Patient-derived Stromal Cells Research Articles

IGFBP2 secretion by mammary adipocytes limits breast cancer invasion.

The progression of noninvasive ductal carcinoma in situ to invasive ductal carcinoma for patients with breast cancer results in a significantly poorer prognosis and is the precursor to metastatic disease. In this work, we have identified insulin-like growth factor-binding protein 2 (IGFBP2) as a potent adipocrine factor secreted by healthy breast adipocytes that acts as a barrier against invasive progression. In line with this role, adipocytes differentiated from patient-derived stromal cells were found to secrete IGFBP2, which significantly inhibited breast cancer invasion. This occurred through binding and sequestration of cancer-derived IGF-II. Moreover, depletion of IGF-II in invading cancer cells using small interfering RNAs or an IGF-II-neutralizing antibody ablated breast cancer invasion, highlighting the importance of IGF-II autocrine signaling for breast cancer invasive progression. Given the abundance of adipocytes in the healthy breast, this work exposes the important role they play in suppressing cancer progression and may help expound upon the link between increased mammary density and poorer prognosis.

A Tumor Microenvironment Model of Pancreatic Cancer to Elucidate Responses toward Immunotherapy (Adv. Healthcare Mater. 14/2023)

3D Cancer Models In article 2201907 by Daniela Loessner and co-workers, the tumor microenvironment of pancreatic cancer is engineered using star poly(ethylene glycol) (PEG)–heparin hydrogels, that are functionalized with integrin-binding and protease-responsive peptides to stimulate cell proliferation and migration. The coculture of cancer cells and patient-derived stromal cells mimics the multicellular composition of tumor tissues and cancer–immune interactions and allows the testing of immunomodulatory treatments in combination with chemotherapy.

Combinatorial Application of Papain and CD66B for Isolating Glioma- Associated Neutrophils.

Stromal cells in the tumor microenvironment play crucial roles in glioma development. Current methods for isolating tumor-associated stromal cells (such as neutrophils) are inefficient due to the conflict between tissue dissociation and cell surface protein protection, which hampers the research on patient-derived stromal cells. Our study aims to establish a novel method for isolating glioma-associated neutrophils (GANs). To observe neutrophil-like polymorphonuclear cells, we performed Hematoxylin-Eosin staining on glioma tissues. For isolating single cells from glioma tissues, we evaluated the efficiency of tissue dissociation with FastPrep Grinder-mediated homogenization or proteases (trypsin or papain) digestion. To definite specific markers of GANs, fluorescence-activated cell sorting (FACS) and immunofluorescence staining were performed. FACS and Ficoll were performed for the separation of neutrophils from glioma tissue-derived single-cell or whole blood pool. To identify the isolated neutrophils, FACS and RT-PCR were carried out. Neutrophil-like cells were abundant in high-grade glioma tissues. Among the three tissue dissociation methods, papain digestion produced a 5.1-fold and 1.7-fold more living cells from glioma mass than physical trituration and trypsin digestion, respectively, and it preserved over 97% of neutrophil surface protein markers. CD66B could be adopted as a unique neutrophil surface protein marker for FACS sorting in glioma. Glioma-derived CD66B+ cells specifically expressed neutrophil marker genes. A combination of papain-mediated tissue dissociation and CD66B-mediated FACS sorting is an effective novel method for the isolation of GANs from glioma tissues.

Inhibition of Tumor Microenvironment Cytokine Signaling Sensitizes Ovarian Cancer Cells to Antiestrogen Therapy.

