Bone Marrow Conditioned Medium Research Articles

Novel Evidence That Alternative Activation Pathway of Complement Cascade (ComC) Regulates Optimal Homing and Engraftment of Hematopoietic Stem/Progenitor Cells (HSPCs) in Reactive Oxygen Species (ROS) - Nlrp3 Inflammasome-Dependent Manner

Background. We reported in the past that activation of the third (C3) and fifth element (C5) of complement cascade (ComC) is required not only for mobilization of hematopoietic stem progenitor cells (HSPCs) but also for their proper homing and engraftment after transplantation to bone marrow (BM) (Leukemia 2012; 26:106-16). The ComC consists of zymogen proteins that become activated in a cascade-mediated manner by the i) classical, ii) mannan-binding lectin (MBL), or iii) alternative pathway. However, it is not clear which of these pathways plays a crucial role in inducing state of sterile inflammation in recipient BM conditioned for transplantation. Interestingly, the alternative pathway of ComC activation, in contrast to the other two pathways, is not triggered by antibodies or specific structures expressed on the surface of invading microorganisms, but is continuously activated and “ticking” by the spontaneous hydrolysis of the third component of the ComC (C3), which is the most abundant complement protein present in blood plasma. This process of C3 hydrolysis is hyperactivated in response to tissue/organ damage and changes the structure of C3 in order to promote binding of factor B (FB) that initiates the amplification process by which more C3b molecules and C3b-Bb convertases are created responsible for activation of the ComC. We also recently demonstrated that homing and engraftment of HSPCs to BM is mediated by activation of innate immunity pattern recognition receptor Nlrp3 inflammasome (Leukemia 2020; 34:1512-1523). On the other hand reactive oxygen species (ROS) are known activators of Nlrp3 inflammasome. Hypothesis. We hypothesized that sensitive and “continuously ticking” in peripheral blood (PB) alternative pathway of ComC activation facilitates homing and engraftment of HSPCs in response to myeloablation triggered sterile inflammation of hematopoietic microenvironment in donor BM. We also hypothesized that this depends on the activation of the Nlrp3 inflammasome in ROS-dependent manner. Materials and Methods. Activation of ComC and Nlrp3 inflammasome in BM of conditioned for hematopoietic transplantation by lethal irradiation mice was evaluated by C5a ELISA assay and immunofluorescence glow assay measuring activation of Nlrp3 inflammasome product that is activated caspase-1, respectively. We also run shotgun proteomic analysis with BM conditioned media and BMMNCs extracts. The release of ROS was measured by a colorimetric assay. To assess the involvement of alternative pathway of ComC activation, we performed homing and engraftment experiments in wild-type (WT) and FB-deficient mice, that have defect in alternative pathway of ComC activation. FB-KO and WT animals were transplanted with WT BMMNC. We also analyzed changes in the BM microenvironment in response to lethal irradiation in WT and FB-KO mice at mRNA and protein level. Results. We demonstrate for a first time that conditioning for transplantation by myeloablative irradiation induces in BM state of sterile inflammation reflected by activation of ComC, the release of reactive oxygen species (ROS), and activation of Nlrp3 inflammasome in ROS-dependent manner. Moreover, as compared to WT animals, all these pathways were significantly inhibited in FB-KO mice. As a result of this FB-KO animals displayed defective homing and engraftment after transplantation of WT BMMNC. This correlated at molecular level by the decreased expression of cell adhesion molecules and group of structural proteins involved in so called “docking structures” necessary for cell migration and homing as well as for Nlrp3 inflammasome complex and caspase 1, 2, 4 and 6. Moreover, FB-KO mice demonstrated lower level of SDF-1 and KL in BM after myeloablative conditioning for transplantation. Conclusions. We provide for a first time an evidence that myeloablative conditioning for transplantation by lethal irradiation activates in ComC-ROS-Nlrp3 inflammasome - dependent manner a state of sterile inflammation in the BM microenvironment, required for optimal homing and engraftment. This data also explains this phenomenon at molecular level and provides an evidence for a crucial involvement of alternative pathway of ComC activation. DisclosuresNo relevant conflicts of interest to declare.

Exposure to Adipose-Enriched Bone Marrow Increased Breast Cancer Cell Expression of Genes that Drive Tumor Metastasis

Background and Hypothesis: Previously thought to be inert, bone marrow adipose tissue (BMAT) has emerged as a significant fat depot that increases with age, radiation, estrogen deficiency, and high fat diet. Because bone is a preferred site for breast cancer metastasis, the lack of understanding regarding the effects of BMAT accrual on tumor cell migration and proliferation represents a significant knowledge gap in the cancer field. We hypothesized that exposure to adipokines secreted from BMAT-enriched bone marrow would induce changes in the tumor cell transcriptome.
 Experimental Design or Project Methods: Twenty-week female C57Bl/6 mice were randomized and treated with control diet (5% kcal% fat) or high fat diet (HFD; 60% kcal% fat) for nine weeks. Relative to control mice, HFD mice had increased body weight, peripheral fat mass, BMAT volume, and circulating adipokines. Bone marrow was flushed and cultured for 24 hours, and control or BMAT-enriched conditioned media was collected. The adipokines leptin and MCP-1 were assayed by ELISA and found to be increased in media obtained from BMAT-enriched cultures. Human MDA-MB-231 breast cancer cells were then cultured in bone marrow conditioned media for 24 hours and tumor cells were processed for RNA sequencing.Results: Exposure to soluble factors derived from BMAT-enriched cultures resulted in significant increase in the expression of genes associated with cancer cell motility and neovascularization, including SEMA4C, PLXNA1, and VEGFA.
 Conclusion and Potential Impact: These data indicate that BMAT contributes to the tumor microenvironment and may drive the progression of breast cancer bone metastases.

