Co-culture Of Bone Marrow Stromal Cells Research Articles

Bone marrow stromal cells in Modic type 1 changes promote neurite outgrowth.

The pain in patients with Modic type 1 changes (MC1) is often due to vertebral body endplate pain, which is linked to abnormal neurite outgrowth in the vertebral body and adjacent endplate. The aim of this study was to understand the role of MC1 bone marrow stromal cells (BMSCs) in neurite outgrowth. BMSCs can produce neurotrophic factors, which have been shown to be pro-fibrotic in MC1, and expand in the perivascular space where sensory vertebral nerves are located. The study involved the exploration of the BMSC transcriptome in MC1, co-culture of MC1 BMSCs with the neuroblastoma cell line SH-SY5Y, analysis of supernatant cytokines, and analysis of gene expression changes in co-cultured SH-SY5Y. Transcriptomic analysis revealed upregulated brain-derived neurotrophic factor (BDNF) signaling-related pathways. Co-cultures of MC1 BMSCs with SH-SY5Y cells resulted in increased neurite sprouting compared to co-cultures with control BMSCs. The concentration of BDNF and other cytokines supporting neuron growth was increased in MC1 vs. control BMSC co-culture supernatants. Taken together, these findings show that MC1 BMSCs provide strong pro-neurotrophic cues to nearby neurons and could be a relevant disease-modifying treatment target.

Anterior cruciate ligament remnant preservation attenuates apoptosis and enhances the regeneration of hamstring tendon graft

The effects of remnant preservation on the anterior cruciate ligament (ACL) and its relationship with the tendon graft remain unclear. We hypothesized that the co-culture of remnant cells and bone marrow stromal cells (BMSCs) decreases apoptosis and enhances the activity of the hamstring tendons and tenocytes, thus aiding ACL reconstruction. The ACL remnant, bone marrow, and hamstring tendons were surgically harvested from rabbits. The apoptosis rate, cell proliferation, and expression of types I and III collagen, transforming growth factor-β (TGF-β), vascular endothelial growth factor (VEGF), and tenogenic genes (scleraxis (SCX), tenascin C (TNC), and tenomodulin (TNMD)) of the hamstring tendons were compared between the co-culture medium (ACL remnant cells (ACLRCs) and BMSCs co-culture) and control medium (BMSCs-only culture). We also evaluated the apoptosis, cell proliferation, migration, and gene expression of hamstring tenocytes with exposure to co-culture and control media. Compared to BMSCs-only culture medium, the co-culture medium showed substantially decreased early and late apoptosis rates, attenuation of intrinsic and extrinsic apoptotic pathways, and enhanced proliferation of the hamstring tendons and tenocytes. In addition, the expression of collagen synthesis, TGF-β, VEGF, and tenogenic genes in the hamstring tendons and tenocytes significantly increased in the co-culture medium compared to that in the control medium. In the presence of ACLRCs and BMSCs, the hamstring tendons and tenocytes significantly attenuated apoptosis and enhanced the expression of collagen synthesis, TGF-β, VEGF, and tenogenic genes. This in vitro study suggests that the ACLRCs mixed with BMSCs could aid regeneration of the hamstring tendon graft during ACL reconstruction.Cite this article: Bone Joint Res2023;12(1):9-21.

Angelica sinensis polysaccharides prevents hematopoietic regression in D-Galactose-Induced aging model via attenuation of oxidative stress in hematopoietic microenvironment.

Extrinsic molecular mechanisms that regulate hematopoietic stem/progenitor cell (HSPC) aging are still poorly understood, and a potential protective medication needs to be explored. The senescent parameters of hematopoietic cells and bone marrow stromal cells (BMSCs) including cell cycle analysis, senescence-associated SA-β-gal staining and signals, hematopoietic factors and cellular junction were analyzed in femur and tibia of rats. Furthermore, Sca-1+ HSPCs and BMSCs co-culture system was established to evaluate the direct effects of BMSC feeder layer to HSPCs. Oxidative DNA damage indicators in Sca-1+ HSCs and senescence-associated secretory phenotype (SASP) of BMSCs, gap junction intercellular communication between BMSCs, osteogenesis/adipogenisis differentiation balance of BMSCs were detected. In the D-gal pre-administrated rats, ASP treatment rescued senescence of hematopoietic cells and BMSCs, reserved CFU-GEMM; also, ASP treatment attenuated stromal oxidative load, ameliorated SCF, CXCL12, and GM-CSF production, increased Connexin-43 (Cx43) expression. BMSCs and Sca-1+ HSPCs co-cultivation demonstrated that ASP treatment prevented oxidative DNA damage response in co-cultured Sca-1+ HSPCs induced by D-gal pre-administration of feeder layer and the underlying mechanism may be related to ASP ameliorating feeder layer dysfunction due to D-gal induced senescence via inhibiting secretion of IL-1, IL-6, TNF-α, and RANTES, enhancing Cx43-mediated intercellular communication, improving Runx2 expression whereas decreasing PPARγ expression in BMSCs. The antioxidant property of ASP may provide a stroma-mediated potential therapeutic strategy for HSPC aging.

