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

Tumor and its embedding microenvironment form a unique, dynamic system, largely orch‐ estrated by cellular players, including fibroblasts and endothelial cells (EC), and surround‐ ing extracellular matrix (ECM) with its distinctive physical, biochemical, and biomechanical properties. There is a general consensus that, beyond genetic mutations and epigenetic mod‐ ifications, the dialogue that occurs between tumor and its microenvironment, through solu‐ ble factors and molecular interactions, may affect tumor cells survival, growth, proliferation, response to chemical/physical factors, and lies the basis for metastatization to distant, specif‐ ic organs. This theory was proposed by Paget in the 1880s [1], who underlined the need, for investigating and targeting tumor, to focus not only on the cancer cell, “the seed”, but also on the “soil” where tumor homes and in which it derives its nutrients, oxygen and signals [2, 3]. Accordingly, tight links between tumor and surrounding microenvironment could de‐ termine the overall sensitivity to anti-cancer drugs and therefore represent an attractive therapeutic target [4].


Introduction
Tumor and its embedding microenvironment form a unique, dynamic system, largely orchestrated by cellular players, including fibroblasts and endothelial cells (EC), and surrounding extracellular matrix (ECM) with its distinctive physical, biochemical, and biomechanical properties.There is a general consensus that, beyond genetic mutations and epigenetic modifications, the dialogue that occurs between tumor and its microenvironment, through soluble factors and molecular interactions, may affect tumor cells survival, growth, proliferation, response to chemical/physical factors, and lies the basis for metastatization to distant, specific organs.This theory was proposed by Paget in the 1880s [1], who underlined the need, for investigating and targeting tumor, to focus not only on the cancer cell, "the seed", but also on the "soil" where tumor homes and in which it derives its nutrients, oxygen and signals [2,3].Accordingly, tight links between tumor and surrounding microenvironment could determine the overall sensitivity to anti-cancer drugs and therefore represent an attractive therapeutic target [4].
Tumor microenvironment plays a critical role also in development and progression of haematological malignancies [5,6].In this regard, Multiple Myeloma (MM) represents a paradigmatic condition [5,6].Indeed, MM plasma cells almost exclusively home and thrive inside Bone Marrow (BM) microenvironment, which confers anti-apoptotic and pro-survival signals and resistance to drugs.In turn, tumor cell interactions with BM cells and matrix re-sult in re-shaping of microenvironment, and architectural changes involve in particular the vascular compartment [7].
The establishment of tight links between MM plasma cells and their microenvironment underlines the need for appropriate models for studying MM biology and predicting the impact of drugs.
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, including animal models and a new three-dimensional (3D), gel-based, in vitro model of human MM microenvironment.Finally, we discuss the potential of RCCS TM bioreactorbased, dynamic 3D model systems (cell and tissue culture) to investigate critical aspects of human MM pathobiology and possible clinical applications.Advantages and limitations of each model, relative to MM investigation and assessment of drug sensitivity, are also considered.

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 approximately 1% of neoplastic diseases and 13% of hematologic cancers.Albeit significant advances 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 (asymptomatic) myeloma and, finally, to symptomatic myeloma.In addition to genetic abnormalities accumulating in MM cells, BM microenvironment actively participates to the pathogenesis and progression of the disease.Indeed, host stromal components profoundly influence many steps of tumor progression, such as tumor proliferation, invasion, angiogenesis, metastasis, and even malignant transformation [9].The BM, where MM cells specifically home, provides a highly specialized microenvironment, which optimally "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, together with the ensuing pathogenetic events, are depicted in Fig. 2 (upper panel).Bone homeostasis is the result of a complex network of stimuli, including hormones, vitamins and physico-mechanical forces.In addition to osteoblats and osteoclasts, which are responsible 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) trigger 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 chemotherapeutic agents [10].In particular, adhesion of MM cells to BMSC and to ECM components 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 Macrophage Inflammatory Protein (MIP)-1α.MM cells have also a unique ability to evade immune 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, dynamic and tightly regulated process, that occurs physiologically during normal growth, wound repair after injury and regeneration [12,13].Angiogenesis is controlled by the balance between positive and negative regulators.In a tumor microenvironment, the exaggerate expression of pro-angiogenic cyto-chemokines starts the ''angiogenic switch'', leading to increased micro vessel density (MVD) [14].The occurrence of an "angiogenic switch", responsible for the transition from the avascular "dormant" phase to the vascular phase of exponential 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 microenvironment, including MMEC, stromal cells and inflammatory cells [17][18][19] (Fig. 2 A, upper panel).Major angiogenic cytokines are VEGF-A, fibroblast growth factor (FGF) and hepatocyte growth factor (HGF).Both EC in general, and in particular MMEC, and MM cells secrete 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, Velcade), 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 bortezomib 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.