Effect of bone marrow mesenchymal stromal cells and conditioned medium in an irreversible unilateral ureteral obstruction murine model.

Background & Aim In recent years, many studies have shown that bone marrow mesenchymal stromal cells (BM-MSCs) promote renal recovery and prevent renal damage. In an irreversible unilateral ureteral obstruction (UUO) model, less damage has been reported in rats treated with BM-MSCs after one or two weeks of administration. However, effects in the contralateral kidney had not been studied. The aim of this study is to evaluate the effect of BM-MSCs and CM in attenuating parenchymal damage in an irreversible unilateral ureteral obstruction model (obstructed and contralateral kidney). Methods, Results & Conclusion Methodology BM-MSCs were obtained by primary culture of bone marrow of male Wistar rats, characterized by flow cytometry (CD45-, CD90+, CD 29+) and then administered in passages 2 to 6. Female Wistar rats were divided in 4 groups for treatment under anesthesia and maximum asepsis: 1) Sham operated: median ventral incision and closure by planes (n=6); 2) UUO: Unilateral ureteral obstruction (n=6); 3) UUO + BM-MSC: Unilateral ureteral obstruction with immediate administration of 1 × 106 BM-MSC, by puncture of inferior vena cava (n=7) and 4) UUO + CM: Unilateral ureteral obstruction with immediate administration of 400 µl of conditioned medium (CM) by puncture of inferior vena cava (n=5). At 2 weeks, blood was extracted from the aorta to evaluate creatinine levels and both kidneys were excised. Semi-quantitative (PAS and H&E) and immunohistochemical (Alpha-smooth-muscle actin, E-cadherin and nitrotyrosine) scores were analyzed to evaluate epithelial-mesenchymal transition and nitro-oxidative injury. Statistical analysis was performed by using the t-tests and one way ANOVA with Tukey post hoc test as appropriate. A p-value of Results The administration of BM-MSC in a model of irreversible unilateral ureteral obstruction is associated with a lower creatinine level and less nitrooxidative injury in the contralateral kidney at day 14 (p Conclusion BM-MSC and CM could effectively preserve kidney function in an unilateral obstruction model by protecting the contralateral kidney against nitro-oxidative injury. These results suggest a potential clinical treatment for chronic kidney diseases. This nephroprotective effect can be attributed to the antioxidant and anti-inflammatory activities of BM-MSC and CM.

Cell-Based Therapy in Cardiac Regeneration: An Overview.

Although pioneering preclinical research on the use of cell therapy for cardiac regeneration was conducted in the last quarter of the 20th century,1,2 a preponderance of advances have occurred in the 21st century, making this a relatively young field. In the first important clinical trial of cardiac cell therapy, begun in 2001, Menasche et al3 implanted autologous skeletal myoblasts into postinfarct scar at the time of coronary artery bypass surgery. Although the transplanted cells remained viable and exhibited contraction, they formed the nidus for serious ventricular tachyarrhythmias, which led to premature discontinuation of the trial. Despite this outcome, the trial energized the field, accelerating both preclinical and clinical research, albeit not with skeletal myoblasts. The extensive progress in cardiac regeneration is reviewed in this Compendium, and as occurs frequently in science, important observations have led to more questions and challenges (Table). View this table: Table. Important Challenges to Cell Therapy for Cardiac Regeneration Many cell types have been evaluated as candidates for cardiac regeneration. Among the earliest clinical trials, Zeiher’s group infused autologous bone marrow-derived progenitor cells into the coronary arteries of patients with acute,4 as well as healed myocardial infarction (MI)5 and reported improvements in left ventricular (LV) function. However, these results have not been fully confirmed by later studies, as pointed out in the review in the Compendium by Banarjee, Bolli, and Hare.6 Pittenger et al7 were among the first to direct attention to bone marrow-derived (stromal) mesenchymal stem cells (MSCs), emphasizing that these cells proliferated extensively in culture and suggesting that they could be attractive candidates for transplantation. In 2004, Chen et al8 reported that intracoronary infusion of autologous bone marrow-derived MSCs improved cardiac function. Zimmet and Hare pointed out that MSCs lack histocompatibility type II markers and elude rejection by …

Differential regulation of cell death pathways by the microenvironment correlates with chemoresistance and survival in leukaemia.

Glucocorticoids (GCs) and topoisomerase II inhibitors are used to treat acute lymphoblastic leukaemia (ALL) as they induce death in lymphoid cells through the glucocorticoid receptor (GR) and p53 respectively. Mechanisms underlying ALL cell death and the contribution of the bone marrow microenvironment to drug response/resistance remain unclear. The role of the microenvironment and the identification of chemoresistance determinants were studied by transcriptomic analysis in ALL cells treated with Dexamethasone (Dex), and Etoposide (Etop) grown in the presence or absence of bone marrow conditioned media (CM). The necroptotic (RIPK1) and the apoptotic (caspase-8/3) markers were downregulated by CM, whereas the inhibitory effects of chemotherapy on the autophagy marker Beclin-1 (BECN1) were reduced suggesting CM exerts cytoprotective effects. GCs upregulated the RIPK1 ubiquitinating factor BIRC3 (cIAP2), in GC-sensitive (CEM-C7-14) but not in resistant (CEM-C1-15) cells. In addition, CM selectively affected GR phosphorylation in a site and cell-specific manner. GR is recruited to RIPK1, BECN1 and BIRC3 promoters in the sensitive but not in the resistant cells with phosphorylated GR forms being generally less recruited in the presence of hormone. FACS analysis and caspase-8 assays demonstrated that CM promoted a pro-survival trend. High molecular weight proteins reacting with the RIPK1 antibody were modified upon incubation with the BIRC3 inhibitor AT406 in CEM-C7-14 cells suggesting that they represent ubiquitinated forms of RIPK1. Our data suggest that there is a correlation between microenvironment-induced ALL proliferation and altered response to chemotherapy.

