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 , 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 .
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 result in re-shaping of microenvironment, and architectural changes involve in particular the vascular compartment .
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,
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 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 .
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 . 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  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).
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 . 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) . 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 [17-19] (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 . Finally, Angiopoietins (Angs, Ang-1 and-2) are important mediators in vasculature homeostasis and their circulating levels are considered of prognostic significance in MM .
Overall, BM angiogenesis in MM contributes to disease progression; accordingly, new anti-myeloma agents target not only MM cells, but also the microenvironment, and in particular vessels . 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.
3. 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)
Table 1 illustrates the principal characteristics of 3D
Lack of 3D physical cues
Nano- and micro-topographies are recreated
|Unidirectional, passive fluid diffusion|
Lack of chemical gradients and reduced gas supply
high ECM stiffness (more than 1 GPa)
|Pluri-directional active fluid diffusion|
Gradients of nutrient and gas can be generated
Efficient waste removal in dynamic bioreactors
ECM stiffness lower than in 2D (variable from 1 to 100 kPa)
|Reduced interactions between neighbouring cells||Increased interactions between neighbouring cells||25|
|Flat: geometrically-constrained baso-apical polarity|
Limited spatial distribution of adhesions to ECM
Limited cell survival rate
|Spheroid: free cell polarity guided by ECM|
Whole cell surface distribution of adhesions to ECM
High cell survival rate
|Lack the major physiological cues (biochemical, chemical, physical, mechanical) of the original tissue|
Low cell differentiation state and function
Absent or abnormal neo- synthesized ECM (qualitatively and quantitatively)
|3D models are closer to the |
High differentiation state and functional competence
ECM characteristics may vary, according to the culture model, from synthetic, natural and de-cellularized ECM, but 3D models are closer to the physiological context
|41, 45,46, 47|
|Organized||Disruption of tissue organization, as in i||50|
|Higher growth-/ metabolic- related gene expression|
Activation of mitochondrial and ribosomal gene clusters
Gene expression is, generally, quite different from
|Growth-arrest related genes are activated|
Closer to tumour tissue
|Low (high)||High (low)||56-58|
|-||+/ +++||56 59,60|
In an effort to reproduce
Within this context, 3D
4. Murine models of MM
4.1. 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 107 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 . 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
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.
4.2. 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 .
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 . 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, trans-endothelial migration and invasion, and also to identify the molecular pairs involved . 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 . 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 .
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.
4.3. SCID-hu and SCID-synth-hu models
In an attempt to “humanize” murine models, in 1997 Urashima established an
More recently, Pierfrancesco Tassone and Filippo Causa developed the so-called “SCID-synth-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  (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 .
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
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.
4.4. Transgenic models
MM cells accumulate a series of somatic mutations in the initiating and progressing phases of the disease , thus justifying development of genetically modified MM murine models, which recapitulate and explore the genetics of MM . Recently, a model has been developed based on the enforced B cell lineage-directed transgene expression of XBP-1s . 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 . 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 .
Another model exploited the deregulated expression of Myc. Myc activation occurs in post-germinal center malignancies, including Burkitt’s lymphoma, and is a common feature in MM; in particular its over-expression is generally considered of prognostic significance . Mice engineered to express c-Myc under the control of mouse immunoglobulin kappa (IgK) light-chain gene–regulatory elements (Vk-Myc mice) were developed . 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 .
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 c-myc oncogene, which prevented its expression  (Fig. 3E). Myc could be then sporadically activated in post-germinal B cells as a result of somatic hypermutation, leading to the transition 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 . The model was found to be highly predictive of the activity of anti-myeloma drugs , including those that target microenvironment, and may potentially help to select new agents for evaluation in clinical trials.
5. Human-derived models of MM
in vitro /ex-vivohuman-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
Kirshner and her group have reconstructed,
5.2. 3D culture of human MM isolated cells and tissue explants in the microgravity-based RCCSTM 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
On this basis, we successfully employed the microgravity-based RCCSTM 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
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
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
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.