The neoplastic bone marrow (BM) milieu plays important roles in the development of multiple myeloma (MM). Specifically, cell-cell crosstalk interactions and regulatory signaling molecules involved in the normal hematopoietic stem cell niche have also been implicated in initiation, development, and progression of MM. Moreover, this “myeloma niche” serves as a sanctuary site for MM stem cells. The increasing knowledge of interactions between complex of MM cells and other normal hematopoietic cells and also non-hematopoietic mesenchymal stem cells and their progeny may enhance our understanding of MM cell biology and pathogenesis as well as provide the basis for novel therapeutic strategies.
In order to develop an experimental system mimicking the specialized properties of the neoplastic BM microenvironment, we used hydrogel based 3-dimensional (3-D) versus 2-dimensional (2-D) models together with MM-derived mesenchymal stem cells (MM-MSCs) to create MM niche-like structures in vitro. In this 3-D model, MM-MSCs grew with the matrix fibers and at day 7 formed stable compact clusters, with fibrous meshwork-like structure among them. Furthermore, we showed tri-lineage differentiation capacity of 3-D comparable to 2D MM-MSCs towards osteogenic, adipogenic, and chondrogenic lineages. Our 3-D MM-MSCs model also showed calcium mineralization of clusters associated with differentiation towards the osteoblastogenic lineage. The phenotypic profile of MM-MSCs grown in the 2-D vs. 3-D model using MSC-specific markers (such as CD166, HLA-ABC, CD73, CD105, and CD90) did not reveal any significant changes, however CD271 was more highly expressed in the 3-D model, while CD146 was overexpressed in 2-D cultures.
Because the stem cell niche is enriched with extracellular matrix (ECM) molecules resulting in an overexpansion of the stem cell pool, we studied whether expression of these molecules was conserved in the 3-D model. Analyzing 30 MM-MSCs vs. 5 normal donor MSCs, we observed that the 3-D MM-MSCs expressed significantly high level of ECM molecules including laminin, fibronectin, collagen I, collagen IV and vitronectin (p<0.001) after 7 days of culture compared to the 2-D model, as determined by flow cytometry and confocal microscopy analysis. Furthermore, the formation of dimeric active integrins (α2β1, α4β1 and α5β1) on the MSCs surface when engaged by these ECM molecules, inducing MM proliferation and survival, was also increased in the 3-D model, associated with transcriptional up-regulation of integrin subunits as confirmed by qRT-PCR. We also investigated the key molecules that play an important role in the MM niche including N-cadherin and nestin, as well as the interactions of MM cells with MSCs in the niche via CXCL12/CXCR4 axis. MM-MSCs cultured in the 3-D vs. 2-D model have higher expression of N-cadherin and CXCL12 and decreased expression of nestin, reflecting the MM BM niche.
To mimic the functional marrow niche of MM in our model, we cultured the tumor cells from MM patients (N=30) with their autologous MM-MSCs in vitro. MM plasma cells (identified as CD38/CD138+ cells) were increased in percentage and proliferation after 14 days in the 3-D vs. 2-D model. Finally, MM cells expressing CXCR4, as well as MM side population with “stem-like” features (Jakubikova J et al., Blood 2011) were also significantly increased in 3-D MM-MSCs vs. 2-D MM-MSCs cultures. Our study shows that hydrogel based 3-D MM-MSCs model forms a functional niche in vitro, and may be useful to identify novel targets and validate therapies directed at MM stem cells in the MM BM niche.
Munshi:Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees; Merck: Consultancy; Oncopep: Patents & Royalties. Richardson:Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees. Anderson:Onyx: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Celgene Corp: Membership on an entity's Board of Directors or advisory committees.
Asterisk with author names denotes non-ASH members.