Spheroid co-culture of BMSCs with osteocytes yields ring-shaped bone-like tissue that enhances alveolar bone regeneration

Oral and maxillofacial bone defects severely impair appearance and function, and bioactive materials are urgently needed for bone regeneration. Here, we spheroid co-cultured green fluorescent protein (GFP)-labeled bone marrow stromal cells (BMSCs) and osteocyte-like MLO-Y4 cells in different ratios (3:1, 2:1, 1:1, 1:2, 1:3) or as monoculture. Bone-like tissue was formed in the 3:1, 2:1, and 1:1 co-cultures and MLO-Y4 monoculture. We found a continuous dense calcium phosphate structure and spherical calcium phosphate similar to mouse femur with the 3:1, 2:1, and 1:1 co-cultures, along with GFP-positive osteocyte-like cells encircled by an osteoid-like matrix similar to cortical bone. Flake-like calcium phosphate, which is more mature than spherical calcium phosphate, was found with the 3:1 and 2:1 co-cultures. Phosphorus and calcium signals were highest with 3:1 co-culture, and this bone-like tissue was ring-shaped. In a murine tooth extraction model, implantation of the ring-shaped bone-like tissue yielded more bone mass, osteoid and mineralized bone, and collagen versus no implantation. This tissue fabricated by spheroid co-culturing BMSCs with osteocytes yields an internal structure and mineral composition similar to mouse femur and could promote bone formation and maturation, accelerating regeneration. These findings open the way to new strategies in bone tissue engineering.

Phagocytosis by stroma confounds coculture studies

SummarySignals provided by the microenvironment can modify and circumvent pathway activities that are therapeutically targeted by drugs. Bone marrow stromal cell coculture models are frequently used to study the influence of the bone marrow niche on ex vivo drug response. Here, we show that mesenchymal stromal cells from selected donors and NKTert, a stromal cell line, which is commonly used for coculture studies with primary leukemia cells, extensively phagocytose apoptotic cells. This could lead to misinterpretation of results, especially if viability readouts of the target cells (e.g. leukemic cells) in such coculture models are based on the relative proportions of dead and alive cells. Future coculture studies which aim to investigate the impact of bone marrow stromal cells on drug response should take into account that stromal cells have the capacity to phagocytose apoptotic cells.

Gene expression profiling of tumor stroma interactions in retinoblastoma

We aimed to identify the critical molecular pathways altered upon tumor stroma interactions in retinoblastoma (RB). In vitro 2 D cocultures of RB tumor cells (Weri-Rb-1 and NCC-RbC-51) with primary bone marrow stromal cells (BMSC) was established. Global gene expression patterns in coculture samples were assessed using Affymetrix Prime view human gene chip microarray and followed with bioinformatics analyses. Key upregulated genes from Weri-Rb-1 + BMSC and NCC-RbC-51 + BMSC coculture were validated using qRT-PCR to ascertain their role in RB progression. Whole genome microarray experiments identified significant (P ≤ 0.05, 1.1 log 2 FC) transcriptome level changes induced upon coculture of RB cells with BMSC. A total of 1155 genes were downregulated and 1083 upregulated in Weri-Rb-1 + BMSC coculture. Similarly, 1865 genes showed downregulation and 1644 genes were upregulation in NCC-RbC-51 + BMSC coculture. The upregulated genes were significantly associated with pathways of focal adhesion, PI3K-Akt signalling, ECM-receptor interaction, JAK-STAT, TGF-β signalling thus contributing to RB progression. Validation of key genes by qRT-PCR revealed significant overexpression of IL8, IL6, MYC and SMAD3 in the case of Weri-Rb-1 + BMSC coculture and IL6 in the case of NCC-RbC-51 + BMSC coculture. The microarray expression study on in vitro RB coculture models revealed the pathways that could be involved in the progression of RB. The gene signature obtained in a stimulated model when a growing tumor interacts with its microenvironment may provide new horizons for potential targeted therapy in RB.

Β-Catenin and Axl Receptor Tyrosine Kinase Modulation in CLL B-Cells with Co-Culture on Marrow Stromal Cells: Implications for Drug Resistance