Soluble bone-derived osteopontin promotes migration and stem-like behavior of breast cancer cells.

Breast cancer is a leading cause of cancer death in women, with the majority of these deaths caused by metastasis to distant organs. The most common site of breast cancer metastasis is the bone, which has been shown to provide a rich microenvironment that supports the migration and growth of breast cancer cells. Additionally, growing evidence suggests that breast cancer cells that do successfully metastasize have a stem-like phenotype including high activity of aldehyde dehydrogenase (ALDH) and/or a CD44+CD24- phenotype. In the current study, we tested the hypothesis that these ALDHhiCD44+CD24- breast cancer cells interact with factors in the bone secondary organ microenvironment to facilitate metastasis. Specifically, we focused on bone-derived osteopontin and its ability to promote the migration and stem-like phenotype of breast cancer cells. Our results indicate that bone-derived osteopontin promotes the migration, tumorsphere-forming ability and colony-forming ability of whole population and ALDHhiCD44+CD24- breast cancer cells in bone marrow-conditioned media (an ex vivo representation of the bone microenvironment) (p≤0.05). We also demonstrate that CD44 and RGD-dependent cell surface integrins facilitate this functional response to bone-derived osteopontin (p≤0.05), potentially through activation of WNK-1 and PRAS40-related pathways. Our findings suggest that soluble bone-derived osteopontin enhances the ability of breast cancer cells to migrate to the bone and maintain a stem-like phenotype within the bone microenvironment, and this may contribute to the establishment and growth of bone metastases.

The Effect of Lenalidomide on Multiple Myeloma Associated Macrophages

Introduction: In multiple myeloma (MM), accessory cells, such as monocytes and macrophages, located in the bone marrow (BM) tumor microenvironment play a crucial role in the fate of malignant cells. Under the influence of the surrounding milieu, monocytes can change their migratory capacity and differentiate into tumor-associated macrophages (TAMs). In the tumor site, TAMs can alter their profile from M1 macrophages with antitumor activities to M2-like macrophages that support tumor growth.Lenalidomide (Len), an immunomodulatory drug, used for MM treatment, is known to target different immune components inducing inflammatory responses; however, its direct influence on monocyte migration, macrophage differentiation and function in the tumor microenvironment is still unclear. The current study has aimed to explore the effect of Len on monocyte recruitment, macrophage polarization and pro-tumor functions.Methods: Monocytes were isolated from peripheral blood mononuclear cells of healthy donors, using anti-CD14 microbeads. To assess their migration capacity, monocytes were allowed to migrate through transwell insert towards the conditioned medium (CM) obtained from the BM of newly diagnosed MM patients or from MM cell lines (RPMI or U266) in the presence or absence of Len (10µM); the percentage of migrating monocytes was determined by FACS. For macrophage generation, monocytes were cultured with M-CSF followed by incubation with IL-4 to obtain M2 macrophages. To generate TAMs, CM obtained from the BM of MM patients was used. Len was added to the culture every 24 hours. The phenotype and functional properties of the generated macrophages were assessed. Endocytosis was evaluated by an antigen uptake assay. Macrophages were incubated with FITC-dextran at 37°C or 4°C, as a control, for 60 minutes, and analyzed by FACS. To test T cell proliferation, autologous lymphocytes labeled with CFSE, were stimulated with PHA and co-cultured with macrophages. T cell (CD3+) division was assessed by FACS. For IFN-γ secretion evaluation, lymphocytes co-cultured with macrophages were stimulated with PMA and ionomycin. The percentage of T cells expressing IFN-γ was quantified by FACS.Results: Monocyte migration towards CM obtained from MM cell lines (RPMI or U266) or from BM of MM patients (80.89%; n=4; p<0.01, 57.17%; n=4; p<0.01 and 42.9%; n=9; p<0.05, respectively) was significantly higher compared to migration towards normal BM CM (25.39%; n=2). Monocytes treated with Len demonstrated significantly decreased migration toward CM of MM cell lines (RPMI or U266) compared to untreated monocytes (45% vs. 80.8%; n=4 and 30.2% vs. 57.1%; n=4, respectively; p<0.01). The effect of Len on monocyte migration toward patient- derived CM was diverse. While 4 samples demonstrated decreased migration compared to untreated cells [51.1% vs. 59.6%; n=4; p<0.01], in 5 samples it increased [39.8% vs. 29.7%; n=5; n=5; p<0.01].To evaluate the effect of Len on macrophage polarization we examined their phenotype and functions. Both M2 macrophages and TAMs treated with Len expressed higher levels of M2 markers CD206, CD163, and cytokine IL-10 compared to untreated macrophages. Functional assays showed that Len increased endocytosis of both M2 macrophages [50% vs. 20%; n=5; P<0.01] and TAMs [47.4% vs. 41.4%; n=6; NS]. Exposure to Len led to suppression of T cell proliferation, when T cells were co-cultured with either autologous M2 macrophages [31% vs. 16%; n=4; p<0.05] or TAMs [39.7% vs. 31.5%; n=3; p<0.01]. In addition, M2 macrophages treated with Len demonstrated a reduction in IFN-γ secretion from T cells compared to untreated M2 macrophages (10.6% vs. 7.1%; n=4; NS).Conclusions: This study has demonstrated that Len has a direct effect on monocyte/macrophage behavior in the microenvironment generated by MM cells. Len is found to reduce monocyte migration, support polarization of macrophages towards the M2 phenotype and promote macrophage immunosuppressive functions, such as endocytosis, reduction of T-cell proliferation and inhibition of IFN-γ production. These findings need to be further investigated in in vivo experiments and could support the benefit of using agents targeting specific pathways associated with TAM development in the treatment of MM patients. DisclosuresNo relevant conflicts of interest to declare.

