**5. Human-derived models of MM**

#### **5.1. 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 re‐ semble the *in vivo* situation in patients. Reliable, human-derived *in vitro* models, able to re‐ produce 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 al‐ lowed 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 cel‐ lular compartments inside a 3D architecture [63].

#### **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 substan‐ tially higher than those needed for the maintenance of traditional cell monolayers (2D cul‐ ture) kept in liquid media under static conditions. Dynamic bioreactors were primarily developed to modulate mass transfer, a crucial element for guaranteeing gas/nutrient sup‐ ply 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 cul‐ ture of functional 3D tissue constructs and explants, were attained only with the introduc‐ tion 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 rele‐ vance 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 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 bio‐ reactor, 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 identifi‐ able with the specific anti-CD38 monoclonal antibody (Fig.4B).

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 predic‐ tive of the activity of anti-myeloma drugs [88], including those that target microenviron‐

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 re‐ semble the *in vivo* situation in patients. Reliable, human-derived *in vitro* models, able to re‐ produce myelomagenesis within the specificity of BM microenvironment, are therefore of

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 al‐ lowed 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 cel‐

**5.2. 3D culture of human MM isolated cells and tissue explants in the microgravity-based**

It is well known that the metabolic requirements of complex 3D cell constructs are substan‐ tially higher than those needed for the maintenance of traditional cell monolayers (2D cul‐ ture) kept in liquid media under static conditions. Dynamic bioreactors were primarily developed to modulate mass transfer, a crucial element for guaranteeing gas/nutrient sup‐ ply 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 cul‐ ture of functional 3D tissue constructs and explants, were attained only with the introduc‐ tion 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 rele‐ vance 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

ment, and may potentially help to select new agents for evaluation in clinical trials.

**5. Human-derived models of MM**

50 Multiple Myeloma - A Quick Reflection on the Fast Progress

extreme value.

**RCCSTM bioreactor**

**5.1. 3D** *in vitro /ex-vivo* **human-derived models of MM**

lular compartments inside a 3D architecture [63].

group, and, namely, in the case of bone [31,47.93].

**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 biop‐ sies (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 show‐ ing plasma cells death. Arrows indicate bone lamellae.

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 ker‐ atin stratum and skin annexes. Moreover, both blood and lymphatic vasculature was identi‐ fiable 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 pe‐ riods 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 indi‐ vidual patients.

**References**

1889;1: 571–573.

2012;21(3): 172-177.

2009;114:3367-375.

2006;20:193-199.

2011;364:1046-1060.

Medicine2003;54:17-28

2000;407:249-257.

vironment 2009;2(Suppl. 1):9-17.

[1] Paget S. The distribution of secondary growths in cancer of the breast. The Lancet

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

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

53

[2] Witz IP. The tumor microenvironment: the making of a paradigm. Cancer Microen‐

[3] Weber CE, Kuo PC. The tumor microenvironment. Journal of Surgical Oncology

[4] Correia AL, Bissell MJ. The tumor microenvironment is a dominant force in multi‐

[5] Tripodo C, Sangaletti S, Piccaluga PP, Prakash S, Franco G, Borrello I, Orazi A, Co‐ lombo MP, Pileri SA.The bone marrow stroma in hematological neoplasms: a guilty

[6] Burger JA, Ghia P, Rosenwald A, Caligaris-Cappio F. The microenvironment in ma‐ ture B-cell malignancies: a target for new treatment strategies Blood

[7] Vacca A, Ribatti D. Bone Marrow angiogenesis in Multiple Myeloma. Leukemia

[8] Palumbo A; Anderson K. Multiple Myeloma. New. English Journal of Medicine

[9] Dvorak HF, Weaver VM, Tlsty TD, Bergers G. Tumor microenvironment and pro‐

[10] Hideshima T, Mitsiades C, Tonon G, Richardson PG, Anderson KC. Understanding multiple myeloma pathogenesis in the bone marrow to identify new therapeutic tar‐

[11] Alsayed Y, Ngo H, Runnels J, Leleu X, Singha UK, Pitsillides CM, Spencer JA, Kim‐ linger T, Ghobrial JM, Jia X, Lu G, Timm M, Kumar A, Côté D, Veilleux I, Hedin KE, Roodman GD, Witzig TE, Kung AL, Hideshima T, Anderson KC, Lin CP, Ghobrial IM. Mechanisms of regulation of CXCR4/SDF-1 (CXCL12)-dependent migration and

[12] Semenza GL. Angiogenesis in ischemic and neoplastic disorders. Annual Review of

[13] Carmeliet P, Jain RK. Angiogenesis in cancer and other diseases. Nature

[14] Moserle L, Amadori A, Indraccolo S. The angiogenic switch: implications in the regu‐

lation of tumor dormancy. Current Molecular Medicine 2009;9:935-941.

drug resistance. Drug Resistance Update 2012;15(1-2):39-49.

bystander. Nature Reviews Clinical Oncology 2011;8(8):456-466.

gression. Journal of Surgical Oncology 2011;103:468-474.

homing in multiple myeloma. Blood. 2007;109(7):2708-17.

gets. Nature Reviews Cancer 2007;7:585-598.
