Metastatic breast cancer induces an osteoblast inflammatory response

Breast cancer preferentially metastasizes to the skeleton, a hospitable environment that attracts and allows breast cancer cells to thrive. Growth factors released as bone is degraded support tumor cell growth, and establish a cycle favoring continued bone degradation. While the osteoclasts are the direct effectors of bone degradation, we found that osteoblasts also contribute to bone loss. Osteoblasts are more than intermediaries between tumor cells and osteoclasts. We have presented evidence that osteoblasts contribute through loss of function induced by metastatic breast cancer cells. Metastatic breast cancer cells suppress osteoblast differentiation, alter morphology, and increase apoptosis. In this study we show that osteoblasts undergo an inflammatory stress response in the presence of human metastatic breast cancer cells. When conditioned medium from cancer cells was added to human osteoblasts, the osteoblasts were induced to express increased levels of IL-6, IL-8, and MCP-1; cytokines known to attract, differentiate, and activate osteoclasts. Similar findings were seen with murine osteoblasts and primary murine calvarial osteoblasts. Osteoblasts are co-opted into creating a microenvironment that exacerbates bone loss and are prevented from producing matrix proteins for mineralization. This is the first study implicating osteoblast produced IL-6, IL-8 (human; MIP-2 and KC mouse), and MCP-1 as key mediators in the osteoblast response to metastatic breast cancer cells.     

Senescence-Associated Secretory Phenotypes Reveal Cell-Nonautonomous Functions of Oncogenic RAS and the p53 Tumor Suppressor

Cellular senescence suppresses cancer by arresting cell proliferation, essentially permanently, in response to oncogenic stimuli, including genotoxic stress. We modified the use of antibody arrays to provide a quantitative assessment of factors secreted by senescent cells. We show that human cells induced to senesce by genotoxic stress secrete myriad factors associated with inflammation and malignancy. This senescence-associated secretory phenotype (SASP) developed slowly over several days and only after DNA damage of sufficient magnitude to induce senescence. Remarkably similar SASPs developed in normal fibroblasts, normal epithelial cells, and epithelial tumor cells after genotoxic stress in culture, and in epithelial tumor cells in vivo after treatment of prostate cancer patients with DNA-damaging chemotherapy. In cultured premalignant epithelial cells, SASPs induced an epithelial–mesenchyme transition and invasiveness, hallmarks of malignancy, by a paracrine mechanism that depended largely on the SASP factors interleukin (IL)-6 and IL-8. Strikingly, two manipulations markedly amplified, and accelerated development of, the SASPs: oncogenic RAS expression, which causes genotoxic stress and senescence in normal cells, and functional loss of the p53 tumor suppressor protein. Both loss of p53 and gain of oncogenic RAS also exacerbated the promalignant paracrine activities of the SASPs. Our findings define a central feature of genotoxic stress-induced senescence. Moreover, they suggest a cell-nonautonomous mechanism by which p53 can restrain, and oncogenic RAS can promote, the development of age-related cancer by altering the tissue microenvironment.     

Enhanced apoptotic cell clearance capacity and B cell survival factor production by IL-10-activated macrophages: implications for Burkitt's lymphoma