Abstract 2377: Bone-derived osteopontin mediates the migration and stem-like properties of breast cancer cells

Abstract Metastatic breast cancer has an affinity for certain organs and tissues, a phenomenon termed organ tropism. Of particular interest is breast cancer's preference for bone, since this is the most common site of metastasis in breast cancer patients. In addition, research suggests that breast cancer cells that do metastasize may have stem-like properties, including the ability to self-renew and differentiate into a heterogeneous tumor. These cells can be identified by their high aldehyde dehydrogenase activity (ALDH) and/or CD44+CD24- phenotype. However, it is unclear whether properties of the organ microenvironment or the stem-like cancer cells (or both) facilitate metastatic organ tropism. In the current study, we tested the hypothesis that bone marrow-conditioned media (an ex vivo representation of the bone microenvironment) contains specific soluble factors that enhance the growth and migration of whole population and ALDHhiCD44+CD24- breast cancer cells. Bone marrow-conditioned media (BMCM) generated from the bones of athymic nude mice was analyzed for the presence and identity of soluble factors using protein arrays. Osteopontin (OPN) was detected in the BMCM in significant amounts by the protein array and confirmed by ELISA. OPN was then depleted from the BMCM using immunoprecipitation and migration of MDA-MB-231 and SUM-159 breast cancer cells to the BMCM was assessed. Results indicate that bone-derived OPN significantly enhances MDA-MB-231 and SUM-159 breast cancer cell migration (P&amp;lt;0.05). Additionally, bone-derived OPN enhances the migration of ALDHhiCD44+CD24- MDA-MB-231 breast cancer stem cells relative to their ALDHlowCD44-CD24+ counterparts (P&amp;lt;0.05). The effect of bone-derived OPN on the tumorsphere forming abilities of whole population and ALDHhiCD44+CD24- MDA-MB-231 was also assessed; bone-derived OPN significantly enhances the tumorsphere forming abilities of whole population and stem-like MDA-MB-231 breast cancer cells (P&amp;lt;0.05). The interaction between bone-derived OPN and its cell surface receptors CD44 and αvβ5 in breast cancer cell migration to BMCM was also investigated using blocking antibodies. We observed that both whole population MDA-MB-231 and SUM-159 breast cancer cells interact with bone-derived OPN via CD44 and αvβ5 (P&amp;lt;0.05). Ongoing studies are investigating the activation of OPN-mediated signaling pathways in breast cancer cells as well as the role of CD44 and other cell surface integrins in the tumorsphere forming abilities of breast cancer cells. Overall, elucidation of the interactions between bone-derived OPN and breast cancer cells could contribute to future development of novel therapeutics that interrupt these interactions in the bone marrow niche, thereby improving breast cancer patient prognosis. Citation Format: Graciella M. Pio. Bone-derived osteopontin mediates the migration and stem-like properties of breast cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2377. doi:10.1158/1538-7445.AM2015-2377

Role of Enhanced Microenvironmental Interleukin-1 (IL-1) Expression and Increased IL-1 Responsiveness in Persistence of Leukemia Stem Cells in TKI Treated CML Patients

