolysis and production of lactate, as a strategy to support energy requirement [26]. Notably, knockdown of lactate restores MM sensitivity to bortezomib, overall suggesting that targeting hypoxia and MM energy metabolism

Overall, the cross-talk between MM cells and their BM microenvironment results in autocrine/paracrine loops of MM survival/proliferation and also promotes the "angiogenic switch", osteoclastogenesis, and defective immune functions [12, 13]. In particular, adhesion of MM cells to ECM components and to BMSC triggers classical survival signaling pathways including, but not limited to, the PI3K/AKT signaling pathway, anti-apoptotic signals and also the release of the pro-survival factor Interleukin (IL)-6 [27]. MM cells-BM interactions also play a key role in disease pathogenesis. In particular, new blood vessel formation is considered a hallmark of MM development and is supported by the histopathological evidence of increased microvessel density (MVD), surrogate parameter endowed with prognostic significance, in the BM of MM patients [28]. Angiogenesis, the sprouting of capillaries from existing blood vessels, is also suggested by the plethora of soluble angiogenic factors in the BM and in the peripheral blood (PB) samples from myeloma patients (vascular endothelial growth factor, VEGF; basic fibroblast growth factor, bFGF; angiopoietins, Angs) [29, 30], whose contribution to the process has been extensively reviewed [31]. Moreover, the finding of an elevated number of circulating endothelial precursor cells (EPC) in MM patients indicates that complementary modalities to build vessels, e.g., vasculogenesis, are engaged [31]. Finally, EC are by nature fine sensors of O2 variations, and the hypoxic microenvironment inside the BM significantly contributes to the induction of the "angiogenic switch" and the maintenance of the pro-angiogenic profile through the transcription of HIF-1α [32]. MM plasma cells and BM stroma also contribute to the pathophysiology of MM-associated bone disease through the activation of signaling pathways regulating osteoclastogenesis, particularly the RANK/RANK-Ligand (RANK-L) and the Wnt pathway, and the release of osteoclast-activating factors, such as IL-1, IL-6, tumor necrosis factor (TNF)-α, IL-8 and Macrophage Inflammatory Protein (MIP)-1α. These factors, together with recently identified dysregulated miRNAs, determine osteoblast suppression with excessive osteoclastic resorption [33]. Finally, MM cells display a unique ability to evade immune surveillance through several mechanisms, including impairment of cytotoxic activity, induction of

dendritic cell dysfunction and recruitment of regulatory cells [34].

*DOI: http://dx.doi.org/10.5772/intechopen.95333*

could alleviate drug resistance [26].

#### *3D Models of Surrogate Multiple Myeloma Bone Marrow Microenvironments: Insights… DOI: http://dx.doi.org/10.5772/intechopen.95333*

Another key feature of the BM microenvironment is hypoxia. In the BM, oxygen (O2) tensions fluctuate throughout two specialized niches, the hypoxic endosteal niche and the oxygenated vascular niche, mapping areas with controlled, physiological O2 gradients, instrumental to hematopoietic stem cells homeostasis [21]. BM homing is a common feature of hematological malignancies, that in proximity of hypoxic niches escape drug-inflicted apoptosis and acquire a drug-resistant phenotype. This is particularly true for MM that develops almost exclusively in the BM, where myeloma cells accumulation and the abnormal vasculature contribute to aggravate hypoxia. BM samples from MM patients as well as circulating MM cells are reported to have a hypoxic phenotype [22] and a strong stabilized expression of the hypoxia master regulator hypoxia-inducible factor (HIF)-1α protein [23]. Notably, HIF-1α suppression in myeloma cells blocks tumor growth *in vivo* and interferes negatively with angiogenesis and bone destruction [24]. In addition to conventional cell contact-dependent and -independent signaling pathways, hypoxia promotes MM survival and drug resistance through alternative mechanisms. Hypoxia is indeed a major regulator of exosomal content and angiogenesis in MM settings [25]. Moreover, hypoxia shifts the metabolic profile of MM cells toward elevated glycolysis and production of lactate, as a strategy to support energy requirement [26]. Notably, knockdown of lactate restores MM sensitivity to bortezomib, overall suggesting that targeting hypoxia and MM energy metabolism could alleviate drug resistance [26].

Overall, the cross-talk between MM cells and their BM microenvironment results in autocrine/paracrine loops of MM survival/proliferation and also promotes the "angiogenic switch", osteoclastogenesis, and defective immune functions [12, 13]. In particular, adhesion of MM cells to ECM components and to BMSC triggers classical survival signaling pathways including, but not limited to, the PI3K/AKT signaling pathway, anti-apoptotic signals and also the release of the pro-survival factor Interleukin (IL)-6 [27]. MM cells-BM interactions also play a key role in disease pathogenesis. In particular, new blood vessel formation is considered a hallmark of MM development and is supported by the histopathological evidence of increased microvessel density (MVD), surrogate parameter endowed with prognostic significance, in the BM of MM patients [28]. Angiogenesis, the sprouting of capillaries from existing blood vessels, is also suggested by the plethora of soluble angiogenic factors in the BM and in the peripheral blood (PB) samples from myeloma patients (vascular endothelial growth factor, VEGF; basic fibroblast growth factor, bFGF; angiopoietins, Angs) [29, 30], whose contribution to the process has been extensively reviewed [31]. Moreover, the finding of an elevated number of circulating endothelial precursor cells (EPC) in MM patients indicates that complementary modalities to build vessels, e.g., vasculogenesis, are engaged [31]. Finally, EC are by nature fine sensors of O2 variations, and the hypoxic microenvironment inside the BM significantly contributes to the induction of the "angiogenic switch" and the maintenance of the pro-angiogenic profile through the transcription of HIF-1α [32].

MM plasma cells and BM stroma also contribute to the pathophysiology of MM-associated bone disease through the activation of signaling pathways regulating osteoclastogenesis, particularly the RANK/RANK-Ligand (RANK-L) and the Wnt pathway, and the release of osteoclast-activating factors, such as IL-1, IL-6, tumor necrosis factor (TNF)-α, IL-8 and Macrophage Inflammatory Protein (MIP)-1α. These factors, together with recently identified dysregulated miRNAs, determine osteoblast suppression with excessive osteoclastic resorption [33]. Finally, MM cells display a unique ability to evade immune surveillance through several mechanisms, including impairment of cytotoxic activity, induction of dendritic cell dysfunction and recruitment of regulatory cells [34].