Simple SummaryAntiestrogen hormonal therapy is a relatively low side effect, orally administered cancer treatment option, yet response rates have been limited in epithelial ovarian cancer despite estrogen receptor expression in many tumors. This suggests that other pathways impact estrogen response. Cytokine signaling from the tumor microenvironment promotes ovarian cancer growth, and crosstalk between cytokine signaling and estrogen signaling has been reported in other tumor types. We therefore aimed to investigate whether cytokine signaling impacts estrogen signaling in high-grade serous ovarian cancer. We demonstrated crosstalk between these two pathways in patient-derived samples, in vitro and in animal studies. We found that inhibiting interleukin-6/leukemia inhibitory factor (IL6/LIF) cytokine signaling activates estrogen signaling and blocking both pathways was synergistic in inhibiting tumor cell growth. These results suggest a potential role for combination therapy for epithelial ovarian cancer patients.Antiestrogen therapy (AET) is an alternative to cytotoxic chemotherapy for recurrent ovarian cancer, yet the often short duration of response suggests mechanisms of resistance. We previously demonstrated that tumor microenvironment interleukin-6/leukemia inhibitory factor (IL6/LIF) cytokines induce tumor cell JAK-STAT signaling to promote cancer growth. Crosstalk between estrogen signaling and cytokine signaling has been reported. Therefore, we sought to characterize the impact of IL6/LIF signaling on estrogen signaling in epithelial ovarian cancer and investigate the efficacy of combination therapy. We first assessed patient tumors for cytokine expression and compared it with response to AET to determine clinical relevance. In vitro, we determined the effect of IL6/LIF on estrogen receptor expression and signaling. Cell viability assays were used to determine the efficacy and potential synergy of cytokine blockade and AET. We then extended studies to animal models, incorporating patient-derived stromal cells. Our results demonstrated shorter progression-free interval on AET in patients with stromal IL6/LIF expression. In vitro, IL6/LIF increased tumor cell estrogen receptor expression and signaling, and combination cytokine blockade and AET resulted in synergistic inhibition of tumor cell growth. The anticancer effect was verified in a mouse model. In conclusion, due to crosstalk between IL6/LIF cytokine signaling and estrogen signaling, dual blockade is a potential new treatment approach for ovarian cancer.

Globally altered epigenetic landscape and delayed osteogenic differentiation in H3.3-G34W-mutant giant cell tumor of bone

The neoplastic stromal cells of giant cell tumor of bone (GCTB) carry a mutation in H3F3A, leading to a mutant histone variant, H3.3-G34W, as a sole recurrent genetic alteration. We show that in patient-derived stromal cells H3.3-G34W is incorporated into the chromatin and associates with massive epigenetic alterations on the DNA methylation, chromatin accessibility and histone modification level, that can be partially recapitulated in an orthogonal cell line system by the introduction of H3.3-G34W. These epigenetic alterations affect mainly heterochromatic and bivalent regions and provide possible explanations for the genomic instability, as well as the osteolytic phenotype of GCTB. The mutation occurs in differentiating mesenchymal stem cells and associates with an impaired osteogenic differentiation. We propose that the observed epigenetic alterations reflect distinct differentiation stages of H3.3 WT and H3.3 MUT stromal cells and add to H3.3-G34W-associated changes.

Histone H3.3 G34 mutations promote aberrant PRC2 activity and drive tumor progression

A high percentage of pediatric gliomas and bone tumors reportedly harbor missense mutations at glycine 34 in genes encoding histone variant H3.3. We find that these H3.3 G34 mutations directly alter the enhancer chromatin landscape of mesenchymal stem cells by impeding methylation at lysine 36 on histone H3 (H3K36) by SETD2, but not by the NSD1/2 enzymes. The reduction of H3K36 methylation by G34 mutations promotes an aberrant gain of PRC2-mediated H3K27me2/3 and loss of H3K27ac at active enhancers containing SETD2 activity. This altered histone modification profile promotes a unique gene expression profile that supports enhanced tumor development in vivo. Our findings are mirrored in G34W-containing giant cell tumors of bone where patient-derived stromal cells exhibit gene expression profiles associated with early osteoblastic differentiation. Overall, we demonstrate that H3.3 G34 oncohistones selectively promote PRC2 activity by interfering with SETD2-mediated H3K36 methylation. We propose that PRC2-mediated silencing of enhancers involved in cell differentiation represents a potential mechanism by which H3.3 G34 mutations drive these tumors.

Stromal-AR influences the growth of epithelial cells in the development of benign prostate hyperplasia.