BCR-ABL tyrosine kinase inhibitors (TKI), although highly effective in the treatment of chronic myelogenous leukemia (CML) patients, fail to eliminate leukemia stem cells (LSC), which remain a potential source of relapse. In previous studies we have shown that altered expression of inflammatory cytokines in the CML bone marrow (BM) microenvironment provides a selective growth advantage to CML compared with normal long term hematopoietic stem cells (LTHSC) (Cancer Cell 2012, 21:577). Our studies suggest an important role for the pivotal pro-inflammatory cytokine Interleukin-1α/β (IL-1α/β) in selectively promoting growth of CML LTHSC. Using a transgenic BCR-ABL mouse model of CML and human CML and normal CD34+CD38- cells, we showed that inhibition of IL-1 signaling using recombinant IL-1 receptor antagonist (IL-1RA) in combination with nilotinib (NIL) resulted in significantly greater inhibition of CML LSC, compared with NIL alone (Blood 2013, abstract 512). To further investigate the mechanisms underlying increased IL-1 sensitivity of CML stem cells, we evaluated expression of the IL-1 receptor components, IL-1 receptor-associated protein (IL-1RAP) and IL-1R1, on CML and normal stem cells using flow cytometry. Expression of both IL-1RAP and IL-1R1 were increased on primary CML CD34+CD38-CD90+ cells compared to their normal counterparts (n=5, p<0.05). Exposure to IL-1α (10ng/ml) resulted in increased expression of p-NF-kB (p65), p-p38 MAPK and p-JNK in CML compared to normal CD34+CD38-CD90+ cells as evaluated by flow cytometry, indicating enhanced sensitivity to IL-1 induced signaling (n=5, p<0.05). The expression of p-NF-kB(p65), p-p38 MAPK and p-JNK in CML CD34+CD38-CD90+ cells cultured in CML BM conditioned medium (CM) was reduced after treatment with NIL or IL-1RA, and further reduced by the combination of NIL and IL-1RA (n=4, p<0.05). Immunohistochemistry (IHC) analysis showed that nuclear NF-κB p65 protein was reduced in NIL and IL-1RA treated CML CD34+CD38-CD90+ cells compared with controls. Treatment with NIL and IL-1RA also significantly reduced expression of the NF-κB target genes NFκB1A, BCL2L1, BIRC3 and CD83 (n=6, p<0.001), and of the inflammatory cytokines IL6, CXCL1, CXCL2, CCL2, CCL3, CCL4 and TNF-α (n=6, p<0.05), as assessed by Q-RT-PCR. We evaluated IL-1 expression in BM samples from CML patients with undetectable minimal residual disease (UMRD) using Q-RT-PCR. Interestingly IL-1α, but not IL-1β, expression was increased in BM samples from CML patients with UMRD compared to normal BM samples (n=12, p<0.05). To evaluate the source of increased IL-1α expression we analyzed selected monocyte (CD45+CD14+), non-monocytic myeloid cell (CD45+CD14-CD33+), T cell (CD45+CD14-CD33-CD3+), B cell (CD45+CD14-CD33-CD19+), endothelial cell (CD45-GPA-CD31+) and mesenchymal cell (CD45-GPA-CD31-) populations from BM samples obtained from CML patients with UMRD and from normal healthy controls. These studies revealed significantly elevated IL-1α expression in BM CD14+ monocytic and CD31+ endothelial cells from CML patients with UMRD compared to normal controls (n=12, p<0.05). Our studies indicate that CML LSC demonstrate increased IL-1 receptor expression and IL-1 induced NF-kB, p38 MAPK and JNK signaling. We also observe enhanced IL-1α expression in BM endothelial and monocytic cells from CML patients achieving UMRD, indicating persistence of an inflammatory microenvironment that may contribute to persistence of residual LSC. Our studies provide a strong rationale for the application of anti-IL-1 directed strategies to inhibit inflammatory signaling and enhance LSC elimination in TKI treated CML patients. DisclosuresNo relevant conflicts of interest to declare.

Anterior gradient protein-2 is a regulator of cellular adhesion in prostate cancer.

Anterior Gradient Protein (AGR-2) is reported to be over-expressed in many epithelial cancers and promotes metastasis. A clear-cut mechanism for its observed function(s) has not been previously identified. We found significant upregulation of AGR-2 expression in a bone metastatic prostate cancer cell line, PC3, following culturing in bone marrow-conditioned medium. Substantial AGR-2 expression was also confirmed in prostate cancer tissue specimens in patients with bone lesions. By developing stable clones of PC3 cells with varying levels of AGR-2 expression, we identified that abrogation of AGR-2 significantly reduced cellular attachment to fibronectin, collagen I, collagen IV, laminin I and fibrinogen. Loss of cellular adhesion was associated with sharp decrease in the expression of α4, α5, αV, β3 and β4 integrins. Failure to undergo apoptosis following detachment is a hallmark of epithelial cancer metastasis. The AGR-2-silenced PC3 cells showed higher resistance to Tumor necrosis factor-related apoptosis- inducing ligand (TRAIL) induced apoptosis in vitro. This observation was also supported by significantly reduced Caspase-3 expression in AGR-2-silenced PC3 cells, which is a key effector of both extrinsic and intrinsic death signaling pathways. These data suggest that AGR-2 influence prostate cancer metastasis by regulation of cellular adhesion and apoptosis.

Abstract 5173: The human homologue of frog (Xenopus laevis) anterior gradient protein promotes metastasis via regulation of cellular adhesion

Abstract Our recent studies indicated a significant up-regulation of anterior gradient protein-2 (AGR-2) in bone metastatic human prostate cancer cells, PC3, following growth in bone marrow conditioned medium. Evidence indicates that AGR2 belongs to the family of protein disulfide isomerases and has been linked to intestinal mucus production, tumor growth and metastasis of various adenocarcinomas and poor survival of patients with breast and prostate cancers. Few recent reports have also reported the regulatory elements of AGR2 function, but its specific role(s) in tumorigenesis and metastasis is still unclear. In the present study, we have analyzed the role of AGR2 in cancer progression and metastasis using prostate cancer as a model. Human prostate cancer tissue microarrays and autopsy tissues, obtained from University of Alabama at Birmingham consortiums were immunostained for AGR-2 expression. Results were also confirmed in a spontaneously developing transgenic adenocarcinoma of mouse prostate (TRAMP) model. Further characterization of AGR-2 function was determined in PC3 cells with targeted silencing of AGR-2 expressions for tumor growth and metastases in vitro and in vivo. Results of our study found AGR-2 expression mainly in the prostate luminar epithelial cells, but no difference was observed between normal prostate gland, BPH and early stages of prostate cancer in both human and TRAMP mouse. Significant reduction in AGR-2 expression was observed in grade IV &amp; V prostate cancers. Interestingly, although declining AGR-2 was evident in higher grade prostate tumors, significantly elevated AGR-2 expressions were observed in the metastatic sites of the same individuals. AGR-2-silenced PC3 cells formed tumors in SCID mice similar to control cells whereas their ability to remain attached to various ECM proteins were greatly compromised. Several key integrins, which mediates ECM binding, were also found to be down-regulated in the AGR-2 silenced PC3 cells, suggesting loss of AGR-2 may be associated with their detachment from the primary tumor and initiation of metastasis. AGR-2 is further up-regulated in metastatic sites indicating its necessity for restoration of integrins for attachment in a new microenvironment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5173. doi:1538-7445.AM2012-5173