Burkitt's lymphoma (BL) is typified by frequent tumor cell apoptosis and significant macrophage infiltration. Since BL cells have an inherent tendency to undergo apoptosis at a high rate, we reasoned that macrophages in BL are functionally enhanced in at least two activities that have implications for tumor pathogenesis: 1) engulfment of apoptotic cells, an anti-inflammatory process known to suppress immune responses, and 2) production of BL cell survival factors that limit the extent of tumor cell apoptosis. In this study, we show that the microenvironment of BL is rich in the pleiotropic cytokine IL-10, which can be produced by both tumor cells and macrophages, and that IL-10-activated human macrophages have enhanced capacity to engulf apoptotic cells in vitro. This was found to be dependent on the macrophage tethering receptor of apoptotic cells, CD14. Furthermore, IL-10-activated macrophages were found to produce markedly higher levels of the B cell survival factor, B cell-activating factor of the TNF family/B lymphocyte stimulator (BAFF/BLyS) than macrophages matured in the absence of IL-10. Coculture of macrophages with BL cells further enhanced BAFF secretion. Significantly, we show that enhancement of BL cell survival by IL-10-activated macrophages is mediated by a BAFF-dependent component and that BAFF is produced at high levels by tumor-associated macrophages in situ. These results indicate that macrophages, regulated by IL-10, have the potential to promote BL pathogenesis, first, through suppression of antitumor immunity following enhanced engulfment of apoptotic tumor cells and, second, through increased production of tumor cell growth/survival factors.     

Tumor-derived MUC1 mucins interact with differentiating monocytes and induce IL-10highIL-12low regulatory dendritic cell

Dendritic cells (DC) initiate immunity by the activation of naive T cells and control immunity through their ability to induce unresponsiveness of lymphocytes by mechanisms that include deletion and induction of regulatory cells. An inadequate presentation to T cells by tumor-induced "regulatory" DC, among several mechanisms, can explain tolerance to tumor-associated Ags. In this study, we show that tumor-derived mucin profoundly affects the cytokine repertoire of monocyte-derived DC and switch them into IL-10(high)IL-12(low) regulatory APCs with a limited capacity to trigger protective Th1 responses. In fact, DC cocultured with pancreatic tumor cell lines in a Transwell system did not reach full maturation, had low immunostimulatory functions, did not produce IL-12, and released high levels of IL-10. The involvement of known tumor-derived immune-suppressive factors (e.g., vascular endothelial growth factor, TGF-beta, IL-6, and IL-10) was considered and excluded. We provide evidence that tumor-derived MUC1 mucins are responsible for the impaired DC maturation and function. DC obtained in the presence of tumor microenvironment preferentially polarized IL-4(+) response. Moreover, T cells primed by these regulatory DC became anergic and behaved as suppressor/regulatory cells. These findings identify mucin secretion as a novel mechanism of tumor escape from immune surveillance and provide the basis for the generation of potentially tolerogenic DC.     

Interleukin 6 mediated recruitment of mesenchymal stem cells to the hypoxic tumor milieu

Mesenchymal stem cells (MSCs) are a heterogeneous population of non-hematopoietic precursor cells predominantly found in the bone marrow. They have been recently reported to home towards the hypoxic tumor microenvironment in vivo. Interleukin-6 is a multifunctional cytokine normally involved in the regulation of the immune and inflammatory response. In addition to its normal function, IL-6 signaling has been implicated in tumorigenesis. Solid tumors develop hypoxia as a result of inadequate O₂ supply. Interestingly, tumor types with increased levels of hypoxia are known to have increased resistance to chemotherapy as well as increased metastatic potential. Here, we present evidence that under hypoxic conditions (1.5% O₂) breast cancer cells secrete high levels of IL-6, which serve to activate and attract MSCs. We now report that secreted IL-6 acts in a paracrine fashion on MSCs stimulating the activation of both Stat3 and MAPK signaling pathways to enhance migratory potential and cell survival. Inhibition of IL-6 signaling utilizing neutralizing antibodies leads to attenuation of MSC migration. Specifically, increased migration is dependent on IL-6 signaling through the IL-6 receptor. Collectively, our data demonstrate that hypoxic tumor cells specifically recruit MSCs, which through activation of signaling and survival pathways facilitate tumor progression.     

