SCID-Synth-Hu: a Novel Multiple Myeloma Model for In Vivo Expansion of Primary Cells

Abstract 452

The critical role of the human bone marrow microenvironment (HuBMM) in the pathogenesis of Multiple Myeloma (MM) has recently allowed the design of novel therapeutical approaches targeting not only MM cells but also their specific HuBMM. However, the lack of adequate mouse models, capable to recapitulate a HuBMM, has restrained large scale in vivo screening of investigational drugs. In fact, only the SCID-hu model, in which human MM cells are grown in human fetal bone chips previously implanted in SCID mice, addresses this specific requirement. However the poor availability of human fetal bone chips, the allogeneic nature of the fetal BM milieu versus MM cells and the heterogeneity of implanted human bone chips are important restrains of this system. Here we report the development of a novel in vivo model of human MM (SCID-synth-hu), which is based on the implant of a three-dimensional (3-D) poly-ε-caprolactone polymeric scaffold (PCLS) into SCID mice as recipient to allow growth of MM cells in a reconstituted HuBMM. This biosynthetic scaffold has been designed to resemble the micro-architecture of a normal human adult bone and was characterized by 3-D interconnected large and small pores suitable for engraftment of bone marrow-derived cells. Human bone marrow stromal cells (BMSCs) were collected from BM aspirates from MM patients and firstly used for coating 3D internal surface of PCLSs. We performed in vitro dynamic seeding of BMSCs into PCLSs using a suspension of 10⁶ cells in 500 μl of growth medium. Before implantation, PCLSs were incubated in complete medium at 37°C, in 5% CO₂ for 24 hours, in order to allow cell adhesion on 3D surfaces. Then, PCLSs were implanted subcutaneously into SCID mice. CD138⁺ immune-selected primary MM cells, obtained by MM patient with a different disease status, were directly injected into PCLSs, which have been previously coated with allogeneic BMSCs 2–3 weeks after the in vivo implant. By this experimental approach, we achieved engraftment of primary MM cells in PCLSs within a non autologous bone marrow milieu. In a subsequent series of experiments, bone marrow mononuclear cells (BMMCs), obtained by Ficoll gradient separation and containing primary unselected CD138⁺ and their autologous BMSCs, were directly seeded in vitro into PCLSs which were implanted in SCID mice after 24 hours of incubation. At different time points, H&E and CD138 or κ/λ staining demonstrated engraftment and filling of 3-D spaces by primary MM cells within the autologous bone marrow milieu in PCLSs retrieved from SCID-synth-hu mice. Neo-synthesized extracellular matrix and angiogenesis were also shown by H&E and immune histochemical staining in retrieved PCLSs. Angiogenesis mostly occurred within areas of MM infiltration, suggesting its role in our system. In vivo MM growth was monitored by ELISA measuring of human monotypic immunoglobulins (Igs) in mouse sera 4 to 10 weeks after cell injection. To demonstrate the usefulness of our SCID-synth-hu model as an experimental platform for in vivo testing of investigational drugs, mice bearing human MM implants were treated intraperitoneally with bortezomib plus dexamethasone (Bort+Dex). As expected, SCID-synth-hu mice treated with Bort+Dex exhibited a significant decrease of monotypic light chains in mice sera and induction of apoptosis of MM cells in retrieved PCLSs, as compared to untreated control mice. Our experimental findings demonstrate that the SCID-synth-hu is the first experimental system which allows the in vivo expansion of human primary MM cells within their autologous adult HuBMM. The unlimited availability and the low cost of PCLSs, as well as the potential for dissecting the biological events within the HuBMM, represent a clear improvement over the available preclinical in vivo models of MM. We conclude that the SCID-synth-hu is a unique tool for large scale in vivo preclinical screening of novel agents targeting MM in its autologous HuBMM, and a novel resource for translational research in the experimental treatment of this still incurable disease.