Introduction: Bone marrow stromal cells (BMSCs), a major component of CLL microenvironment influences CLL B-cell survival. We have utilized an in vitro BMSC model system and found unique alterations in CLL B-cells with short term BMSC co-culture that point to previously unidentified biologic changes in the CLL B-cells that may influence CLL B-cell signaling and their drug resistance. Methods: Purified primary CLL B-cells (n=41) from previously untreated CLL patients were cultured alone or co-cultured with primary BMSCs from either normal individuals (n=24) or CLL patients (n=18) at a 50:1 ratio in AIMV medium. After 48 hours, separated CLL B-cells or BMSCs were examined by immunoprecipitation/Western blot analyses to assess the presence of intracellular signal proteins and real time PCR to determine the RNA level expression. In separate experiments to assess CLL B-cell response in co-culture with BMSC, purified CLL B-cells were treated with the following agents at sub lethal doses; fludarabine, chlorambucil, ibrutinib and venetoclax as single agents. Results: Consistent and significant increase in expression of Axl receptor tyrosine kinase for both mRNA and protein levels was observed in CLL B-cells co-cultured with BMSCs compared to CLL B-cells cultured alone. We also detected significantly increased expression of β-catenin at the protein level in CLL B-cells co-cultured with BMSCs. In contrast there was no significant change in β-catenin or Axl protein expression in the co-cultured BMSCs. Co-culturing of CLL B-cells with BMSCs using transwells confirmed that the upregulation of both Axl and β-catenin is dependent on the direct contact of CLL B-cells with BMSCs. Experiments showed that the increased Axl level in co-cultured CLL B-cells was independent of Axl kinase activity. Furthermore, the CLL B-cells from co-culture also had clear upregulation of downstream P-ERK-1/2 but no change in P-AKT(Ser473). Next we reasoned that if increases in Axl and β-catenin are related to drug resistance then CLL B-cells should have increases in those proteins when cultured with drugs and in presence of BMSCs. Treatment with chemotherapeutic or targeted therapy drugs, (i.e. fludarabine, chlorambucil, ibrutinib, venetoclax) with sublethal doses was found to lead to increase in expression levels of both β-catenin and Axl in co-cultured CLL B-cells. However these increases were significantly over that seen with simple co-culture of CLL B-cells with BMSCs. Since CLL B-cells were less sensitive to these latter chemotherapy drugs in presence of BMSCs, this suggests to us a role for both Axl and β-catenin in stromal mediated CLL B-cell drug resistance to these agents. Moreover, high nuclear β-catenin and P-ERK-1/2 levels were also detected in co-cultured CLL B-cells. It is also known that ERK associates with and inactivates GSK-3β resulting in the up-regulation of β-catenin. We found upregulation in P-GSK-3β (Ser9) an inactive molecule which can result in increasing accumulation of β-catenin. Inhibition of P-ERK-1/2 using the ERK inhibitor PD98059 in co-cultured CLL B-cells inhibited β-catenin as well as Axl expression levels. Furthermore, it has been shown that Axl expression can be regulated by c-Jun activity. In that regard we have observed upregulation of P-c-Jun(Ser73) which can enhance Axl level in co-cultured CLL B-cells and inhibition of c-Jun activity using SP600125 (c-jun upstream JNK inhibitor), as well inhibited the Axl expression. Finally, we studied 5 CLL patients before and while being treated with ibrutinib/chemo-immuno therapy for the expression of CLL B-cell Axl, β-catenin, P-ERK-1/2, P-c-Jun(Ser73) levels. We found that 2 patients had increase in Axl expression, 4 patients had increased β-catenin and P-ERK-1/2 levels and 3 patients showed increase in P-c-Jun(S73) level after the therapy. Conclusion: Here we show that marrow stromal cell contact with CLL B-cells consistently mediate increased expression in both β-catenin and Axl in CLL B-cells (Figure1). The mechanism for this may, in part, via activated ERK and c-Jun levels (Figure1). We believe that these changes in both molecules are associated with leukemic B-cell survival and drug resistance (Figure1). These studies suggest that a further understanding of the roles of Axl and β-catenin in the CLL B-cells mediated by contact with BMSC will help to develop potential strategies for the management of CLL disease resistant to various drugs. Disclosures Warner: Tolero Pharmaceuticals: Employment. Bearss:Tolero Pharmaceuticals: Employment. Kay:Agios: Other: DSMB; Infinity Pharmaceuticals: Other: DSMB; Celgene: Other: Data Safety Monitoring Board; MorphoSys: Other: Data Safety Monitoring Board.

Enhanced Expression of Beta-Catenin and Axl Receptor Tyrosine Kinase (RTK) in Chronic Lymphocytic Leukemia (CLL) B-Cells with Co-Culture on Marrow Stromal Cells: Implications for Leukemic Cell Drug Resistance