Signaling pathways bridging microbial-triggered inflammation and cancer

Microbial-triggered inflammation protects against pathogens and yet can paradoxically cause considerable secondary damage to host tissues that can result in tissue fibrosis and carcinogenesis, if persistent. In addition to classical pathogens, gut microbiota bacteria, i.e. a group of mutualistic microorganisms permanently inhabiting the gastrointestinal tract and which plays a key role in digestion, immunity, and cancer prevention, can induce inflammation-associated cancer following the alterations of their microenvironment. Emerging experimental evidence indicates that microbiota members like Escherichia coli and several other genotoxic and mutagenic pathogens can cause DNA damage in various cell types. In addition, the inflammatory response induced by chronic infections with pathogens like the microbiota members Helicobacter spp., which have been associated with liver, colorectal, cervical cancers and lymphoma, for instance, can also trigger carcinogenic processes. A microenvironment including active immune cells releasing high amounts of inflammatory signaling molecules can favor the carcinogenic transformation of host cells. Pivotal molecules released during immune response such as the macrophage migration inhibitory factor (MMIF) and the reactive oxygen and nitrogen species' products superoxide and peroxynitrite, can further damage DNA and cause the accumulation of oncogenic mutations, whereas pro-inflammatory cytokines, adhesion molecules, and growth factors may create a microenvironment promoting neoplastic cell survival and proliferation. Recent findings on the implication of inflammatory signaling pathways in microbial-triggered carcinogenesis as well as the possible role of microbiota modulation in cancer prevention are herein summarized and discussed.     

Cancer Associated Fibroblasts Promote Tumor Growth and Metastasis by Modulating the Tumor Immune Microenvironment in a 4T1 Murine Breast Cancer Model

Background

Local inflammation associated with solid tumors commonly results from factors released by tumor cells and the tumor stroma, and promotes tumor progression. Cancer associated fibroblasts comprise a majority of the cells found in tumor stroma and are appealing targets for cancer therapy. Here, our aim was to determine the efficacy of targeting cancer associated fibroblasts for the treatment of metastatic breast cancer.

Methodology/Principal Findings

We demonstrate that cancer associated fibroblasts are key modulators of immune polarization in the tumor microenvironment of a 4T1 murine model of metastatic breast cancer. Elimination of cancer associated fibroblasts in vivo by a DNA vaccine targeted to fibroblast activation protein results in a shift of the immune microenvironment from a Th2 to Th1 polarization. This shift is characterized by increased protein expression of IL-2 and IL-7, suppressed recruitment of tumor-associated macrophages, myeloid derived suppressor cells, T regulatory cells, and decreased tumor angiogenesis and lymphangiogenesis. Additionally, the vaccine improved anti-metastatic effects of doxorubicin chemotherapy and enhanced suppression of IL-6 and IL-4 protein expression while increasing recruitment of dendritic cells and CD8⁺ T cells. Treatment with the combination therapy also reduced tumor-associated Vegf, Pdgfc, and GM-CSF mRNA and protein expression.

Conclusions/Significance

Our findings demonstrate that cancer associated fibroblasts promote tumor growth and metastasis through their role as key modulators of immune polarization in the tumor microenvironment and are valid targets for therapy of metastatic breast cancer.     

Th17 cells are involved in the local control of tumor progression in primary intraocular lymphoma

Background

Th17 cells play an important role in the pathogenesis of many autoimmune diseases, but despite some reports of their antitumor properties, too little is known about their presence and role in cancers. Specifically, knowledge is sparse about the relation of Th17 to lymphoma microenvironments and, more particularly, to the microenvironment of primary intraocular B-cell lymphoma (PIOL), an aggressive lymphoma with a poor prognosis.

Methods and Principal Findings

In this work, we investigated the presence of Th17 cells and their related cytokines in a syngeneic model of PIOL, a subtype of non-Hodgkin lymphoma. The very small number of lymphocytes trafficking in normal eyes, which represent a low background as compared to tumor-bearing eyes, allows us to develop the present model to characterize the different lymphocyte subsets present when a tumor is developing. IL-21 mRNA was expressed concomitantly with IL-17 mRNA in tumor-bearing eyes and intracellular expression of IL-17A and IL-21 in infiltrating CD4⁺ T lymphocytes. Interestingly, IL-17A production by T cells was negatively correlated with tumor burden. We also showed that IL-21 but not IL-17 inhibits tumor cell proliferation in vitro.