Activation of epithelial-AR signaling is identified as the major cause of hyperproliferation of the cells during benign and malignant prostate conditions. However, the contribution of stromal-AR is also precarious due to its secretory actions that contribute to the progression of benign and malignant tumors. The present study was aimed to understand the influence of stromal-AR mediated actions on epithelial cells during BPH condition. The secretome (conditioned media-CM) was collected from AR agonist (testosterone-propionate-TP) and antagonist (Nilutamide-Nil) treated BPH patient-derived stromal cells and exposed to BPH epithelial cells. Epithelial cells exhibited increased cell proliferation with the treatment of CM derived from TP-treated stromal cells (TP-CM) but did not support the clonogenic growth of BPH epithelial cells. However, CM derived from Nil-treated stromal cells (Nil-CM) depicted delayed and aggressive BPH epithelial cell proliferation with increased clonogenicity of BPH epithelial cells. Further, decreased AR levels with increased cMyc transcripts and pAkt levels also validated the clonogenic transformation under the paracrine influence of inhibition of stromal-AR. Moreover, the CM of stromal-AR activation imparted positive regulation of basal/progenitor pool through LGR4, β-Catenin, and ΔNP63α expression. Hence, the present study highlighted the restricted disease progression and retains the basal/progenitor state of BPH epithelial cells through the activation of stromal-AR. On the contrary, AR-independent aggressive BPH epithelial cell growth due to paracrine action of loss stromal-AR directs us to reform AR pertaining treatment regimes for better clinical outcomes.

Transforming growth factor β1-mediated functional inhibition of mesenchymal stromal cells in myelodysplastic syndromes and acute myeloid leukemia.

Mesenchymal stromal cells are involved in the pathogenesis of myelodysplastic syndromes and acute myeloid leukemia, but the underlying mechanisms are incompletely understood. To further characterize the pathological phenotype we performed RNA sequencing of mesenchymal stromal cells from patients with myelodysplastic syndromes and acute myeloid leukemia and found a specific molecular signature of genes commonly deregulated in these disorders. Pathway analysis showed a strong enrichment of genes related to osteogenesis, senescence, inflammation and inhibitory cytokines, thereby reflecting the structural and functional deficits of mesenchymal stromal cells in myelodysplastic syndromes and acute myeloid leukemia on a molecular level. Further analysis identified transforming growth factor β1 as the most probable extrinsic trigger factor for this altered gene expression. Following exposure to transforming growth factor β1, healthy mesenchymal stromal cells developed functional deficits and adopted a phenotype reminiscent of that observed in patient-derived stromal cells. These suppressive effects of transforming growth factor β1 on stromal cell functionality were abrogated by SD-208, an established inhibitor of transforming growth factor β receptor signaling. Blockade of transforming growth factor β signaling by SD-208 also restored the osteogenic differentiation capacity of patient-derived stromal cells, thus confirming the role of transforming growth factor β1 in the bone marrow microenvironment of patients with myelodysplastic syndromes and acute myeloid leukemia. Our findings establish transforming growth factor β1 as a relevant trigger causing functional inhibition of mesenchymal stromal cells in myelodysplastic syndromes and acute myeloid leukemia and identify SD-208 as a candidate to revert these effects.

Abstract 5005: Inhibition of E-selectin or E-selectin together with CXCR4 resensitizes multiple myeloma to treatment

Abstract INTRODUCTION: Multiple myeloma (MM) is a plasma cell malignancy localized in the bone marrow. Selectins and CXCR4 play a pivotal role in homing, adhesion, and proliferation of MM. Herein, we tested the role of E-selectin in the pathophysiology of MM by using a specific E-selectin antagonist (GMI-1271) and a dual E-selectin/CXCR4 antagonist (GMI-1359) on the proliferation and cell trafficking of MM in vitro and in vivo. PROCEDURES: Endothelial cells (HUVECs), stromal cells (HS5), MM patient-derived stromal cells (MSP1), and MM cell lines (MM1s, H929, U266 and RPMI8226) were tested for expression of E-selectin, CLA (E-selectin ligand) and CXCR4 by flow cytometry. The effect of GMI-1271 and GMI-1359 was studied on MM cell adhesion to stroma; chemotaxis to stromal media or SDF1; and MM cell survival by MTT assay, where MM cells were treated with lenalidomide or carfilzomib; in the presence, or absence of GMI-1271 or -1359. In a xenograft mouse model, the sensitivity of MM was tested in mice treated with vehicle, lenalidomide, GMI-1271, or a combination of GMI-1271 and lenalidomide (n=10 mice/group); and tumor growth was monitored weekly using bioluminescence imaging. SUMMARY: Expression of E-selectin was high in HUVECs, MSP1 and HS5; CLA was high only in RPMI8226; and CXCR4 was high in all MM cells. MM1s cell adhesion to stroma was decreased by GMI-1271 and even more profoundly decreased by GMI-1359 in a dose-dependent manner. Chemotaxis of MM1s cells towards conditioned media or SDF1 was modestly affected by GMI-1271, but greatly reduced by GMI-1359. MM cell proliferation was significantly inhibited by GMI-1359, only when MM cells were co-cultured with stroma, but not alone. Stroma-induced drug resistance of MM cells to carfilzomib and lenalidomide was reduced in the presence of the GMI compounds, while the effect of GMI-1359 was stronger than GMI-1271. In vivo, GMI-1271 and lenalidomide as single agents delayed tumor growth compared to vehicle by 40% and 35%, respectively; however, a combination treatment delayed tumor growth more profoundly than vehicle by 64% (p=0.04). CONCLUSIONS: GMI-1271 and GMI-1359 disrupt the interaction between MM cells and bone marrow microenvironment as determined by reduction of adhesion, chemotaxis and microenvironment-induced drug resistance in vitro. However, the dual inhibition of E-selectin and CXCR4 produced more profound effects compared to E-selectin alone. Moreover, GMI-1271 sensitized MM cells to lenalidomide by alleviating chemoresistance and delayed tumor growth in vivo. Citation Format: Barbara Muz, Henna Y. Bazai, Anita Sekula, William E. Fogler, Ted Smith, John L. Magnani, Abdel Kareem Azab. Inhibition of E-selectin or E-selectin together with CXCR4 resensitizes multiple myeloma to treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5005. doi:10.1158/1538-7445.AM2017-5005