IntroductionCLL remains an incurable disease and represents a significant health problem in the western world. Increasing evidence highlights that the impact of marrow stromal cells is a key component influencing CLL B-cell survival. We have utilized an in vitro bone marrow stromal cell (BMSC) model system and found unique alterations in CLL B-cells with BMSC co-culture that point to previously unidentified biologic changes in the CLL B-cells that may influence CLL B-cell signaling and drug resistance.MethodsPurified primary CLL B-cells (n= 39) from previously untreated CLL patients were cultured alone or co-cultured with primary BMSCs from either normal individuals (n=26) or CLL patients (n=17) at a 50:1 ratio in AIMV medium. After 48 hours, separated CLL B-cells or BMSCs were examined by immunoprecipitation/Western blot analyses and where needed real time PCR was done to assess the presence of intracellular proteins. In separate experiments to assess CLL B-cell killing, purified CLL B-cells were treated with TP-0903, fludarabine, chlorambucil and ibrutinib as single agents with or without BMSC co-culture.ResultsWe observed significant increases in expression of Axl for both mRNA and protein levels in CLL B-cells co-cultured with BMSCs compared to CLL B-cells cultured alone. We also detected significantly increased expression of β-catenin at the protein level in CLL B-cells co-cultured with BMSC. But, we did not see any significant change in β-catenin or Axl protein expression in BMSCs co-cultured with CLL B-cells. Co-culturing of CLL B-cells with BMSCs using transwells confirmed that the upregulation of both Axl and β-catenin is dependent on the direct contact of CLL B-cells with BMSCs. The CLL B-cells from co-culture also had upregulation in phosphorylated (P)-ERK-1/2 but no change in P-AKT(Ser473). High nuclear β-catenin and P-ERK-1/2 levels were also detected in co-cultured CLL B-cells. ERK associates with and inactivates GSK-3β resulting in the up-regulation of β-catenin. We next checked for P-GSK-3β (Ser9) in co-cultured CLL B-cells. Upregulation in P-GSK-3β (Ser9) detected in co-cultured CLL B-cells suggests inactivation of GSK-3β and increasing β-catenin accumulation in co-cultured CLL B-cells. Moreover, inhibition of P-ERK-1/2 with inhibitor PD98059 in CLL B-cells cultured with BMSCs inhibited β-catenin as well as Axl expression levels. We also determined the phosphorylation status of Axl in CLL B-cells in co-culture with BMSC but found no change either at Y702 (Axl kinase domain) or total tyrosine phosphorylation levels for Axl in CLL B-cells. Thus, we assume that the role of Axl in co-cultured leukemic B-cells is independent of its kinase activity. Next we determined the effect of the highly specific Axl inhibitor TP-0903 on CLL B-cell status of Axl and b-catenin while in BMSC co-culture. Interestingly, both Axl and β-catenin protein expression levels were found to be further upregulated in CLL B-cells exposed to sub-lethal doses of TP-0903 in co-culture with BMSC. Treatment with chemotherapeutic or targeted therapy drugs, (i.e. fludarabine, chlorambucil or ibrutinib) also led to increase in expression levels of both β-catenin and Axl CLL B-cells co-cultured with BMSC. Of interest CLL B-cells were less sensitive to the chemotherapy drugs in presence of BMSCs, suggesting a role for both Axl and β-catenin in stromal mediated CLL B-cell drug resistance to these agents. This was not true for the Axl inhibitor as TP-0903 was able to induce robust cell death by targeting P-Axl and overcome BMSC mediated protection even in the presence of increased Axl and b-catenin. We also found that TP-0903 decreased P-Axl as well as the Axl downstream mediator, P-Akt(S473) and reduced Mcl-1 expression in CLL B-cells in BMSC co-culture.ConclusionsHere we show that marrow stromal cell mediated increased expression in both β-catenin and Axl in CLL B-cells is associated with leukemic B cell survival and drug resistance. The mechanism for this may in part be via activated ERK levels that also occur when CLL B-cells contact BMSC. The BMSC resistance appears to be more profound for chemotherapeutic agents since Axl inhibitor can still induce CLL B-cell killing with BMSC co-culture. These studies suggest that a further understanding of the roles of Axl and β-catenin in the resistance status of CLL B-cells mediated by contact with BMSC are warranted. [Display omitted] DisclosuresWarner:Tolero Pharmaceuticals: Employment. Bearss:Tolero Pharmaceuticals, Inc: Employment. Kay:Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Cytomx Therapeutics: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Infinity Pharm: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Acerta: Research Funding; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Agios Pharm: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees.

Targeting EIF4E signaling with ribavirin in infant acute lymphoblastic leukemia.

The poor outcomes in infant acute lymphoblastic leukemia (ALL) necessitate new treatments. Here we discover that EIF4E protein is elevated in most cases of infant ALL and test EIF4E targeting by the repurposed antiviral agent ribavirin, which has anticancer properties through EIF4E inhibition, as a potential treatment. We find that ribavirin treatment of actively dividing infant ALL cells on bone marrow stromal cells (BMSCs) at clinically achievable concentrations causes robust proliferation inhibition in proportion with EIF4E expression. Further, we find that ribavirin treatment of KMT2A-rearranged (KMT2A-R) infant ALL cells and the KMT2A-AFF1 cell line RS4:11 inhibits EIF4E, leading to decreases in oncogenic EIF4E-regulated cell growth and survival proteins. In ribavirin-sensitive KMT2A-R infant ALL cells and RS4:11 cells, EIF4E-regulated proteins with reduced levels of expression following ribavirin treatment include MYC, MCL1, NBN, BCL2 and BIRC5. Ribavirin-treated RS4:11 cells exhibit impaired EIF4E-dependent nuclear to cytoplasmic export and/or translation of the corresponding mRNAs, as well as reduced phosphorylation of the p-AKT1, p-EIF4EBP1, p-RPS6 and p-EIF4E signaling proteins. This leads to an S-phase cell cycle arrest in RS4:11 cells corresponding to the decreased proliferation. Ribavirin causes nuclear EIF4E to re-localize to the cytoplasm in KMT2A-AFF1 infant ALL and RS4:11 cells, providing further evidence for EIF4E inhibition. Ribavirin slows increases in peripheral blasts in KMT2A-R infant ALL xenograft-bearing mice. Ribavirin cooperates with chemotherapy, particularly L-asparaginase, in reducing live KMT2A-AFF1 infant ALL cells in BMSC co-cultures. This work establishes that EIF4E is broadly elevated across infant ALL and that clinically relevant ribavirin exposures have preclinical activity and effectively inhibit EIF4E in KMT2A-R cases, suggesting promise in EIF4E targeting using ribavirin as a means of treatment.

Neutrophils Inhibit Synthesis of Mineralized Extracellular Matrix by Human Bone Marrow-Derived Stromal Cells In Vitro.