Conclusions

These data demonstrate that IL-17A and IL-21-producing CD4⁺ T cells, referred as Th17 cells, infiltrate this tumor locally and suggest that Th17-related cytokines may counteract tumor progression via IL-21 production. Thus, Th17 cells or their related cytokines could be considered to be a new therapeutic approach for non-Hodgkin B-cell lymphomas, particularly those with an ocular localization.     

Cytokine production and inflammation drive autophagy in the tumor microenvironment: Role of stromal caveolin-1 as a key regulator

Recently, we proposed a new paradigm for understanding the role of the tumor microenvironment in breast cancer onset and progression. In this model, cancer cells induce oxidative stress in adjacent fibroblasts. This, in turn, results in the onset of stromal autophagy, which produces recycled nutrients to “feed” anabolic cancer cells. However, it remains unknown how autophagy in the tumor microenvironment relates to inflammation, another key driver of tumorigenesis. To address this issue, here we employed a well-characterized co-culture system in which cancer cells induce autophagy in adjacent fibroblasts via oxidative stress and NFκB-activation. We show, using this co-culture system, that the same experimental conditions that result in an autophagic microenvironment, also drive in the production of numerous inflammatory mediators (including IL-6, IL-8, IL-10, MIp1α, IFNγ, RANTES (CCL5) and GMCSF). Furthermore, we demonstrate that most of these inflammatory mediators are individually sufficient to directly induce the onset of autophagy in fibroblasts. To further validate the in vivo relevance of these findings, we assessed the inflammatory status of Cav-1 (−/−) null mammary fat pads, which are a model of a bonafide autophagic microenvironment. Notably, we show that Cav-1 (−/−) mammary fat pads undergo infiltration with numerous inflammatory cell types, including lymphocytes, T-cells, macrophages and mast cells. Taken together, our results suggest that cytokine production and inflammation are key drivers of autophagy in the tumor microenvironment. These results may explain why a loss of stromal Cav-1 is a powerful predictor of poor clinical outcome in breast cancer patients, as it is a marker of both (1) autophagy and (2) inflammation in the tumor microenvironment. Lastly, hypoxia in fibroblasts was not sufficient to induce the full-blown inflammatory response that we observed during the co-culture of fibroblasts with cancer cells, indicating that key reciprocal interactions between cancer cells and fibroblasts may be required.Key words: caveolin-1, oxidative stress, cytokine production, inflammation, tumor microenvironment, autophagy, breast cancer     

Pro-inflammatory cytokines IL-6 and CCL2 suppress expression of circadian gene Period2 in mammary epithelial cells

Chronic inflammation is known to contribute to tumor initiation and cancer progression. In breast tissue, the core circadian gene Period (PER)2 plays a critical role in mammary gland development and possesses tumor suppressor function. Interleukin (IL)-6 and C-C motif chemokine ligand (CCL) 2 are among the most abundant cytokines in the inflammatory microenvironment. We found that acute stimulation by IL-6/CCL2 reduced PER2 expression in non-tumorigenic breast epithelial cells. Longer term exposure to IL-6/CCL2 suppressed PER2 to an even lower level. IL-6 activated STAT3/NFκB p50 signaling to recruit HDAC1 to the PER2 promoter. CCL2 activated the PI3K/AKT pathway to promote ELK-1 cytoplasm-to-nucleus translocation, recruit HDAC1 to the proximal PER2 promoter and facilitate DNMT3-EZH2-PER2 promoter association. Ectopic expression of PER2 inhibited IL-6 or CCL2 induced mammosphere forming ability and reduced sphere size indicating that PER2 repression in breast epithelial cells can be crucial to activate tumorigenesis in an inflammatory microenvironment. The diminished expression of PER2 can be observed over a time scale of hours to weeks following IL-6/CCL2 stimulation suggesting that PER2 suppression occurs in the early stage of the interaction between an inflammatory microenvironment and normal breast epithelial cells. These data show new mechanisms by which mammary cells interact with a cancerous microenvironment and provide additional evidence that PER2 expression contributes to breast tumorigenesis.     

