**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) *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‐

**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

42 Multiple Myeloma - A Quick Reflection on the Fast Progress

clude, in addition to tissue-specific multiple cellularity, biochemical and mechanical proper‐ ties, also the three-dimensionality [25,26]. Since the pioneering studies of Bissell and colleagues [27], different groups, including ours, have demonstrated that significant differ‐ ences exist between the biological behaviour and gene expression profiles of normal and transformed/tumor-derived cells maintained in culture with traditional (2D) culture meth‐ ods, and that of cells kept in 3D culture (see, for example, 28-31), proving that 3D models can mimic *in vivo* conditions better than 2D systems [26,32,33].

**Characteristics of**

**2D conformation (on flat glass or plastic**

Absent or abnormal neosynthesized ECM (qualitatively

**Cell organization** Organized Disruption of tissue organization,

Higher growth-/ metabolicrelated gene expression Activation of mitochondrial and ribosomal gene clusters Gene expression is, generally, quite different from *in vivo*

tumours

and quantitatively)

**3D conformation**

**supports)**

context


activated

+/- ++ 54,55

+ ++ 55


**(sensitivity)** Low (high) High (low) 56-58

as in i*n vivo* tumours

Growth-arrest related genes are

Closer to tumour tissue *in vivo*

**(cell spheroids, 3D artificial**

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

from synthetic, natural and decellularized ECM, but 3D models are closer to the physiological

**References**

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

45

50

51-53

**substrates)**

*in vitro* **models**

**ECM-related cell motility and mechanobiology, compared to the in vivo situation**

**Gene expression**

**Capability to reproduce specific morphological and behavioural characteristics of in vivo malignant**

**cells**

**Responsiveness to surviving signal from ECM**

**Drug resistance**

**Capability to reproduce the complexity of tumour**

**Table 1.**

**microenvironment**

*In vitro models of tumor tissues*

**the**

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.



**Table 1.**

clude, in addition to tissue-specific multiple cellularity, biochemical and mechanical proper‐ ties, also the three-dimensionality [25,26]. Since the pioneering studies of Bissell and colleagues [27], different groups, including ours, have demonstrated that significant differ‐ ences exist between the biological behaviour and gene expression profiles of normal and transformed/tumor-derived cells maintained in culture with traditional (2D) culture meth‐ ods, and that of cells kept in 3D culture (see, for example, 28-31), proving that 3D models

Table 1 illustrates the principal characteristics of 3D *versus* conventional 2D *in vitro* models

**3D conformation**

**supports)**

Multilayer

diffusion

are recreated

be generated

**(cell spheroids, 3D artificial**

Nano- and micro-topographies

Gradients of nutrient and gas can

Pluri-directional active fluid

Efficient waste removal in dynamic bioreactors

ECM stiffness lower than in 2D (variable from 1 to 100 kPa)

Increased interactions between

Spheroid: free cell polarity guided

Whole cell surface distribution of

3D models are closer to the *in vivo* condition and number of *in vivo* cell/tissue features can be

High differentiation state and functional competence ECM characteristics may vary, according to the culture model,

neighbouring cells

adhesions to ECM High cell survival rate

reproduced

by ECM

**References**

34-37

26, 38-41

25

3142-44

41, 45,46, 47

of differentiated and tumoral tissues, and their relevance to the *in vivo* situation.

can mimic *in vivo* conditions better than 2D systems [26,32,33].

**2D conformation (on flat glass or plastic**

Lack of 3D physical cues

Unidirectional, passive fluid

reduced gas supply

neighbouring cells

baso-apical polarity

adhesions to ECM Limited cell survival rate

original tissue

and function

Lack of chemical gradients and

high ECM stiffness (more than 1

Reduced interactions between

Flat: geometrically-constrained

Limited spatial distribution of

Lack the major physiological cues (biochemical, chemical, physical, mechanical) of the

Low cell differentiation state

**substrates)**

44 Multiple Myeloma - A Quick Reflection on the Fast Progress

diffusion

GPa)

*In vitro models of differentiated tissues*

**Architecture** Monolayer

**Characteristics of**

*in vitro* **models**

**Cell-milieu interaction**

**Cell-cell interactions**

**viability**

**in vivo**

**Cell morphology/**

**Ability to mimic the physiological behaviour of cells**

**the**

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 nor‐ mal 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 devel‐ opment and behaviour [60-62]. Recently, this experimental approach has been also exploited for the study of MM-cell biology and sensitivity to therapeutic agents [63].

Murine models of MM, including the 5TMM model, contribute to overcome this latter

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

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

47

**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.

limitation.

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.
