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D. Belloni et al.
(ATM), 13q14.3 (D13S319), 13q34 (LSI13q34), 17p13 (TP53) (Multi-color Probe, Abbott Molecular) and IGH gene rearrange- ments (DAKO). Microscope observation was performed using a Nikon Eclipse 90i (Nikon Instruments, Japan) and analyzed by Genikon software (Nikon).
Statistical analysis
Statistical analysis was performed using Student t-test or one- way ANOVA. *P≤0.05; **P≤0.01; ***P≤0.001.
Results
Generation of a functional MM 3D microenvironment in RCCSTM bioreactor requires supportive stroma
3D reconstruction of a surrogate MM microenviron- ment was based upon the proper combination of a scaf- fold and stromal cells, according to the procedure depicted in Figure 1A. As scaffold we used a commercially avail- able gelatin biomaterial, which was selected according to several parameters (Online Supplementary Figure S1),
including efficiency of cell seeding and recovery, and espe- cially its ultrastructure, resembling that of adult bone (Figure 1B). An efficient scaffold seeding with either the HS-5 cell line or HUVEC was achieved in the RCCSTM bioreactor, as indicated by the overall number of viable recovered cells upon 18 h 3D culture (Figure 1C). As a final step, MM cells were added to the bioreactor vessels (Figure 1A) resulting in the successful formation of a homogeneously populated, dense construct where both tumor cells and stroma conserved lineage specific markers (Figure 1D).
We established our model with MM1.S and RPMI.8266 MM cell lines. These cell lines differ remarkably in the expression of β1-integrin and very late antigen-4 (VLA-4) (Figure 2A) which, through the interaction with its ligand VCAM1, is involved in MM adhesion to BM stroma.23 As a result, adhesion to HS-5 or L-VCAM transfectant paral- lels these differences (Figure 2B). Stromal cells were also required for MM cell permanence inside the scaffold, as indicated by the significantly higher number of MM cells populating the scaffold in the presence versus the absence
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Figure 3. Pro-survival signals are delivered to multiple myeloma (MM) cell lines. (A) Western blot analyses of pAkt, Akt, survivin and actin in 3D versus 2D co-cul- tures of HS-5 and RPMI.8266 or MM1.S cells and of primary bone marrow stromal cells (BMSC) and MM1.S cells (one representative experiment out of three) and also on isolated HS-5 cells. (B) Mean±Standard Error of Mean (SEM) of pAkt/totalAkt ratios from three independent experiments. β2-microglobulin (C), IL-6, angiopoi- etin-2 (Ang-2), VEGF and FGF (D) levels in 3D culture supernatants. *P≤0.05.
haematologica | 2018; 103(4)