Abstract 5261: Role of anterior gradient protein-2 in prostate cancer bone metastasis

Abstract Skeletal metastasis is common in late stage prostate cancer, which leads to significant decline in the quality of life and mortality. In a recent study, we identified over-expression of anterior gradient protein (AGR2) in the PC3, bone metastatic prostate cancer cells following in vitro growth in bone marrow-conditioned medium. AGR2 is a secreted protein and originally reported for its role in the formation of forebrain and mucus-secreting cement gland in the frog Xenopus laevis. Role of AGR2 in carcinogenesis and metastasis is recently being investigated and reports indicate it is over-expressed in various adenocarcinomas and linked to poor survival. But no study has elucidated the role of AGR2 in bone metastasis. When AGR2 expression was compared between human prostate cancer cell lines obtained from bone (PC3), lymph node (LnCap) and brain (Du145) by RT-PCR and Western blot, highest level of AGR2 was observed in PC3 cells. To further confirm, PC3 cells expressing firefly luciferase were injected in athymic nude mice via intra-cardiac route and cells were retrieved from bone, adrenal, lung and heart 3 weeks later following organ dissemination. RT-PCR analysis indicated highest AGR2 expression in PC3 cells isolated from bone microenvironment, suggesting that AGR2 may have a potential role in prostate cancer bone metastasis. To correlate the relevance of this finding in humans, AGR2 immunohistochemistry was performed on human primary and metastatic prostate cancer tissue sections. Preliminary results indicate higher AGR2 expression in metastasized bone sections compared to primary prostate cancers. Additionally, AGR2 was stably silenced in the PC3 cells utilizing a short hairpin RNA and growth kinetics of the cell line was tested in vivo following intra-cardiac injection in a preclinical mouse model. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5261. doi:10.1158/1538-7445.AM2011-5261

A Novel Paradigm In Stem Cell Trafficking: The Ratio of Peripheral Blood Sphingosine-1 Phosphate (S1P) to Bone Marrow Ceramide-1 Phosphate (C1P) Regulates Mobilization and Homing of Hematopoietic Stem Cells

AbstractAbstract 554The stromal derived factor-1 (SDF-1)–CXCR4 axis plays an unquestioned role in developmental migration of hematopoietic stem cells (HSPCs) and their retention in the bone marrow (BM). However, changes in the SDF-1 gradient between BM and peripheral blood (PB) do not always support its having a crucial role as chemoattractant for mobilization or homing of HSPCs. As demonstrated by others (e.g., Bone Marrow Transplantation 2003; 31:651–654, and Transfus Apher Sci 2009;40:159) and us (Leukemia 2010;24:976–985) the plasma SDF-1 level does not correlate with mobilization of HSPCs. On the other hand, there is increasing doubt about an exclusive role for SDF-1 in homing of HSPCs in BM. This is based on evidence that i) CXCR4−/− fetal liver HSPCs may home to BM in an SDF-1–independent manner (Immunity 1999;10:463-471), ii) homing of murine HSPCs made refractory to SDF-1 by incubation and co-injection with a CXCR4 receptor antagonist is normal or only mildly reduced (Science 2004;305:1000), and finally iii) HSPCs in which CXCR4 has been knocked down by means of an SDF-1 intrakine strategy also engraft in lethally irradiated recipients (Blood 2000;96:2074–,2080). All this strongly suggests the existence of other factors involved in the mobilization and homing of HSPCs. Moreover, while SDF-1 is a potent chemoattractant for HSPCs when employed at supraphysiological concentrations in vitro, as a peptide it is highly susceptible to degradation by proteases that are elevated, for example, in PB during stem cell mobilization or in the BM microenvironment after myeloablative conditioning for transplantation. Employing ELISA for detection in the present study, we observed insignificant changes in SDF-1 level both in PB during mobilization and in BM after myeloablative conditioning. We also found that mobilized PB (mPB) plasma as well as conditioned media (CM) from lethally irradiated mice chemoattract HSPCs in an SDF-1–independent manner as demonstrated by i) normal chemotaxis of AMD3100 pre-treated cells and ii) preservation of chemotactic activity of plasma and BM-derived CM following heat inactivation. However, the chemotactic activity of mPB plasma and BM CM was inhibited after stripping by activated charcoal. This suggested the involvement of small molecule bioactive lipids. It is known that sphingolipids, which are important components of cell membranes, give rise to two bioactive derivatives, sphingosine-1 phosphate (S1P) and ceramide-1 phosphate (C1P), with S1P already identified as a chemoattractant for HSPCs (Ann N Y Acad Sci. 200;1044:84–89). To our surprise, we found that C1P is also a strong chemoattractant for human and murine HSPCs. In addition, we observed that at physiological concentrations both these bioactive lipids i) activate phosphorylation of MAPKp42/44 and AKT in HSPCs, ii) induce expression of matrix metalloproteinases (MMPs), and iii) modulate adhesion to stroma and endothelium. Interestingly, by employing ELISA and/or mass spectophotometry we found that, while the S1P level increases in PB during mobilization, the C1P level increases in BM after myeloablative conditioning for transplantation. Based on these findings, we propose a new paradigm in which the S1P:C1P ratio plays a role in mobilization and homing of HSPCs. While S1P is a major chemoattractant that directs egress of HSPCs from BM into PB, C1P released from damaged cells in BM after myeloablative conditioning creates a homing gradient for circulating HSPCs. We also postulate that the S1P:C1P ratio plays a more universal role and is involved in regulating migration of other types of stem cells, such as circulating mesenchymal stem cells (MSCs), endothelial progenitor cells (EPCs), and very small embryonic-like (VSEL) stem cells. Accordingly, while S1P plays a role in egress of stem cells into PB, C1P released from damaged cells (e.g., in infarcted myocardium or brain tissue after stroke) chemoattracts circulating stem cells for potential repair. Disclosures:No relevant conflicts of interest to declare.

