Mesenchymal stem cells expressing osteoprotegerin variants inhibit osteolysis in a murine model of multiple myeloma by Jerome T. Higgs, Joo Hyoung Lee, Hong Wang, Vishnu C. Ramani, Diptiman Chanda, Cherlene Y. Hardy, Ralph D. Sanderson, and Selvarangan Ponnazhagan BloodAdv Volume 1(25):2375-2385 November 28, 2017 © 2017 by The American Society of Hematology

Jerome T. Higgs et al. Blood Adv 2017;1:2375-2385 © 2017 by The American Society of Hematology

Characterization of human MSCs Characterization of human MSCs. hMSCs were isolated from remnants of bone marrow transplant bags and cultured in DMEM with 10% FCS. Upon confluence, the cells were harvested and stained with antihuman CD11b to exclude the population that was positive for this marker. Characterization of human MSCs. hMSCs were isolated from remnants of bone marrow transplant bags and cultured in DMEM with 10% FCS. Upon confluence, the cells were harvested and stained with antihuman CD11b to exclude the population that was positive for this marker. The remaining cells were sorted as positive for CD90, CD73, and CD44, and negative for CD45 (A) and cultured to confluence. The pluripotency of MSCs was determined by adipogenic and osteogenic differentiation assays using ADM and ODM, respectively. Fourteen days later, Oil-Red-O staining was performed for lipid droplets, and Alizarin red staining was performed for calcium deposition. Replicates of cells, grown in ODM, were also subjected to von Kossa staining on day 21 for mineralization. Original magnification ×100 (B). Jerome T. Higgs et al. Blood Adv 2017;1:2375-2385 © 2017 by The American Society of Hematology

Establishment of a mouse myeloma model with skeletal dissemination of tumor and osteolysis. Establishment of a mouse myeloma model with skeletal dissemination of tumor and osteolysis. Two different mouse models were tested for myeloma osteolytic dissemination. Groups of BALB/c mice were challenged with 5 × 105 MPC-11 cells, and SCID mice were challenged with 5 × 105 CAGHep cells via tail vein. Based on constitutive expression of firefly luciferase in both cell lines, mice were monitored by noninvasive imaging to monitor tumor growth within the tibia, femur, and spine. Cohorts of mice from each group were euthanized at the indicated time points, and bones were subjected to histology (A-B; original magnification ×100, hematoxylin and eosin stain) and micro-CT (C). Representative data indicate aggressive tumor growth and skeletal damage in the BALB/c-MPC-11 model by 2 weeks. However, the growth of myeloma cells in the SCID-CAGHep model was significantly delayed, allowing a window of >6 weeks before aggressive bone destruction was noted. Jerome T. Higgs et al. Blood Adv 2017;1:2375-2385 © 2017 by The American Society of Hematology

Noninvasive bioluminescence imaging as a measure of therapeutic potential of OPG variants as compared with WT OPG. Groups of SCID mice were challenged with 5 × 105 CAGHep cells via tail vein followed by noninvasive imaging for establishment of tumor cells around day 14. Noninvasive bioluminescence imaging as a measure of therapeutic potential of OPG variants as compared with WT OPG. Groups of SCID mice were challenged with 5 × 105 CAGHep cells via tail vein followed by noninvasive imaging for establishment of tumor cells around day 14. Cohorts of mice were then left untreated or administered with hMSCs modified to express WT OPG (OPGwt), or OPG variants: OPGY49R/OPGF107A. Tumor growth following therapy was monitored by noninvasive imaging on day 7 (day 21 after tumor challenge), and day 14 (day 28 after tumor challenge) after initiation of therapy. Jerome T. Higgs et al. Blood Adv 2017;1:2375-2385 © 2017 by The American Society of Hematology

Micro-CT and quantitative analysis of tibia and spine following hMSC-OPG therapy. Micro-CT and quantitative analysis of tibia and spine following hMSC-OPG therapy. Four weeks after tumor challenge, a time established as midpoint for the degree of osteolytic damage, cohorts of mice from untreated and indicated treatment groups were euthanized, and tibia (A) and spine (B) were subjected to micro-CT analysis. Data obtained from micro-CT were used in 3D reconstruction images and quantitative analysis of respective bones for connectivity density, trabecular number, and trabecular spacing (*P < .05; **P < .01, compared with untreated mice following tumor challenge). Jerome T. Higgs et al. Blood Adv 2017;1:2375-2385 © 2017 by The American Society of Hematology

Osteoclast staining in tibia and spine following hMSC-OPG therapy. Osteoclast staining in tibia and spine following hMSC-OPG therapy. Bone tissues were harvested from mice in control and indicated treatment groups 28 days following tumor challenge. The bones were decalcified, and 5-µM sections were made. The slides were stained with TRAP stain on both tibia and spine for osteoclast activity within the bone microenvironment. Original magnification ×100. Jerome T. Higgs et al. Blood Adv 2017;1:2375-2385 © 2017 by The American Society of Hematology

ELISA for OPG levels. ELISA for OPG levels. Forty-eight hours after transduction of hMSCs with AAV-OPGwt or AAV-OPGY49R or AAV-OPGF107A culture, supernatants were analyzed by ELISA for OPG levels using a commercial kit (A). OPG levels were also quantitated in mouse serum following therapy. Blood samples were obtained from untreated and hMSC-OPG–treated mice both prior to initiating hMSC therapy and 14 days after the treatment and similarly analyzed by ELISA for OPG levels (B). Data presented are mean ± standard error from triplicate experiments (*P < .05; **P < .01). Jerome T. Higgs et al. Blood Adv 2017;1:2375-2385 © 2017 by The American Society of Hematology