Stromelysin-2 (matrix metalloproteinase 10) is inducible in lymphoma cells and accelerates the growth of lymphoid tumors in vivo

Matrix metalloproteinase (MMP) 10 (stromelysin-2) is known to degrade various components of the extracellular matrix; however, the signals that regulate its expression and its role in lymphoma growth remain unknown. In the present work, we report the up-regulated expression of MMP10 in T lymphoma cells following contact with endothelial cells. The induction of MMP10 was found to be dependent on the specific interaction between LFA-1 and ICAM-1, which play a central role in regulating the expression of genes involved in the rate-limiting steps of lymphoma development. MMP10, but not MMP3 (stromelysin-1), was also up-regulated in human B lymphoma cells following exposure to IL-4, IL-6, and IL-13, but not to IL-1. To gain further insight into the role of MMP10 in lymphoma development, we generated lymphoma cell lines constitutively expressing high levels of MMP10 and studied these cells for their ability to form thymic lymphoma in vivo. Mice injected with lymphoma cells constitutively expressing MMP10 developed thymic lymphoma more rapidly than those injected with control lymphoma cells. These results provide the first in vivo evidence that overexpression of MMP10 promotes tumor development, and indicate that MMP10 induction is an important pathway activated not only upon ICAM-1/LFA-1-mediated intercellular contact, but also following activation of tumor cells with inflammatory cytokines.     

TRAF Family Member-associated NF-κB Activator (TANK) Inhibits Genotoxic Nuclear Factor κB Activation by Facilitating Deubiquitinase USP10-dependent Deubiquitination of TRAF6 Ligase

DNA damage-induced NF-κB activation plays a critical role in regulating cellular response to genotoxic stress. However, the molecular mechanisms controlling the magnitude and duration of this genotoxic NF-κB signaling cascade are poorly understood. We recently demonstrated that genotoxic NF-κB activation is regulated by reversible ubiquitination of several essential mediators involved in this signaling pathway. Here we show that TRAF family member-associated NF-κB activator (TANK) negatively regulates NF-κB activation by DNA damage via inhibiting ubiquitination of TRAF6. Despite the lack of a deubiquitination enzyme domain, TANK has been shown to negatively regulate the ubiquitination of TRAF proteins. We found TANK formed a complex with MCPIP1 (also known as ZC3H12A) and a deubiquitinase, USP10, which was essential for the USP10-dependent deubiquitination of TRAF6 and the resolution of genotoxic NF-κB activation upon DNA damage. Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated deletion of TANK in human cells significantly enhanced NF-κB activation by genotoxic treatment, resulting in enhanced cell survival and increased inflammatory cytokine production. Furthermore, we found that the TANK-MCPIP1-USP10 complex also decreased TRAF6 ubiquitination in cells treated with IL-1β or LPS. In accordance, depletion of USP10 enhanced NF-κB activation induced by IL-1β or LPS. Collectively, our data demonstrate that TANK serves as an important negative regulator of NF-κB signaling cascades induced by genotoxic stress and IL-1R/Toll-like receptor stimulation in a manner dependent on MCPIP1/USP10-mediated TRAF6 deubiquitination.     