Abstract A05: Proteomic analysis of exosome content changes induced by gemcitabine in pancreatic cancer stromal cells

Abstract Despite recent advances in multimodality therapy, pancreatic ductal adenocarcinoma (PDAC) remains the 3rd leading cause of cancer-related deaths in the United States with a 5-year survival rate of 8%. One hallmark of this disease is desmoplasia that consist largely of stromal cells and extracellular matrix (ECM) proteins. The abundant stromal cells provide a unique tumor microenvironment that aid tumorigenesis. Unfortunately, there is little data on the mechanism underlying the stromal cells’ ability to impact chemoresistance in neighboring cancer cells in response to drug treatment. Exosomes are small vesicles secreted into the extracellular environment from cells and evidence is accumulating that exosomes play a key role in intercellular signaling. Researchers have focused on exosomes secreted from cancer cells, but the function of stromal exosomes has not been investigated, especially in pancreatic cancer. As stromal cells comprise the majority of the pancreatic tumor bulk, we hypothesize that stromal cell-derived exosomes help pancreatic cancer cells resist chemotherapy. Thus, it is critical to characterize the complex composition of stromal exosomes. In this study, we analyzed the proteomic content of exosomes collected from stromal cell lines created from PDAC patients. Furthermore, we also determined the change in exosomal proteomic composition after the patient-derived stromal cells were treated with gemcitabine, the current standard of care chemotherapy for patients with PDAC. Our studies have identified possible diagnostic and prognostic biomarkers and suggest novel molecular mechanism responsible for the tumor microenvironment’s ability to regulate chemoresistance in neighboring pancreatic cancer cells. Citation Format: Wei Huang, Peter Feist, Amanda Hummon, Reginald Hill.{Authors}. Proteomic analysis of exosome content changes induced by gemcitabine in pancreatic cancer stromal cells. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr A05.

Molecular Pathways That Determine the Activation State of Macrophages within the Myeloma Niche