Although controlled local inflammation is essential for adequate bone regeneration, several studies have shown that hyper-inflammatory conditions after major trauma are associated with impaired fracture healing. These hyper-inflammatory conditions include the trauma-induced systemic inflammatory response to major injury, open fractures, and significant injury to the surrounding soft tissues. The current literature suggests that increased or prolonged influx of neutrophils into the fracture hematoma may mediate impairment of bone regeneration after hyper-inflammatory conditions. The underlying mechanism remains unclear. We hypothesize that high neutrophil numbers inhibit synthesis of mineralized extracellular matrix (ECM) by bone marrow stromal cells (BMSCs). We therefore studied the effect of increasing concentrations of neutrophils on ECM synthesis by human BMSCs in vitro. Moreover, we determined how high neutrophil concentrations affect BMSC cell counts, as well as BMSC osteogenic activity determined by alkaline phosphatase (ALP) expression and ALP activity. Co-culture of BMSCs with neutrophils induced a 52% decrease in BMSC cell count (p < 0.01), a 64% decrease in the percentage of ALP+ cells (p < 0.001), a 28% decrease in total ALP activity (p < 0.01), and a significant decrease in the amount of mineralized ECM [38% decrease after 4 weeks (p < 0.05)]. Co-cultures with peripheral blood mononuclear cells and neutrophils within transwells did not induce a significant decrease in ALP activity. In conclusion, our data shows that a decreased amount of mineralized ECM became synthesized by BMSCs, when they were co-cultured with high neutrophil concentrations. Moreover, high neutrophil concentrations induced a decrease in BMSC cell counts and decreased ALP activity. Clarifying the underlying mechanism may contribute to development of therapies that augment bone regeneration or prevent impaired fracture healing after hyper-inflammatory conditions.

Tension-loaded bone marrow stromal cells potentiate the paracrine osteogenic signaling of co-cultured vascular endothelial cells.

ABSTRACTCo-culture of bone marrow stromal cells (BMSCs) and vascular endothelial cells (VECs) is a promising strategy for better osteogenesis and pre-vascularization in bone tissue engineering. Recent reports have shown that mechanical stretching further promotes osteogenesis in BMSC/VEC co-culture systems, but the underlying mechanism of this process remains unclear. In this study, noncontact co-cultures of rat primary BMSCs and VECs were employed to interrogate paracrine cell-to-cell communications in response to tension. Exposure of VECs to 6% tension for 48 h elicited neither ALP activity nor mRNA expression of OCN and OPN in BMSCs incubated in a shared culture medium. Instead, BMSCs subjected to tension induced robust VEGF release, and its conditioned medium enhanced the proliferation and tubular formation of VECs with a concurrent increase in BMP-2 and IGF-1 production. Conditioned medium from activated VECs in turn promoted expression of osteogenic genes in BMSCs, followed by an increase in matrix mineralization. The addition of VEGF-R inhibitor Tivozanib to these systems abrogated the tension-induced paracrine effects on VECs and subsequently impaired BMSC osteogenesis. These results clearly demonstrate that the response of BMSCs to tension potentiates paracrine osteogenic signaling from VECs; this positive feedback loop is initiated by VEGF release.

Overexpression of heme oxygenase-1 in bone marrow stromal cells promotes microenvironment-mediated imatinib resistance in chronic myeloid leukemia

Neoplasm cells from patients with chronic myeloid leukemia (CML) interact with stromal cells of the surrounding microenvironment. Bone marrow stromal cells (BMSCs) represent the main population in CML marrow stroma, which may play a key role in disease support and progression. Heme oxygenase-1 (HO-1) is a key enzyme of antioxidative metabolism that is associated with cell proliferation and resistance to apoptosis. We herein up-regulated HO-1 expression of BMSCs and evaluated whether BMSCs influenced K562 cells survival. BMSCs were isolated from the bone marrow of normal people and CML patients. Following co-culture of BMSCs and K562 cells, up-regulating HO-1 expression in bone marrow stromal cells increased the imatinib (IM) resistance of K562 cells, whereas the apoptosis of K562 cells was effectively promoted without BMSCs co-culture. The protection may be mediated by CXCL12 (stromal derived factors 1, SDF-1)/CXCR4 signaling. The CXCL12/CXCR4 interaction significantly enhanced the phosphorylation of AKT. As far as drug resistance was concerned, BMSCs counteracted the cytotoxic effect of IM administration in vitro, and they protected K562 cells from the apoptosis induced by kinase inhibitor IM. The regulated HO-1 expression of BMSCs provides a new putative target for CML therapy.

Abstract 69: Stem Cells Harvested from Bone Marrow and Adipose Tissue Demonstrate Equivalent Healing but Through Different Mechanisms in a Murine Model of Irradiated Mandibular Fracture Healing

PURPOSE: The difficulty of harvest and relative scarcity of bone marrow stromal cells (BMSCs) has limited the widespread use and clinical application of this technology, thereby necessitating inquiry into other therapies including adipose-derived stromal cells (ASCs). The goal of this study was to compare the ability of ASCs and BMSCs to heal mandibular defects and understand the mechanism through which this occurs. We hypothesize that ASCs will enhance fracture healing by improving vasculogenesis, while BSMCs will directly affect osteogenesis. METHODS: Male Lewis rats were radiated (35Gy), and subsequently underwent mandibular osteotomy with external fixation with implantation of two million BMSCs (n=12) or ASCs (n=16) marked with Green fluorescent protein (GFP). After 40 days, union rates were evaluated using microCT. Confocal microscopy visualized the contribution of ASCs/BMSCs to the bone regenerate. Quantitative polymerase chain reaction of ASCs/BMSCs compared expression of osteogenic and vasculogenic genes. Coculture of ASCs (n=3) or BMSCs (n=3) with human umbilical vein endothelial cells (HUVECs) was performed in vitro in transwells to measure tubule formation as a marker of vasculogenesis. RESULTS: ASC-implantation resulted in higher union rates than BMSC-implantation (union rate: 94% vs. 66%). These cells contribute indirectly to fracture healing, as GFP was not visualized at the site. BMSCs expressed osteogenic genes including osteopontin to a significantly greater degree than did ASCs, while ASCs expressed greater levels of vascular endothelial growth factor. This translated to greater tubule formation among HUVECs co-cultured with ASCs than with BMSCs (64.3 ± 7.3 vs. 23.3 ± 2.6, p=0.0008), and increased vasculogenesis in vivo in mandibles after ASC implantation. CONCLUSIONS: ASCs heal fracture defects better than BMSCs. This effect is likely mediated by indirect modulation of vasculogenesis, rather than by a direct effect on osteogenesis. Clinicians interested in cell-based therapies for irradiated bone injury should consider ASCs as a promising option, given their abundance, ease of acquisition, and improved fracture healing.