Advantages of models which mimic tumor microenvironment exploiting the third dimension
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 physical cues coming from BM ECM and cell components.
Traditional two-dimensional (2D) in vitro models (static culture of single cells kept as monolayer 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 physiological 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-clude, in addition to tissue-specific multiple cellularity, biochemical and mechanical properties, also the three-dimensionality [25,26].Since the pioneering studies of Bissell and colleagues [27], different groups, including ours, have demonstrated that significant differences exist between the biological behaviour and gene expression profiles of normal and transformed/tumor-derived cells maintained in culture with traditional (2D) culture methods, and that of cells kept in 3D culture (see, for example, [28][29][30][31], proving that 3D models can mimic in vivo conditions better than 2D systems [26,32,33]. Table 1 illustrates the principal characteristics of 3D versus conventional 2D in vitro models of differentiated and tumoral tissues, and their relevance to the in vivo situation.In an effort to reproduce in vitro the 3D specific microenvironment of the parental tissue, taking advantage of the rapid development of new technologies and tissue engineering techniques, an extremely wide variety of tissue models have been produced.The latter have already been successfully applied for investigating critical aspects of in vivo behaviour of a number of normal and tumoral cells (reviewed and discussed in 26).On this basis, 3D culture systems have been proposed as the most physiologically relevant in vitro models to investigate tumor development and behaviour [60][61][62].Recently, this experimental approach has been also exploited for the study of MM-cell biology and sensitivity to therapeutic agents [63].
Within this context, 3D in vitro (cell-based)/ex-vivo (tissue-based) human-derived culture systems represent important tools to generate new approaches to the understanding of the molecular mechanisms of MM progression, essential prerequisites for the development of more effective interventional, diagnostic and prognostic strategies.

Subcutaneous xenograft models
The simplest way to generate an animal model of cancer consists in the injection of tumor cells into an immune-deficient mouse.This approach, known as the xenograft model, has been extensively employed for solid tumors [64,65] and then extended to MM.The xenograft model of MM consists in the subcutaneous injection of 1-2 x 10 7 human myeloma cells (from RPMI-8226, U266, ARH-77 or OPM-2 cell lines) into the flanks of Severe Combined Immune-Deficient (SCID), nonobese diabetic (NOD), SCID/NOD and SCID/beige, mice [66,67] (Fig. 3A).The resulting plasmacytoma is palpable, and tumor burden measurable with a pair of caliper or, when lines are transduced with the eGFP-luc fusion gene, by bioluminescence imaging [68].After harvesting, tumor mass is suitable for histological examination, allowing identification of vasculature and determination of cell proliferation/apoptosis.The model is currently used to assess the activity of new drugs on MM tumor growth and to establish the effective, minimally toxic, dose.As an example, this model has been employed to investigate the in vivo anti-myeloma effect induced by the mTOR inhibitor CCI-779 [69].
More recently, the efficacy of new inhibitors of the CXCR4/CXCL12 axis (AMD3100 and BKT140) [70], and of stressors of the endoplasmic reticulum (spicamycin analogue, KRN5500) [71], which inflict death of MM cells, have been demonstrated using the xenograft model.Besides mono-therapies, the model is suitable to evaluate the maximal effect, in terms of tumor volume reduction, obtainable with combined molecules [72].
While the xenograft model is extremely practical, particularly for drug testing, it still suffers from several limitations.In fact, it does not accurately mimic human disease, since myeloma cell lines do not behave as primary myeloma cells, more closely resembling the aggressive stage of plasma cell leukemia.More importantly, it fails to recapitulate the reciprocal interactions between MM cells and their microenvironment, which follow MM cell localization and retention inside the BM.As a result, drug efficacy can be over-estimated, lacking implanted MM cells the specific, proper human context of ECM and non-malignant accessory cells.
Murine models of MM, including the 5TMM model, contribute to overcome this latter limitation.