Abstract Abstract 443 Benefit from cytotoxic therapy in myeloma may be limited by persistence of residual tumor cells nested within favorable niches. However, the contribution of macrophages to the regulation of myeloma niches is still incompletely understood. We have previously shown that macrophages provide growth and anti-apoptotic signals to myeloma cells when grown in co-culture. These results prompted us to investigate the regulation of primary monocytes/macrophages that reside within myeloma niches in the bone marrow. Unmanipulated CD14+ monocytic cells, freshly explanted from myeloma bone marrows, displayed a pre-dominantly pro-inflammatory transcriptomic profile when compared to normal monocytes. We found enhanced transcription of genes encoding pro-inflammatory mediators, known to support growth and survival of myeloma cells, such as TNFalpha, IL-1beta, IL-6, IL-8 and TACI. Downregulation of TGFbeta was also consistent with a pro-inflammatory (M1) signature. Interestingly, we also found concurrent transcription of some genes characteristic of “alternative macrophage activation” (M2 phenotype) such as IL-10 and IL1-receptor antagonist (IL-1RN). These results suggest that myeloma-associated macrophages, while being predominantly pro-inflammatory, display significant plasticity between the M1-M2 phenotypic extremes. To obtain insights into the underlying mechanisms, we examined the role of TPL2 (Cot, MAP3K8), a serine/threonine kinase with central and non-redundant roles in regulating innate immune responses in macrophages following activation by Toll receptor (TLR) ligands and members of the TNF ligand superfamily. In myeloma-associated macrophages, we found constitutive activation of a TPL2 kinase-dependent, ERK-mediated pathway that promotes synthesis and processing of pro-inflammatory cytokines, including TNFalpha and IL-1beta. We also discovered constitutive activation of AKT at Ser473, a site dependent for its phosphorylation on TPL2 activity in macrophages responding to TLR signaling. Notably, the Akt/mTOR pathway limits the magnitude and duration of macrophage activation, in part through synthesis of the anti-inflammatory cytokine IL-10. These events involve signaling through STAT3. Accordingly, we discovered constitutive phosphorylation of STAT3 at a site regulated by TPL2 in activated macrophages. In addition to non-tumor cell autonomous roles in regulating myeloma through macrophages, we showed a tumor cell-autonomous, growth-regulatory role of TPL2 kinase. Treatment of myeloma cells with a TPL2 small molecule inhibitor resulted in apoptosis that was not rescued by the presence of patient-derived stromal cells. We postulate that TPL2 inhibition interferes with growth signaling in myeloma cells because TPL2 has been shown to substitute for RAF proteins in growth signal transduction. Interestingly, we found that TPL2 was activated by phosphorylation as cells entered G2/M. Treatment with nocodazole increased the proportion of cells that co-expressed phosphorylated TPL2 and phosphorylated histone H3. Moreover, we found that TPL2 activity was required for MAPK pathway signal transduction in response to TNF receptor stimulation in myeloma cells. Taken together, our results provide important novel insights into the regulation of macrophages within primary myeloma niches in the bone marrow. Plasticity between M1 and M2 phenotypes may correlate tightly with the actions of TPL2 kinase. In the myeloma niche, TPL2 activity helps to fine-tune macrophage activation by promoting synthesis and release of pro-inflammatory cytokines required by the myeloma tumor cell while engaging counter-regulatory mechanisms to prevent tissue destruction mediated by activated macrophages. Additionally, we described a growth regulatory role of TPL2 in the tumor cell itself. Thus, TPL2 blockade may disrupt crucial macrophage-tumor cell interactions within myeloma niches. Disclosures: No relevant conflicts of interest to declare.

Wnt/β-catenin signaling is differentially regulated by Gα proteins and contributes to fibrous dysplasia

Skeletal dysplasias are common disabling disorders characterized by aberrant growth of bone and cartilage leading to abnormal skeletal structures and functions, often attributable to defects in skeletal progenitor cells. The underlying molecular and cellular mechanisms of most skeletal dysplasias remain elusive. Although the Wnt/β-catenin signaling pathway is required for skeletal progenitor cells to differentiate along the osteoblastic lineage, inappropriately elevated levels of signaling can also inhibit bone formation by suppressing osteoblast maturation. Here, we investigate interactions of the four major Gα protein families (Gα(s), Gα(i/o), Gα(q/11), and Gα(12/13)) with the Wnt/β-catenin signaling pathway and identify a causative role of Wnt/β-catenin signaling in fibrous dysplasia (FD) of bone, a disease that exhibits abnormal differentiation of skeletal progenitor cells. The activating Gα(s) mutations that cause FD potentiated Wnt/β-catenin signaling, and removal of Gα(s) led to reduced Wnt/β-catenin signaling and decreased bone formation. We further show that activation of Wnt/β-catenin signaling in osteoblast progenitors results in an FD-like phenotype and reduction of β-catenin levels rescued differentiation defects of FD patient-derived stromal cells. Gα proteins may act at the level of β-catenin destruction complex assembly by binding Axin. Our results indicate that activated Gα proteins differentially regulate Wnt/β-catenin signaling but, importantly, are not required core components of Wnt/β-catenin signaling. Our data suggest that activated Gα proteins are playing physiologically significant roles during both skeletal development and disease by modulating Wnt/β-catenin signaling strength.