Cytokine production and inflammation drive autophagy in the tumor microenvironment

Recently, we proposed a new paradigm for understanding the role of the tumor microenvironment in breast cancer onset and progression. In this model, cancer cells induce oxidative stress in adjacent fibroblasts. This, in turn, results in the onset of stromal autophagy, which produces recycled nutrients to “feed” anabolic cancer cells. However, it remains unknown how autophagy in the tumor microenvironment relates to inflammation, another key driver of tumorigenesis. To address this issue, here we employed a well-characterized co-culture system in which cancer cells induce autophagy in adjacent fibroblasts via oxidative stress and NFκB-activation. We show, using this co-culture system, that the same experimental conditions that result in an autophagic microenvironment, also drive in the production of numerous inflammatory mediators (including IL-6, IL-8, IL-10, MIP1a, IFNg, RANTES (CCL5) and GMCSF). Furthermore, we demonstrate that most of these inflammatory mediators are individually sufficient to directly induce the onset of autophagy in fibroblasts. To further validate the in vivo relevance of these findings, we assessed the inflammatory status of Cav-1 (-/-) null mammary fat pads, which are a model of a bonafide autophagic microenvironment. Notably, we show that Cav-1 (-/-) mammary fat pads undergo infiltration with numerous inflammatory cell types, including lymphocytes, T-cells, macrophages and mast cells. Taken together, our results suggest that cytokine production and inflammation are key drivers of autophagy in the tumor microenvironment. These results may explain why a loss of stromal Cav-1 is a powerful predictor of poor clinical outcome in breast cancer patients, as it is a marker of both (1) autophagy and (2) inflammation in the tumor microenvironment. Lastly, hypoxia in fibroblasts was not sufficient to induce the full-blown inflammatory response that we observed during the co-culture of fibroblasts with cancer cells, indicating that key reciprocal interactions between cancer cells and fibroblasts may be required.     

Gadd45a and Gadd45b protect hematopoietic cells from UV-induced apoptosis via distinct signaling pathways, including p38 activation and JNK inhibition

Gadd45a, Gadd45b, and Gadd45g (Gadd45/MyD118/CR6) are genes that are rapidly induced by genotoxic stress and have been implicated in genotoxic stress-induced responses, notably in apoptosis. Recently, using myeloid-enriched bone marrow (BM) cells obtained from wild-type (WT), Gadd45a-deficient, and Gadd45b-deficient mice, we have shown that in hematopoietic cells Gadd45a and Gadd45b play a survival function to protect hematopoietic cells from DNA-damaging agents, including ultra violet (UV)-induced apoptosis. The present study was undertaken to decipher the molecular paths that mediate the survival functions of Gadd45a and Gadd45b against genotoxic stress induced by UV radiation. It is shown that in hematopoietic cells exposed to UV radiation Gaddd45a and Gadd45b cooperate to promote cell survival via two distinct signaling pathways involving activation of the GADD45a-p38-NF-kappaB-mediated survival pathway and GADD45b-mediated inhibition of the stress response MKK4-JNK pathway.     

IL-6 Undergoes Transition from in vitro Autocrine Growth Factor toin vivo Growth Inhibitor of B Lymphoma Cells

IL-6 is a multifunctional cytokine involved in differentiation and proliferation of immune cells. Moreover, it has diverse effects on the proliferation of tumor cells in vivo and in vitro. Although stimulating cell growth of multiple myeloma cells, it inhibits the proliferation of B16 melanoma cells and lung cancer cells. B9.55 cells, B-cell lymphoma, are IL-6-dependent cells, definitely requiring exogenous IL-6 for growth. When the cDNA for IL-6 was transfected into B9.55 cells, they began growing in an autocrine pattern without exogenous IL-6. To investigate the effects of IL-6 on B9.55 lymphoma in vivo, IL-6-transfected B9.55 cells (B9.G7) or neotransfected B9.55 cells (B9.vec) were injected subcutaneously into syngeneic mice. Initially, B9.G7 outgrew B9.vec, but after 3 weeks, B9.G7 grew slower than B9.vec. In addition, 5 µg of recombinant human IL-6 was injected daily into the tumor site. Reduced tumor sizes of IL-6-treated rats, similar to those observed in mice which received B9.G7, indicated that IL-6 itself is the mediator of tumor regression. When B9.G7 cells were injected into the irradiated normal mice, tumor regression was released compared with the untreated normal control, suggesting that radiosensitive host components were involved in the regression of B9.G7 cell growth. However, the tumor regression of B9.G7 cells was not released in SCID mice. Histologically, B9.G7 tumor demonstrated severe necrosis and apoptotic cells with infiltration of host inflammatory cells. Above data indicate that IL-6 functions as an autocrine growth factor for B9.G7 cells in vitro, but behaves as an autocrine inhibiting factor in vivo. These contrasting effects of IL-6 on tumor cells in vitro and in vivo will be facilitative in understanding the interaction of cytokines and host immune systems.They have contributed equally to this work.     