5TMM models
The 5T model has been developed in the late seventies upon injection of mice with syngeneic murine MM cells, spontaneously arising in elderly C57BL/KaLwRij mice [73,74].The group of MM murine models collectively indicated as 5TMM mice comprises different types of mice, each bearing different tumor cells and having distinct characteristics (Figure 3B).The most commonly used, the 5T2MM and the 5T33MM models, display selective localization of cells in the BM, the presence of a serum M component and increased BM angiogenesis.The first one is characterized by moderate growth and development of osteolytic lesions more closely reproducing the human disease, while the second one displays a more aggressive behaviour with rapid growth [75].
Studies based on these models, substantially contributed by Karin Vanderkerken's group, have provided valuable insights into MM biology, and in particular on the mechanisms responsible for bone disease, MM-associated neoangiogenesis, and MM cell homing to the BM [75].Indeed, taking advantage from these models, it has been possible to dissect the single steps which participate to the homing process, including chemo-attraction, adhesion, transendothelial migration and invasion, and also to identify the molecular pairs involved [75].Moreover, these models allow the assessment of the impact of drugs on MM cells inside their proper microenvironment.In particular, the 5T2MM model allowed to unravel the anti-tumor activity, in addition to prevention of bone resorption, of the amino-biphosphonate zolendronic acid [76].More recently, the novel 'second-generation' pyrimidyl-hydroxamic acid-based histone deacetylase inhibitor JNJ-26481585 was found to reduce tumor burden and also to affect angiogenesis and osteolysis [77].
A major limitation of the model is represented by the limited availability of different 5T cell lines, which fails to recapitulate the high variability both in terms of genetics and of tumor behaviour which characterize MM developing in humans.Moreover, the results obtained with 5T models should be interpreted with caution, given the potential differences in the biology of human vs murine myeloma.

SCID-hu and SCID-synth-hu models
In an attempt to "humanize" murine models, in 1997 Urashima established an in vivo model of human MM using SCID mice bilaterally implanted with human fetal bone grafts (SCIDhu mice) [78].The purpose was to study the role of adhesion molecules which participate to human MM-BMSC interactions and regulate MM cell homing.The original experimental design consisted in the injection of MM cell lines (ARH-77, OCI-My5, U-266 or RPMI-8226) (1x10 4 -10 5 ) into the BM cavity of the left bone implants in irradiated mice (Fig. 3C).Human monoclonal MM cells grew within the human BM replacing the stroma and metastatized to the controlateral right bone implant, but not to murine bones or other murine organs [79], suggesting the existence of species-specific interactions.In myeloma-bearing mice, circulating human Ig were detectable and mice developed tubular nephropathy, due to light chains deposition, closely mirroring MM clinical manifestations and physiopathology [79].The model was successfully employed to study the efficacy of thalidomide as an anti-myeloma drug, disclosing its anti-angiogenic properties [80].The engraftment of IL-6-dependent INA-6 cells in SCID-hu mice, but not in SCID mice, as well as their sensitivity to anti-myeloma agents, has also been documented [81].
More recently, Pierfrancesco Tassone and Filippo Causa developed the so-called "SCIDsynth-hu" model (Figure 3D), based on the implantation of artificial bone scaffolds repopulated with human BMSC into SCID mice, followed by injection of purified MM cells from patients [82] (Fig. 3D).This model represents a further advancement over the previously described SCID-hu mouse (Fig. 3C).In fact, the use of 3D poly-β-caprolactone polymeric scaffolds, closely reproducing the micro-architecture of a human bone, overcomes the restricted availability of human fetal bones for implant, and also allows to perform studies in the context of an autologous setting [82].
As for SCID-hu models, in SCID-synth-hu mice injected human MM cells were found to optimally engraft the implanted "niche" and to interact with the human bone milieu, as demonstrated not only by histological and immunohistochemical analyses of the retrieved implants, but also by demonstration of immungloglobulin production in vivo [82].
Both systems thereby offer the possibility to investigate human MM cells-BM microenvironment interactions and to perform pre-clinical testing of anti-MM drugs in a clinically relevant context.