Increased signaling through p62 in the marrow microenvironment increases myeloma cell growth and osteoclast formation

Adhesive interactions between multiple myeloma (MM) cells and marrow stromal cells activate multiple signaling pathways including nuclear factor kappaB (NF-kappaB), p38 mitogen-activated protein kinase (MAPK), and Jun N-terminal kinase (JNK) in stromal cells, which promote tumor growth and bone destruction. Sequestosome-1 (p62), an adapter protein that has no intrinsic enzymatic activity, serves as a platform to facilitate formation of signaling complexes for these pathways. Therefore, we determined if targeting only p62 would inhibit multiple signaling pathways activated in the MM microenvironment and thereby decrease MM cell growth and osteoclast formation. Signaling through NF-kappaB and p38 MAPK was increased in primary stromal cells from MM patients. Increased interleukin-6 (IL-6) production by MM stromal cells was p38 MAPK-dependent while increased vascular cell adhesion molecule-1 (VCAM-1) expression was NF-kappaB-dependent. Knocking-down p62 in patient-derived stromal cells significantly decreased protein kinase Czeta (PKCzeta), VCAM-1, and IL-6 levels as well as decreased stromal cell support of MM cell growth. Similarly, marrow stromal cells from p62(-/-) mice produced much lower levels of IL-6, tumor necrosis factor-alpha (TNF-alpha), and receptor activator of NF-kappaB ligand (RANKL) and supported MM cell growth and osteoclast formation to a much lower extent than normal cells. Thus, p62 is an attractive therapeutic target for MM.

CNTO328 Abrogates Glucocorticoid (GC) Resistance Conferred by Interleukin (IL)-6 and the Bone Marrow Microenvironment in Multiple Myeloma (MM).

Abstract Given the critical role that IL-6 plays in MM cell proliferation, survival, and resistance to GCs, we evaluated the ability of CNTO328, a chimeric monoclonal IL-6 neutralizing antibody, to overcome GC resistance in cell line models of human MM. In the presence of IL-6, the MM cell lines ANBL-6 and KAS-6 were resistant to the cytotoxic activity of dexamethasone (Dex) as assessed by cell viability assays both in suspension culture and in the context of patient-derived stromal cells. Resistance to dexamethasone was readily reversed by CNTO328, but not an isotype control antibody, in suspension culture. For example, in the case of the ANBL-6 model, viability was reduced by 12% with CNTO328 alone, 8% with Dex, but 74% with the combination, consistent with a synergistic interaction Given the ability of other growth factors in the bone marrow microenvironment to confer GC resistance in preclinical models of MM, we evaluated the activity of the CNTO328 and Dex combination in ANBL-6 and KAS-6 cells using a physiologically-relevant MM cell/patient-derived bone marrow stromal cell co-culture system. Importantly, bone marrow stromal cells rendered ANBL-6 and KAS-6 cells resistant to Dex in cell viability assays, and CNTO328 was able to reestablish Dex sensitivity, thus confirming a central role of IL-6 in bone marrow stroma-mediated GC resistance. Furthermore, treatment of ANBL-6 and KAS-6 cells with Dex alone did not induce apoptosis in this co-culture system, whereas the combination of CNTO328 and Dex led to a synergistic induction of apoptosis. In KAS-6 cells, IL-6-mediated Dex resistance was not overcome using pharmacologic inhibitors to p38, PI-3 kinase, mTor or MEK, suggesting that other IL-6 signaling pathways are likely involved. In contrast, the mTor inhibitor rapamycin was capable of sensitizing ANBL-6 cells to Dex in the presence of IL-6, suggesting that this pathway may be relevant to IL-6-mediated GC resistance in these cells. Induction of the pro-apoptotic Bcl-2 family member, Bim, has been shown to play an important role in GC-mediated cell death in lymphocytes as well as preclinical lymphoma and acute lymphoblastic leukemia models. Interestingly, although treatment of ANBL-6 cells in the presence of IL-6 with either CNTO328 or dexamethasone did not lead to induction of Bim, the combination led to a 3.3-fold increase in its expression. Taken together, the above data demonstrate that inhibition of IL-6 signaling with CNTO328 can effectively overcome IL-6-mediated GC resistance even in the presence of bone marrow stroma, and provide a compelling rationale for translation of this combination into clinical trials for patients suffering from MM. Furthermore, we show that the ability of CNTO328 to overcome GC resistance may be mediated in part by its ability to reverse IL-6-mediated repression of GC-induced Bim expression. Studies evaluating the relevance of Bim modulation in IL-6-mediated GC resistance and the molecular pathways that mediate this effect are on-going.