Abstract 231: Mechanism of bone metastasis in prostate cancer: Role of anterior gradient homolog 2

Abstract Prostate cancer commonly metastasizes to the skeleton in advanced stages and leads to significant decline in life expectancy. In a recent study we demonstrated enhanced proliferation of the human prostate cancer cell line PC3 when cultured in the presence of bone marrow conditioned medium (BMCM). In order to identify key molecules involved in increasing proliferation rate, total RNA was isolated from PC3 cells grown in either BMCM or regular medium and subjected to cDNA microarray analysis. Results indicated a significant up-regulation of anterior gradient homolog 2 (AGR2) in the PC3 cells cultured in BMCM compared to those grown in regular medium. AGR2 is a secreted protein and originally reported for its involvement in the formation of forebrain and mucus-secreting cement gland in the frog Xenopus laevis. AGR2 is also highly expressed in human adenocarcinomas of the prostate, breast, esophagus and the pancreas and a study indicated poor survival of prostate cancer patients with higher AGR2 expression. We compared AGR2 gene expression by RT-PCR in different metastatic prostate cancer cell lines, isolated from the bone (PC3 and C4-2B), brain (Du145) and the lymph node (LnCap). Highest AGR2 expression was observed in bone metastatic cell lines, mainly PC3 cells and least expression was observed in Du145 cells. PC3 cells expressing firefly luciferase were also injected via intra-cardiac route into 6 week-old SCID mice and widespread metastases were observed in the lung, heart, adrenal and bone. Three weeks after the inoculation of the PC3 cells, the mice were sacrificed, tumor cells were harvested from various metastatic sites, total RNA isolated and subjected to RT-PCR analysis for AGR2 expression. Interestingly, highest AGR2 expression was observed in PC3 cells isolated from bone microenvironment, suggesting that AGR2 may have a potential role in prostate cancer bone metastasis. To test this hypothesis, we developed a subclone of PC3 cells with targeted knock down of AGR2 transcripts using lentivirus shRNA vector and tested the cell clone in vitro by implanting intra-tibially or subcutaneously in SCID mice. Results of this study indicated a failure of tumor development in AGR2 knocked down PC3 clones. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 231.

Both CD133+Cells and Monocytes Provide Significant Improvement for Hindlimb Ischemia, Although They do not Transdifferentiate Into Endothelial Cells

To address a number of questions regarding the experimental use of bone marrow (BM) stem cells in hindlimb ischemia, including which is the best cell type (e.g., purified hematopoietic stem cell or monocytes), the best route of delivery [intramuscular (IM) or intravenous (IV)], and the mechanism of action (transdifferentiation or paracrine effects), we have compared the neovascularization capacities of CD133(+) stem cells and monocytes (CD11b(+)) from the BM of Tie2-GFP mice either via IV or IM in a murine severe hindlimb ischemia model. To test the effect of cytokine administration, an extra group received BM conditioned medium. Peripheral blood flow as well as capillary density and GPF-positivity detection in ischemic muscles was evaluated 7, 14, and 21 days postinjection. In addition, CD133(+) and CD11b(+) cells from transgenic animals were cultured in vitro with angiogenic media for 7, 14, and 21 days to assess GFP expression. In all four cell-treated groups, blood flow and capillary density significantly recovered compared with the mice that received no cells or conditioned medium. There were no differences with respect to cell types or administration routes, with the exception of a faster flow recovery in the CD133(+)-treated cell group. We did not find GFP(+) cells in the ischemic muscles and there was no GFP expression after in vitro proangiogenic culture. Our study shows that both purified CD133(+) stem cells and myeloid mononuclear cells, either IM or IV administered, have similar neoangiogenic ability. Nevertheless, transdifferentiation into endothelial cells is not the mechanism responsible for their beneficial effect.

Chromosomal Instability in Unirradiated Hemaopoietic Cells Induced by Macrophages Exposed In vivo to Ionizing Radiation

The tumorigenic potential of ionizing radiation has conventionally been attributed to DNA damage in irradiated cells induced at the time of exposure. Recently, there have been an increasing number of reports of damage in unirradiated cells that are either neighbors or descendants of irradiated cells, respectively, regarded as bystander effects and genomic instability and collectively termed nontargeted effects. In this study, we show that descendants of normal murine hemaopoietic clonogenic stem cells exposed to bone marrow-conditioned medium derived from gamma-irradiated mice exhibit chromosomal instability unlike the descendants of directly gamma-irradiated cells. The instability is expressed in bone marrow cells of the radiation-induced acute myeloid leukemia (r-AML) susceptible strain (CBA/Ca) but not in mice resistant to r-AML (C57BL/6). Furthermore, crossgenetic experiments show the induction of the instability phenotype requires both the producer and responder cells to be of the susceptible CBA/Ca genotype. Macrophages are the source of the bystander signals, and the signaling mechanism involves tumor necrosis factor-alpha, nitric oxide, and superoxide. The findings show a genotype-dependent chromosomal instability phenotype induced by radiation-induced macrophage-mediated bystander signaling. As the majority of accidental, occupational, and therapeutic exposures to ionizing radiation are partial body exposures, the findings have implications for understanding the consequences of such exposure.

