1. Immunomodulatory drugs lenalidomide and pomalidomide inhibit multiple myeloma- induced osteoclast formation and the RANKL/OPG ratio in the myeloma microenvironment targeting the expression of adhesion molecules 
Marina Bolzoni, Paola Storti, Sabrina Bonomini, Katia Todoerti, Daniela Guasco, Denise Toscani, Luca Agnelli, Antonino Neri, Vittorio Rizzoli, Nicola Giuliani 
Experimental Hematology 
Volume 41, Issue 4, Pages e1 (April 2013)
DOI: /j.exphem
Copyright © 2013 ISEH - Society for Hematology and Stem Cells Terms and Conditions

2. Figure 1
LEN and POM inhibit CCL3/MIP1α production by HMCLs without a significant effect on cell proliferation and survival. The HMCLs, JJN3, and XG-1 and the purified CD138+ MM cells were incubated in presence of LEN or POM at concentration ranging from 1–100 μmol/L or vehicle (DMSO 0.1%) for 24–72 hours. Cell proliferation (A) and viability (B) were evaluated after 72 hours of treatment. Lines represent the median percent variation versus control of four independent experiments performed in six replicates. (C) MIP1A/CCL3 gene expression was evaluated using real-time PCR in XG-1 after 24 hours of treatment. Graphs represent the median MIP1A/CCL3 mRNA expression levels of three independent experiments performed in triplicate. (D) Conditioned media of XG-1 incubated in the presence or absence of PreOB were collected after 48 hours of treatment and analyzed by enzyme-linked immunosorbent assay to measure CCL3/MIP1α levels. Graphs represent the mean ± SD of CCL3/MIP1α levels of three independent experiments performed twice.
Experimental Hematology  , e1DOI: ( /j.exphem )
Copyright © 2013 ISEH - Society for Hematology and Stem Cells Terms and Conditions

3. Figure 2
LEN and POM blunted RANKL/OPG imbalance in PreOB cocultured with HMCLs. Human BM PreOB obtained from primary BMSC in osteogenic differentiation medium for two weeks were cocultured with or without XG-1 (A, B) or JJN3 (C) in a cell-to-cell contact system in the presence of LEN, POM, or vehicle (DMSO at 0.1%) for 24 hours for mRNA extraction (A) and 48 hours for CM collection (B, C). RANKL and OPG mRNA levels were evaluated by real-time PCR. Graphs represent the median of RANKL and OPG mRNA levels and the RANKL/OPG mRNA ratio of three independent experiments performed in triplicate (A). RANKL and OPG protein levels were detected with enzyme-linked immunosorbent assay. Graphs represent the mean ± SD of RANKL and OPG levels and the RANKL/OPG protein ratio of three independent experiments performed twice (B, C).
Experimental Hematology  , e1DOI: ( /j.exphem )
Copyright © 2013 ISEH - Society for Hematology and Stem Cells Terms and Conditions

4. Figure 3
LEN and POM does not affect the production of pro-osteoclastogenic cytokines by PBMCs and T lymphocytes. PBMCs were obtained from buffy coats by Ficoll-Hypaque density sedimentation. CD3+ and CD8+ cells were isolated from PBMCs using anti-CD3 or anti-CD8 monoclonal Ab–coated microbeads. PBMCs, CD3+, and CD8+ cells were activated by ionomycin (1 μM) and phorbol myristic acetate (5 ng/mL) for 48–72 hours and then treated with LEN or POM (2–100 μmol/L) or vehicle for an additional 24 hours for RNA extraction and 48 hours for CM collection. RANKL and OPG protein levels were measured by ELISA in the CM of PBMCs, and RANKL/OPG protein ratio was calculated. The graph represents the mean ± SD RANKL/OPG ratio of three independent experiments performed twice (control = vehicle) (A). RANKL mRNA levels were evaluated in activated CD3+ and CD8+ T lymphocytes by real time PCR. Graphs represent the median fold change of RANKL mRNA of treated cells compared with the control (control = vehicle) (B). CCL3/MIP1α and IL-3 protein levels were detected in the CM of PMBC treated for 48 hours with vehicle or LEN or POM. Graphs represent the mean ± SD of CCL3/MIP1α (C) and IL-3 (D) protein levels of three independent experiments performed twice. CON = control.
Experimental Hematology  , e1DOI: ( /j.exphem )
Copyright © 2013 ISEH - Society for Hematology and Stem Cells Terms and Conditions