Transgenic models
MM cells accumulate a series of somatic mutations in the initiating and progressing phases of the disease [10], thus justifying development of genetically modified MM murine models, which recapitulate and explore the genetics of MM [83].Recently, a model has been developed based on the enforced B cell lineage-directed transgene expression of XBP-1s [84].XBP-1 is a major regulator of the Unfolded Protein Response (UPR) and plasma cell differentiation.Moreover, XBP-1 over-expression has been implicated in human carcinogenesis and tumor growth in solid tumors and also in MM [84].XBP-1 transgenic mice spontaneously develop MGUS which progresses to MM, exhibiting remarkable clinical features common to human MM.In particular, BM involvement with clonal MM cells, serum M spike, bone lytic lesions and renal Ig deposition could be demonstrated [84].
Another model exploited the deregulated expression of Myc.Myc activation occurs in postgerminal center malignancies, including Burkitt's lymphoma, and is a common feature in MM; in particular its over-expression is generally considered of prognostic significance [85].
Mice engineered to express c-Myc under the control of mouse immunoglobulin kappa (IgK) light-chain gene-regulatory elements (Vk-Myc mice) were developed [86].Myc is a strong oncogene, and its constitutive expression in early B cells of Vk-Myc mice led to a very aggressive lymphoma, with extra-medullary localization [86].
To create a transgenic mouse model more closely resembling human MM, in their elegant work Chesi and co-workers selected the C57Bl6 strain, genetically predisposed to develop MGUS, and generated a vector (Vk*Myc) containing a stop codon insertion in the human cmyc oncogene, which prevented its expression [87] (Fig. 3E).Myc could be then sporadically activated in post-germinal B cells as a result of somatic hypermutation, leading to the transi-tion from the spontaneous monoclonal gammopathy to a disease that fully recapitulate the biological and clinical features of human MM.In fact, Vk*Myc mice are characterized by the accumulation of slowly proliferating plasma cells exclusively inside the BM.Moreover, high levels of monoclonal antibody are detectable and end-organ damage develops, including anemia, kidney failure and lytic bone disease [87].The model was found to be highly predictive of the activity of anti-myeloma drugs [88], including those that target microenvironment, and may potentially help to select new agents for evaluation in clinical trials.

3D in vitro /ex-vivo human-derived models of MM
Due to inter-species differences, animal models have incomplete predictive value for human MM disease and drug response.New models are, therefore, needed that more closely resemble the in vivo situation in patients.Reliable, human-derived in vitro models, able to reproduce myelomagenesis within the specificity of BM microenvironment, are therefore of extreme value.
Kirshner and her group have reconstructed, in vitro, human BM microenvironment, through the proper overlay of matrix components, on which isolated cells from BM aspirate of MM patients were seeded [63].Cells spontaneously redistributed throughout the gel-matrix 3D substrate, mimicking human BM architecture and BM-MM interactions, thus providing a powerful tool for understanding the biology of MM [89].Strikingly, reconstructed BM allowed the expansion of primary myeloma cells, including the putative stem cell fraction.Moreover, the model allowed the assessment of the impact of anti-MM drugs on distinct cellular compartments inside a 3D architecture [63].