Differential Gene Expression Associated with Migration of Mesenchymal Stem Cells to Conditioned Medium from Tumor Cells or Bone Marrow Cells

Distinct signals that guide migration of mesenchymal stem cells (MSCs) to specific in vivo targets remain unknown. We have used rat MSCs to investigate the molecular mechanisms involved in such migration. Rat MSCs were shown to migrate to tumor microenvironment in vivo, and an in vitro migration assay was used under defined conditions to permit further mechanistic investigations. We hypothesized that distinct molecular signals are involved in the homing of MSCs to tumor sites and bone marrow. To test this hypothesis, gene expression profiles of MSCs exposed in vitro to conditioned medium (CM) from either tumor cells or bone marrow were compared. Analysis of the microarray gene expression data revealed that 104 transcripts were upregulated in rat MSCs exposed to CM from C85 human colorectal cancer cells for 24 hours versus control medium. A subset of 12 transcripts were found to be upregulated in rat MSCs that were exposed to tumor cell CM but downregulated when MSCs were exposed to bone marrow CM and included CXCL-12 (stromal cell-derived factor-1 [SDF-1]), CXCL-2, CINC-2, endothelial cell specific molecule-1, fibroblast growth factor-7, nuclear factor-kappaB p105, and thrombomodulin. Exposure to tumor cell CM enhanced migration of MSCs and correlated with increased SDF-1 protein production. Moreover, knockdown of SDF-1 expression in MSCs inhibited migration of these cells to CM from tumor cells, but not bone marrow cells, confirming the importance of SDF-1 expression by MSCs in this differential migration. These results suggest that increased SDF-1 production by MSCs acts in an autocrine manner and is required for migratory responses to tumor cells.

“In vitro” evaluation of bone marrow angiogenesis in myelodysplastic syndromes: a morphological and functional approach

In myelodysplastic syndromes (MDS), ineffective hematopoiesis and cytopenia may arise either from increased susceptibility to apoptosis of hematopoietic cells, or possibly from alterations of bone marrow stroma that contributes to defective development of marrow lineages. In order to test the significance of the endothelial growth in MDS patients, two in vitro assays are presented in this study: a long-term culture method for the detection of human bone marrow endothelial colonies (CFU-En) (77 patients) and a human primary model for the evaluation of the influence of a “bone marrow conditioned medium” on the formation of new vessels in a culture matrix (“angio-kit assay”) (in 24 out 77 patients). The in vitro growth pattern of bone marrow CFU-En as well as the generation of microvessels in the angio-kit system was increased in RA, RARS and RAEB in comparison with controls. No CFU-En were detected in RAEB-T. The occurrence of large islands, formed by clusters of endothelial cells, unable to generate microcapillaries was also reported. Chromosomal abnormalities did not correlate with the angiogenetic pattern. These in vitro assays may constitute an useful approach to the analysis of angiogenesis in MDS.

Effects of bone marrow conditioned media on proliferation of stromal clonogenic cellsin vitro

Fibroblast growth factors aFGF, bFGF, IGF-I, IGF-II, TGF-β, EGF, and PDGF did not stimulate the formation of stromal fibroblast colonies (CFC-F-colonies) in cultured murine adhesive bone marrow cells. It means that colony-stimulating activity of the bone marrow feeder cells with respect to the formation of stromal fibroblast colonies does not depend on the known growth factors. A scheme and conditions of culturing are developed for preparing conditioned media from bone marrow cell cultures which can replace the CFC-F colonystimulating activity of the bone marrow feeder cellsin vitro. Primary separation of conditioned media by ultrafiltration reveals that only the fraction with molecular weight of more than 65 kD exhibits CFC-F colony-stimulating activity.

Insulin-Like Growth Factor-1 Acts as a Chemoattractant Factor for 5T2 Multiple Myeloma Cells

AbstractThe chemotactic and growth-stimulatory effect of insulin-like growth factor 1 (IGF-1) was investigated in the experimental mouse 5T2 multiple myeloma (MM) model. Chemotaxis was analyzed by classical checkerboard analysis. Bone marrow fibroblasts–conditioned medium exhibited a chemotactic effect on 5T2 MM cells that could be neutralized by adding a blocking antibody to IGF-1. On the other hand, exogenously added IGF-1 also had a chemotactic effect on the 5T2 MM cells. Moreover, in vitro analysis demonstrated that transmigrated 5T2 MM cells have a higher expression of IGF-1 receptor, both in bone marrow–conditioned medium and in IGF-1–induced chemotaxis, in comparison to cells before migration. When analyzed in vivo, 18 hours after injection of the heterogeneous 5T2 MM population, 5T2 MM cells present in the bone marrow show a higher expression of the IGF-1 receptor than their counterparts before injection. When the proliferative effect of IGF-1 was analyzed, no stimulation was observed, which is in contrast to the influence of bone marrow–conditioned medium and interleukin-6. Our results suggest a causal relationship between the presence of IGF-1 in the bone marrow and the chemotaxis of MM cells to and their subsequent presence in the bone marrow.