Vascular endothelial growth factor-transfected adipose-derived stromal cells enhance bone regeneration and neovascularization from bone marrow stromal cells.

Vascular endothelial growth factor (VEGF)-transfected adipose-derived stromal cells (ADSCsVEGF ) were devised to promote bone regeneration and neovascularization of bone marrow stromal cells (BMSCs). ADSCsVEGF were added to BMSCs and cocultured in variable proportions. ADSCsVEGF alone or ADSCsVEGF with BMSCs (BMSCs:ADSCsVEGF ratio of 1:0.025-0.5) induced significantly greater tube formation in human umbilical vein endothelial cells than untransfected ADSCs. The cocultures of BMSCs and ADSCsVEGF at ratios of 1: 0.025-0.1 showed significantly greater osteogenic differentiation and mineralization than BMSCs alone in vitro. Osteogenic markers COL1A1, OCN and BSP were most effectively induced at the BMSC: ADSCVEGF ratio of 1:0.05. Of angiogenesis-related genes, upregulation of cathepsin Z and downregulation of early growth response 1 were observed while two osteogenesis-related genes, osteoactivin and tetranectin, were upregulated in BMSCs/ADSCsVEGF compared to BMSCs/ADSCs. When critical size calvarial defects in rats were implanted with mixture of BMSCs and ADSCsVEGF along with hydroxyapatite/β-tricalcium phosphate granules, BMSCs and ADSCsVEGF at the ratio of 1:0.05 showed better bone regeneration that BMSCs alone. The cotransplantation of ADSCsVEGF with BMSCs significantly increased neovascularization on the regenerated bone of the repaired defect than BMSCs alone. In conclusion, ADSCsVEGF added in small proportion to BMSCs effectively promote bone regeneration and neovascularization. Copyright © 2017 John Wiley & Sons, Ltd.

Preclinical activity of DCZ3301, a novel aryl-guanidino compound in the therapy of multiple myeloma

We synthesized a novel aryl-guanidino compound, DCZ3301, and found that it has potent cytotoxicity against multiple human cancer cell lines. The anticancer activity was most potent against multiple myeloma (MM). DCZ3301 induced cytotoxicity in MM cell lines, as well as patient myeloma cells, in part by decreasing mitochondrial membrane potential to induce apoptosis. In contrast, DCZ3301 had no cytotoxic effect on normal cells. DCZ3301 also inhibited cell cycling and caused a G2/M accumulation that corresponded with downregulation of Cdc25C, CDK1, and Cyclin B1. DCZ3301 retained its activity against MM cells in the presence of exogenous cytokines (IL-6 or VEGF) or bone marrow stromal cells (BMSCs) and reduced activity of multiple signaling pathways (STAT3, NFκB, AKT, ERK1/2) in MM but not normal cells. The STAT3 pathway played an important role in modulating DCZ3301-mediated cytotoxicity. Knockdown of STAT3 using siRNA in MM cells enhanced DCZ3301-induced cytotoxicity, whereas overexpression of STAT3 in MM cells partially protected them from apoptosis. In addition, DCZ3301 inhibited VEGF and IL-6 secretion in a dose-dependent fashion in a co-culture of MM cells and BMSCs. Combining DCZ3301 with bortezomib induced synergistic cytotoxicity in MM cell lines and primary MM cells. Finally, in vivo efficacy of DCZ3301 was confirmed in an MM xenograft mouse model. Together, these results provide a rationale for translation of this small-molecule inhibitor, either alone or in combination, to the clinic against MM.

Myeloma Cells Induce High Level of TAF12 Expression in Bone Marrow Stromal Cells, Resulting in Increased Osteoclastogenesis and Myeloma Cell Growth in Response to 1,25(OH)2D3