Mast Cells Mobilize Myeloid-Derived Suppressor Cells and Treg Cells in Tumor Microenvironment via IL-17 Pathway in Murine Hepatocarcinoma Model

Tumor immunosuppression is commonly braided with chronic inflammation during tumor development. However, the relationship between immunosuppression and inflammation in tumor microenvironment is still unclear. We have demonstrated that mast cells are accumulated and exacerbate the inflammation and immunosuppression in tumor microenvironment via SCF/c-kit signaling pathway. Here, we further elucidate the underlying mechanism, which involves both myeloid-derived suppressor cells (MDSCs) and regulatory T (Treg) cells. Our data showed that mast cells mobilized the infiltration of MDSCs to tumor and induced the production of IL-17 by MDSCs; MDSCs-derived IL-17 indirectly attracted Treg cells, enhanced their suppressor function, and induced the IL-9 production by Treg cells; in turn, IL-9 strengthened the survival and protumor effect of mast cells in tumor microenvironment. Our findings disclose a closed loop among mast cells, MDSCs and Treg cells in tumor microenvironment, which provides a new insight into the paralleled developments of inflammation and immunosuppression in tumor microenvironment. Based on these findings, we propose that targeting tumor inflammation might be a potential strategy to reverse the immunosuppression of tumor microenvironment, thus facilitating cancer immunotherapy.     

Melphalan exposure induces an interleukin-6 deficit in bone marrow stromal cells and osteoblasts

Bone marrow stromal cells (BMSC) and osteoblasts are critical components of the microenvironment that support hematopoietic recovery following bone marrow transplantation. Aggressive chemotherapy not only affects tumor cells, but also influences additional structural and functional components of the microenvironment. Successful reconstitution of hematopoiesis following stem cell or bone marrow transplantation after aggressive chemotherapy is dependent upon components of the microenvironment maintaining their supportive function. This includes secretion of soluble factors and expression of cellular adhesion molecules that impact on development of hematopoietic cells. In the current study, we investigated the effects of chemotherapy treatment on BMSC and human osteoblast (HOB) expression of interleukin-6 (IL-6) as one regulatory factor. IL-6 is a pleiotropic cytokine which has diverse effects on hematopoietic cell development. In the current study we demonstrate that exposure of BMSC or HOB to melphalan leads to decreases in IL-6 protein expression. Decreased IL-6 protein is the most pronounced following melphalan exposure compared to several other chemotherapeutic agents tested. We also observed that melphalan decreased IL-6 mRNA in both BMSC and HOB. Finally, using a model of BMSC or HOB co-cultured with myeloma cells exposed to melphalan, we observed that IL-6 protein was also decreased, consistent with treatment of adherent cells alone. Collectively, these observations are of dual significance. First, suggesting that chemotherapy induced IL-6 deficits in the bone marrow occur which may result in defective hematopoietic support of early progenitor cells. In contrast, the decrease in IL-6 protein may be a beneficial mechanism by which melphalan acts as a valuable therapeutic agent for treatment of multiple myeloma, where IL-6 present in the bone marrow acts as a proliferative factor and contributes to disease progression. Taken together, these data emphasize the responsiveness of the microenvironment to diverse stress that is important to consider in therapeutic settings.