5. Figure 4
LEN and POM inhibit MM-induced in vitro osteoclastogenesis. (A) CD14+ cells were incubated with rhM-CSF (25 ng/mL) and rhRANKL (60 ng/mL) in α-MEM at 10% of FBS in presence of POM or LEN (2 and 100 μmol/L) or vehicle (0.1% DMSO) or Zoledronic acid (ZOL) (2 and 100 μmol/L) in 96-well microtiter plates for 28 days replacing medium every 3–4 days. (B) CD14+ cells were incubated in α-MEM at 10% of FBS or CM/α-MEM at 10% FBS (1:4 ratio) from PreOB cocultured with HMCLs pretreated with IMiDs or vehicle for 48–72 hours, with rhM-CSF (25 ng/mL) and with or without rhRANKL (60 ng/mL) for 28 days replacing the medium every 5 days. OCs were identified at the end of culture period as multinucleated (>3) cells positive for tartrate resistant acid phosphatase (TRAP) assay. Graphs represent the mean ± SD of OC number per well of two independent experiments performed in six replicates. Images were visualized using an Eclipse TE 300 microscope with a digital DS-U1 sight (Nikon Instruments, Florence, Italy). For all micrographs, original magnification is ×200. (*p = 0.05; **p = 0.01).
Experimental Hematology  , e1DOI: ( /j.exphem )
Copyright © 2013 ISEH - Society for Hematology and Stem Cells Terms and Conditions

6. Figure 4
LEN and POM inhibit MM-induced in vitro osteoclastogenesis. (A) CD14+ cells were incubated with rhM-CSF (25 ng/mL) and rhRANKL (60 ng/mL) in α-MEM at 10% of FBS in presence of POM or LEN (2 and 100 μmol/L) or vehicle (0.1% DMSO) or Zoledronic acid (ZOL) (2 and 100 μmol/L) in 96-well microtiter plates for 28 days replacing medium every 3–4 days. (B) CD14+ cells were incubated in α-MEM at 10% of FBS or CM/α-MEM at 10% FBS (1:4 ratio) from PreOB cocultured with HMCLs pretreated with IMiDs or vehicle for 48–72 hours, with rhM-CSF (25 ng/mL) and with or without rhRANKL (60 ng/mL) for 28 days replacing the medium every 5 days. OCs were identified at the end of culture period as multinucleated (>3) cells positive for tartrate resistant acid phosphatase (TRAP) assay. Graphs represent the mean ± SD of OC number per well of two independent experiments performed in six replicates. Images were visualized using an Eclipse TE 300 microscope with a digital DS-U1 sight (Nikon Instruments, Florence, Italy). For all micrographs, original magnification is ×200. (*p = 0.05; **p = 0.01).
Experimental Hematology  , e1DOI: ( /j.exphem )
Copyright © 2013 ISEH - Society for Hematology and Stem Cells Terms and Conditions

7. Experimental Hematology 2013 41, 387-397. e1DOI: (10. 1016/j. exphem
Copyright © 2013 ISEH - Society for Hematology and Stem Cells Terms and Conditions

8. Experimental Hematology 2013 41, 387-397. e1DOI: (10. 1016/j. exphem
Copyright © 2013 ISEH - Society for Hematology and Stem Cells Terms and Conditions

9. Figure 5
LEN and POM reduced HMCL adhesion to fibronectin and CD49d expression by HMCLs. (A) HMCLs have been pretreated with LEN or POM or vehicle for 48 hours or with a blocking anti-CD49 Ab for 2 hours. After washing, HMCL adhesion to fibronectin was evaluated by a commercially available kit according to manufacturer’s protocols. Graphs represent the mean ± SD of the cell adhesion to fibronectin. OD = optical density. (B) LEN or POM (10 pM–100 μmol/L) or vehicle (control) has been added to JJN3 cultured alone or in the presence of BMSCs and to XG-1 for 48 hours. CD49d expression by HMCLs was evaluated by flow cytometry at the end of culture period. (∗p < 0.05; ∗∗p < 0.01).
Experimental Hematology  , e1DOI: ( /j.exphem )
Copyright © 2013 ISEH - Society for Hematology and Stem Cells Terms and Conditions

10. Supplementary Figure E1
The HMCLS JJN3 and XG-1 and fresh purified CD138+ MM cells were treated with LEN or POM or vehicle for 24 hours. ITGA8 and ICAM2 mRNA levels were evaluated by real-time PCR. Graphs represent the median ITGA8 and ICAM2 mRNA levels of three independent experiments performed twice in JJN3 (A) and in XG-1 and CD138+ MM cells (B). (C) CD102 expression has been evaluated by flow cytometry after 48 hours treatment with LEN or POM or vehicle in JJN3 incubated in the absence or the presence of BMSC in a cell-to-cell contact system, in XG-1 and in fresh purified CD138+ MM cells. Control = vehicle; *p = 0.05; **p = 0.01.
Experimental Hematology  , e1DOI: ( /j.exphem )
Copyright © 2013 ISEH - Society for Hematology and Stem Cells Terms and Conditions