1,25 dihydroxy vitamin D3 (1,25D) plays multiple roles in normal and malignant cell functions, including cell differentiation and proliferation, and is a regulator of bone metastasis. However, the role of 1,25D in multiple myeloma (MM) is poorly characterized. We previously showed that TAF12, a member of the TFIID transcription complex, acts as a co-activator for vitamin D receptor (VDR) mediated transcription to increase 1,25D responsivity. Further, we reported that IL-6 can increase TAF12 levels in bone marrow stromal cells (BMSC). Since IL-6 is upregulated in the MM microenvironment, we assessed TAF12 levels in BMSC from 13 MM patients and 10 normals. TAF12 protein levels were significantly increased (25x) in MM BMSC compared to normal. Further, co-culture of BMSC from 3 normals with primary human MM cells significantly increased TAF12 levels in the normal BMSC. Similarly, when 5TGM-1 murine MM cells were inoculated intratibially into C57BL/KaLwRij mice and bone marrow cells collected 3 weeks later, TAF12 protein levels in MM exposed BMSC were significantly increased compared to PBS injected control mice. We then assessed if increased TAF12 levels persisted in normal BMSC (SAKA-T cells) after they were co-cultured with JJN3 MM cells and subsequently cultured in the absence of MM cells. We found that increased TAF12 levels persisted for at least 2 weeks after removal of MM cells, consistent with our findings that BMSC from MM patients express elevated TAF12 levels in the absence of MM cells. To determine if IL-6 contributes to the increased TAF12 levels induced by MM cells, we co-cultured SAKA-T cells with JJN3 MM cells in the presence or absence of anti-IL-6. Anti-IL-6 blocked the increase in TAF12 levels in these co-cultures.We then tested if the increased TAF12 levels enhance the 1,25D responsivity of BMSC. Addition of 1,25D (10-10 - 10-8 M) greatly increased expression of RANKL mRNA and VCAM-1 protein in MM BMSC compared to normals. The increased RANKL and VCAM-1 in MM BMSC treated with 1,25D correlated with increased osteoclast (OCL) formation, MM cell growth and adhesion of MM cells. When human OCL precursors and MM or normal BMSC were co-cultured with 1,25D with/without osteoprotegerin (OPG), OCL numbers were significantly increased in co-cultures of MM BMSC treated with 1,25D compared to normal, and the increase was blocked by OPG. Further, co-culture of JJN3 cells with normal or MM BMSC and 1,25D for 6 hours followed by removal of non-adherent MM cells, demonstrated that the number of MM cells adherent to BMSC was significantly increased with 1,25D treatment of MM BMSC co-cultures compared to normal BMSC. In addition, when JJN3 cells were co-cultured with normal or MM BMSC and 1,25D, JJN3 cells numbers were significantly increased in co-cultures with MM BMSC compared to normal BMSC. Finally, we determined if high levels of TAF12 in MM BMSC increased their 1,25D responsivity. We knocked-down (k/d) TAF12 with a siRNA in normal and MM BMSC, and found that TAF12 k/d in MM or normal BMSC decreased the levels of VCAM 1 induced by 1,25D compared to control siRNA transduced cells. To further investigate this effect, we assessed if TAF12 levels affected the stability of VDR in the presence of 1,25D, using TAF12 heterozygous or WT mouse BMSC. VDR half-life was decreased approximately 50% in TAF12 heterozygous BMSC compared with WT.These results show that MM cells increase expression of TAF12 in BMSC that is mediated by IL-6. BMSCs expressing high TAF12 levels have greater responsivity to 1,25D that enhances RANKL and VCAM-1 expression to induce osteoclastogenesis, MM cell growth and MM cell adhesion. These results further suggest that blocking TAF12 -VDR interactions may be a potential therapeutic target for MM bone disease. DisclosuresRoodman:Amgen: Consultancy.

FLT3 TKI Are Differentially Affected By Both Plasma Binding and Stromal Survival Factors

The FMS-like tyrosine kinase 3 (FLT3) is the most frequently mutated gene in acute myeloid leukemia (AML). As such it represents an attractive target for tyrosine kinase inhibitors (TKI) for the treatment of AML, a number of which have entered clinical trials. However, to date, these trials have demonstrated limited clinical efficacy. Evidence indicates that failure to achieved adequate inhibitory activity against FLT3, at least partially as a result of plasma protein binding, as well as survival factors provided by stroma, are among the reasons for the limited efficacy of FLT3 TKI.In this study we have characterized the effects of plasma binding and stromal survival factors on TTT-3002, a novel TKI that has demonstrated potent inhibition of the internal tandem duplication (FLT3-ITD) as well as a large panel of clinically described kinase mutants (FLT3-KD), and compared it to the effects these same conditions have on other FLT3 TKI.Using plasma from pediatric/young adult patients of a variety of ages (n = 24, median 7.2 years, 4.1-36.9 years), the impact of human plasma binding on the activity of TTT-3002 against FLT3-ITD dependent proliferation. 50% human plasma results in a moderate decrease in activity of TTT-3002 (IC50 45.2 nM in human plasma, 2.4 nM in media with 10% FCS). This is a markedly smaller shift than several TKIs currently or previously in clinical trials including sorafenib, CEP-701, PKC-412, and AC-220 (50 to >500-fold increase in IC50). The data demonstrate a clear linear relationship between plasma alpha-1 acidglycoprotein (AGP), an acute-phase reactant, and fold-increase in IC50 of TTT-3002 (1.7 fold-increase per 10 mg/dL AGP). The data also demonstrate a wide variability in patient plasma AGP concentration that is dependent upon age, but also appears to be significantly impacted by position in treatment (median 117 mg/dL, 58-354 mg/dL). Plasma AGP strongly inhibits several FLT3 inhibitors including CEP-701 and PKC-412 (>100-fold increase in IC50); whereas other FLT3 inhibitors such as sorafenib and AC-220 are inhibited by human plasma in an AGP-independent manner. These data indicate the presence of several human plasma proteins whose binding leads to drug inhibition that TTT-3002 is nonetheless resistant to, in comparison to other clinically relevant FLT3 inhibitors. The identification of these proteins is ongoing.Using primary human bone marrow stromal cell co-culture with two FLT3-ITD cell lines increases the IC50 of TTT-3002 but to a lower extent than that of AC-220 (MV4-11: 1.1-1.9 fold versus 2.3-2.7 fold, MOLM-14: 2.2-2.3 fold versus 3.5-3.6 fold). The data are consistent across stromal cells cultured from several different pediatric patients (n = 5).These data suggest that TTT-3002 may be more resistant to some of the inhibitory effects that have led to disappointing results with other similar FLT3-targeted TKIs. As such, TTT-3002 represents an appealing candidate for clinical studies. DisclosuresNo relevant conflicts of interest to declare.