3D culture of human MM isolated cells and tissue explants in the microgravity-based RCCS TM bioreactor
It is well known that the metabolic requirements of complex 3D cell constructs are substantially higher than those needed for the maintenance of traditional cell monolayers (2D culture) kept in liquid media under static conditions.Dynamic bioreactors were primarily developed to modulate mass transfer, a crucial element for guaranteeing gas/nutrient supply and waste elimination, essential factors for maintaining cell viability within large 3D cell/tissue masses.Despite a wide array of fluid-dynamic bioreactors has been devised [47,90], the low-shear environment and optimal mass transfer, needed for the long-term culture of functional 3D tissue constructs and explants, were attained only with the introduction of the microgravity-based Rotary Cell Culture System (RCCS™, Synthecon Inc., USA) bioreactors (91,92; a vast literature is also available at http://www.synthecon.com).The relevance of this technology in enabling the long-term culture of complex tissue-like engineered 3D bio-constructs and tissue explants of various origin has been demonstrated also by our group, and, namely, in the case of bone [31,47.93].
On this basis, we successfully employed the microgravity-based RCCS TM technology for the generation (and long-term maintenance) of viable human-derived MM tissue explants and 3D cell constructs.Fig. 4 shows the culture chamber of the RCCS™ microgravity-based bioreactor, and histo-morphological images of the ex-vivo models of human MM developed by our group.Isolated cells from the RPMI myeloma cell line, kept in Bioreactor, spontaneously self-aggregated forming spheroid-like structures which retained viability and were identifiable with the specific anti-CD38 monoclonal antibody (Fig. 4B).The suitability of our method for the culture of human tissue samples was, firstly, proved by using skin biopsies, which retained intact epidermal and dermal architecture, including keratin stratum and skin annexes.Moreover, both blood and lymphatic vasculature was identifiable and exhibited normal morphology, in particular patent lumen and complete endothelial lining (Fig. 4C).The 3D culture of thick sections of human MM tissue explants fully preserved tissue architecture and microenvironment integrity (Fig. 4D) for extended periods of time.Moreover, the system was suitable for the assessment of drug sensitivity, not only of tumor compartment, but also of angiogenic vessels (Fig. 4D).Indeed, quantification of MVD in treated specimens could represent a unique method to assess the anti-angiogenic effect of a drug in human samples ex vivo.Finally, specialized functions of both MM cells and their microenvironment, including beta-2 microglobulin and cytokine release and met-alloproteases activities, could be also assessed (M.Ferrarini et al., submitted).Overall, these observations suggest that the 3D culture model in Bioreactor can be exploited as a novel translational tool, allowing prospective pre-clinical toxicity and drug efficacy testing in individual patients.

Conclusions
A major challenge in cancer biology and cancer therapy relies in the availability of suitable models that recapitulate the complex tumor-host interplay and responsiveness to drugs.This is especially true for MM, where the existence of tight links between MM cells and BM microenvironment has hampered for long the development of adequate animals and in vitro models.Recently, innovative murine and chimeric in vivo models have been developed, which allowed both to investigate MM physiopathology and to perform drugs testing.On the other hand, the exploitation of novel technologies for ex-vivo 3D culturing of human MM samples is emerging as a tool to properly investigate its pathogenetic mechanisms (and interactions) within a human context, and also to predict response to drugs in individual patients.
The availability of more and more sophisticated systems is expected to pave the way to a deeper understanding of pathogenetic events and also to development of novel patients-tailored therapeutic strategies.

Figure 1 .
Figure 1.Bone Marrow microenvironment.Bone homeostasis is the result of a complex network of stimuli, including hormones, vitamins and physico-mechanical forces.In addition to osteoblats and osteoclasts, which are responsible 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).

Figure 2 .
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 proliferation/survival, angiogenesis, osteoclastogenesis and defective immune function are depicted.Lower panel illustrates the major growth factor receptors and adhesion molecules used by MM plasma cells to interact with ECM and cellular components of BM microenvironment closer to the in vivo condition and number of in vivo cell/tissue features can be reproduced High differentiation state and functional competence ECM characteristics may vary, according to the culture model, 41, 45,46, 47 Multiple Myeloma -A Quick Reflection on the Fast Progress

Figure 3 .
Figure 3. Schematic representation of currently available MM animal models.The major murine (m) and murinehuman(hu) models together with their main advantages and limitations are depicted.Synth = synthetic polymeric scaffold; BMSC= Bone Marrow Stromal Cells; SCID=severe combined immune deficient.

Figure 4 .
Figure 4. RCCS TM -based 3D ex-vivo models of MM developed by our group.A: Detail of the culture chamber of the RCCS™ microgravity-based bioreactor; B: Monotypic 3D multi-cellular spheroids (RPMI cell line) cultured for 1 week in the RCCS™ bioreactor (H&E staining, left panels; CD38 staining, right panels); C: 3D tissue culture of skin biopsies (1 week) showing intact architecture and identifiable blood and lymphatic (D2-40+) vessels D: MM tissue explants cultured for 3 days in the RCCS™ bioreactor (H&E staining), in the absence or presence of Bortezomib, the latter showing plasma cells death.Arrows indicate bone lamellae.

Table 1 .
Innovative Models to Assess Multiple Myeloma Biology and the Impact of Drugs http://dx.doi.org/10.5772/54312