Investigating osteogenic differentiation in multiple myeloma using a novel 3D bone marrow niche model

Abstract

Clonal proliferation of plasma cells within the bone marrow (BM) affects local cells, such as mesenchymal stromal cells (MSCs), leading to osteolysis and fatality in multiple myeloma (MM). Consequently, there is an urgent need to find better mechanisms of inhibiting myeloma growth and osteolytic lesion development. To meet this need and accelerate clinical translation, better models of myeloma within the BM are required. Herein we have developed a clinically relevant, three-dimensional (3D) myeloma BM coculture model that mimics bone cell/cancer cell interactions within the bone microenvironment. The coculture model and clinical samples were used to investigate myeloma growth, osteogenesis inhibition, and myeloma-induced abnormalities in MM-MSCs. This platform demonstrated myeloma support of capillary-like assembly of endothelial cells and cell adhesion-mediated drug resistance (CAM-DR). Also, distinct normal donor (ND)- and MM-MSC miRNA (miR) signatures were identified and used to uncover osteogenic miRs of interest for osteoblast differentiation. More broadly, our 3D platform provides a simple, clinically relevant tool to model cancer growth within the bone-useful for investigating skeletal cancer biology, screening compounds, and exploring osteogenesis. Our identification and efficacy validation of novel bone anabolic miRs in MM opens more opportunities for novel approaches to cancer therapy via stromal miR modulation.

Figure 1

Development of an in vitro 3D BM niche model. (A) Confocal images of calcein-labeled ND-MSCs, passage 2, (calcein/live cells, green; silk scaffold, red) at 2 and 37 days of culture in osteogenic media. The scale bar represents 100 μm. (B) Confocal images of RFP⁺HUVECs (red) ± GFP⁺MM1S cells (green) on scaffolds (blue) (days 3 and 9; scale bar = 100 μm). Representative image of 3 experiments is shown here, cultured in endothelial growth media. (C) Confocal images at day 30 of culture of GFP⁺MM1S alone (left, green), DiD-labeled MSCs alone (middle, red), and cocultures (right) in 50-50 medium with bortezomib (top, 5 nM) or without bortezomib (bottom) on autofluorescent scaffolds (blue). (Scale bar = 100 μm.) (D) Confocal images of primary patient CD138⁺ MM cells (green with DiI) at day 7 seeded onto ND-MSCs (red with DiD). Channels show the myeloma cell (arrow) as alive (calcein⁺, blue), DiI⁺ (green), and DiD^– (red). Overlay of green and blue appears cyan and demonstrates colocalization of calcein and DiI staining. Samples cultured in 50-50 media (n = 3); the scale bar represents 20 μm.

Figure 2

Inhibited osteogenesis induced by myeloma in a 3D bone model. (A) Fluorescent imaging at week 5 (TurboRFP⁺MSCs, red; GFP⁺MM1S, green; scaffold, blue). Overlaid channels (merge) shows increased pore infiltration, elongation, and proliferation by MSCs when grown in the absence of myeloma cells (left) compared with when grown with MM1S (right). The scale bar represents 200 μm. (B) Confocal images of TurboRFP⁺MSCs (red) and GFP⁺MM1S (green), alone or in coculture, on silk scaffolds (blue) from 1 to 5 weeks of culture in osteogenic media. The scale bar represents 100 μm. (C) Histologic analysis of scaffolds after 5 weeks of osteogenesis for MSCs alone (top) or in coculture with GFP⁺MM1S (bottom) stained for mineralization (Alizarin Red, right) or hematoxylin and eosin (H&E) (left). Black arrow indicates mineralization found only in MSCs cultured alone. Yellow arrows indicate silk scaffold. Red arrow indicates stromal cells, which are found throughout the MSC alone samples and sparsely through coculture samples. Green arrows indicate MM1S plasma cells found only in coculture samples. The scale bar represents 50 μm.

Figure 3

Alterations in MSC miRs in 3D modeling and patient vs normal data, and the resulting changes in MSCs after mimic-induced increased expression of miR-199a. (A) Heat map of the 53 mRNAs identified from nanoString analysis from the 3D model samples that are significantly different between cocultured (Co-culture) and monocultured ND-MSCs (MSCs). Filtering was done on original 800 miRs based on high expression (>25 average counts), significance between myeloma vs normal donor groups (P < .05), and high fc threshold (fc >1.5). (B) Heat map of 41 miRs identified from nanoString analysis that are significantly different between patient samples (myeloma [MM] and normal donor [ND] sample MSCs). Filtering was done on original 800 miRs based on high expression (>25 average counts), with significance between myeloma vs normal donor groups (P < .05), and high fc threshold (fc >1.5). (C) Alizarin Red staining quantification of mineralization produced by MSCs transfected with negative control mimic or miR-199a-5p mimic after 10 days in osteogenic no-dexamethasone media. Data plotted as mean ±SEM, n ≥3 different donors. (D) Alizarin Red staining representative images showing mineralization of MM-MSCs in 6-well plates transfected with negative control mimics or miR-199a-5p mimics to increase miR-199a-5p expression after 10 days in osteogenic no-dexamethasone media. Images are representative of n ≥3 different donors. The scale bars represent 200µm (original magnification ×4), 100 µm (×10), 50 µm (×20), and 20 µm (×40). (E) MM-MSCs transfected to increase expression of miR-199a-3p (E) or 199a-5p (F) demonstrate increased expression of osteogenic markers after 10 days of culture, measured by q-RT-PCR, gene expression normalized to negative control (Control) for each gene. ALPL, alkaline phosphatase; BSP, integrin-binding sialoprotein; Col1a1, collagen type I α 1; OP, osteopontin; OC, osteocalcin; RUNX2, runt-related transcription factor 2. Data plotted as mean ± SEM and analyzed with 1-way ANOVA and a post hoc Fisher least significance difference test for multiple comparisons (each gene vs negative control). Day 10 after transfection with miR mimics or controls and grown in osteogenic-no dexamethasone medium. n ≥ 3 different donors, **P < .05, **P < .01.