Bone Marrow Microenvironment Niche Regulates miR-221/222 in Acute Lymphoblastic Leukemia.

Niche-influenced miR-221/222 may define a novel therapeutic target in ALL to be combined with existing cytotoxic agents to more effectively eradicate refractory disease that contributes to relapse. Mol Cancer Res; 14(10); 909-19. ©2016 AACR.

Pim-1, Beyond an Oncogene, in Acute Myeloid Leukemia

Background and Objective:There are 3 members in Pim family, Pim-1, Pim-2 and Pim-3. They are serine/threonine kinase coding proto-oncogene. It is reported that Pim-1 plays a role in solid tumor, leukemia and polycythemia vera, et al. Pim-3, as a newly cloned oncogene has discovered to functioning in hepatic carcinoma, pancreatic cancer, et al. We are trying to find out the roles of Pim1 in acute myeloid leukemia.Methods:1 Evaluate the expressions of Pim family genes in acute myeloid leukemia patients and analysis the profile of Pim expressions with clinical characteristics.2 Up-regulating the expression of Pim1 in AML cell lines by transient transfection or long-term infection through GFP-expressing plasmids and lenti-virus system and analyze the proliferation, apoptosis and chemotaxis features of the transfected AML cell lines.Results:1 Our investigation showed that Pim-1 is up-regulated in around 15% of acute myeloid leukemia (AML) patients.2 As shown in growth curves and apoptosis assays, over expression of Pim-1induces growth up-regulation and anti-apoptosis. We hypothesized that phenomenon could be originated from the enhanced expression of c-myc, cyclin D1 and Bad phosphorylation shown in western blotting analysis.3 Our chemotaxis assay and bone marrow stromal cell co-culture model demonstrated that overexpression of Pim-1can enhance the AML cells chemotactic movement toward SDF-1¦Á, with calcium influx increment and phosphorylation of CXCR4.4 Flow cytometry analysis and confocal immunofluorescence observation demonstrated the up-regulated CXCR4 expression and internalization induced by SDF-1¦Á.5 We also checked the angiogenesis and adhesion molecule during the process but did not show any contribution.Conclusion:Pim-1, beyond as an oncogene, can promote growth or anti-apoptosis of AML cells, can interact with microenviroment through SDF-1¦Á-CXCR4 axis. The interplay between AML cells and microenviroment mediated by Pim-1 can make sense in AML target therapy and make a good adjunctive for transplantation conditioning regimen. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Abstract 139: Interaction between monocytes and bone marrow microenvironment in pathogenesis of multiple myeloma

Abstract Multiple myeloma (MM) is an incurable haematological malignancy characterized by the clonal proliferation of malignant plasma cells within the bone marrow. The current data on MM disease progression indicate that bone marrow microenvironment plays crucial role in pathogenesis of MM. Myeloma cells contacts with bone marrow stromal cells (BMSCs), which secrete factors/cytokines, promoting tumor cell growth and survival. Paracrine secretion of cytokines, such as IL-6, insulin-like growth factor-1, inflammatory protein-1a in BMSCs promotes MM cell proliferation and protects against drug-induced cytotoxicity. MM lytic bone disease is caused by osteoclast activation and osteoblast inhibition. Disease-related bone complications result in significant morbidity due to pain, pathologic fractures and spinal cord compression. The bone microenvironment creates a supportive niche for MM progression. Osteoclasts and BMSCs, along with extracellular matrix and cytokines stimulate myeloma cell proliferation and confer chemoresistance. Therefore, the reciprocal interactions among tumor cells, osteoclasts, osteoblasts, and bone marrow stromal cells impact both the establishment and progression of MM. In current study, monocyte can directly promote osteogenic differentiation of mesenchymal stem cells through cell contact interactions and production of osteogenic factors. This mechanism is mediated by the activation of STAT3 signaling pathway in the mesechymal stem cells that leads to the upregulation of osteoblasts-associated genes such as Runx2 and alkaline phosphatase (ALP), and the downregulation of osteoblast inhibitors such as DKK1 to drive the differentiation of mesechymal stem cells into osteoblasts. In this study, we examined the role of monocyte, a component of bone marrow microenvironment, in the MM progression. We investigated the proliferation of MM cell lines cultured alone or co-cultured with BMSCs and/or monocytes of MM patients. Consistently, we observed increased proliferation of MM cell lines in the presence of either BMSCs or monocytes compared to cell line-only control. Furthermore, the co-culture of BMSCs plus monocytes induced the greatest degree of proliferation of myeloma cells. In addition to increased proliferation, BMSCs and monocytes decreased the rate of apoptosis of myeloma cells. Our results therefore suggest that highlights the role of monocyte as an important component of the bone marrow microenvironment. Citation Format: Hiroshi Ikeda, Yuka Aoki, Toshiaki Hyayashi, Yumiko Maruyama, Tadao Ishida, Takashi Tokino, Yasuhisa Shinomura, Yasushi Sasaki. Interaction between monocytes and bone marrow microenvironment in pathogenesis of multiple myeloma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 139. doi:10.1158/1538-7445.AM2014-139