Stromal-derived Growth Factor Research Articles

Cellular regenerative therapy in stress urinary incontinence: new frontiers?-a narrative review.

Even if treatment with stem cells has been shown to be safe and effective in many patients with stress urinary incontinence (SUI), there is still room for improvement using other regenerative medicine alternatives. Since the beneficial effects of stem cells are probably mediated by secretion of factors rather than by the cells themselves there is a good rationale for further exploring the therapeutic effects of the secretome and/or its components. However, homing factors such as stromal derived growth factor 1 (SDF-1; CXCL12), stimulation of stem cell growth and stem cell mobilization in vivo using low intensity shock wave therapy (Li-ESWT) or regenerative electrical stimulation (RES), are also promising approaches. A literature search was performed based on PubMed, Scopus and Google Scholar. The search criteria included original basic science articles, systematic reviews and randomized control trials. All studies were published between 2000 and 2023. Selected, peer-reviewed studies were further analyzed to identify those of relevance. Keywords searched included: "female stress incontinence", "homing factors", "CXCL12", "secretome", "low intensity shockwave therapy" and "regenerative electrical stimulation". The peer-reviewed publications on the key word subjects that contained a novel addition to the existing body of literature were included. There is evidence from studies on non-human primates (NHPs) with experimental urinary sphincter injury that CXCL12 can restore sphincter structure and function. Studies with homing factors in human patients with SUI are still to be performed. A large number of clinical studies on the use of secretome or secretome products from mesenchymal stem cells (MSCs) on indications other than human SUI are already available. However, controlled clinical trials on patients with SUI, have to the best of our knowledge, not yet been performed. Also, RES has not been studied in patients with SUI. In contrast, there is clinical evidence that Li-ESWT may improve female SUI. Treatment with homing factors, MSC secretome/secretome components, Li-ESWT and RES are promising frontiers in the treatment of human SUI caused by sphincter damage.

Anti-Fibrotic Effect of SDF-1β Overexpression in Bleomycin-Injured Rat Lung

Rational: Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disease and is associated with high mortality due to a lack of effective treatment. Excessive deposition of the extracellular matrix by activated myofibroblasts in the alveolar space leads to scar formation that hinders gas exchange. Therefore, selectively removing activated myofibroblasts with the aim to repair and remodel fibrotic lungs is a promising approach. Stromal-derived growth factor (SDF-1) is known to stimulate cellular signals which attract stem cells to the site of injury for tissue repair and remodeling. Here, we investigate the effect of overexpression of SDF-1β on lung structure using the bleomycin-injured rat lung model. Methods: Intratracheal administration of bleomycin was performed in adult male rats (F344). Seven days later, in vivo electroporation-mediated gene transfer of either SDF-1β or the empty vector was performed. Animals were sacrificed seven days after gene transfer and histology, design-based stereology, flow cytometry, and collagen measurement were performed on the tissue collected. For in vitro experiments, lung fibroblasts obtained from IPF patients were used. Results: Seven days after SDF-1β gene transfer to bleomycin-injured rat lungs, reduced total collagen, reduced collagen fibrils, improved histology and induced apoptosis of myofibroblasts were observed. Furthermore, it was revealed that TNF-α mediates SDF-1β-induced apoptosis of myofibroblasts; moreover, SDF-1β overexpression increased alveolar epithelial cell numbers and proliferation in vivo and also induced their migration in vitro. Conclusions: Our study demonstrates a new antifibrotic mechanism of SDF-1β overexpression and suggests SDF-1β as a potential new approach for the treatment of lung fibrosis.

Role Of Growth Factors In Smooth Muscle Cell Migration In Tissue Engineered Vascular Grafts

INTRODUCTION AND OBJECTIVES This study sought to utilize growth factors to promote the migration of smooth muscle cells (SMC) into an electrospun vascular scaffold. The hypothesis was that the use of growth factors would allow for acellular tissue-engineered vascular grafts that are capable of recruiting smooth muscle cellular proliferation and infiltration in situ. METHODS Scaffolds electrospun with poly(ε-caprolactone) (PCL)/collagen polymers were surface modified through the passive adsorption of platelet-derived growth factor with two beta subunits (PDGF-BB) and stromal derived growth factor 1α (SDF-1α) at various concentrations, individually and together. Single-factor studies investigated each factor at concentrations of 100 ng/mL, 250 ng/mL, and 500 ng/mL. Dual-factor studies investigated the combined effects of 100 ng/mL PDGF-BB and 500 ng/mL of SDF-1α. RESULTS Compared to unmodified controls, scaffolds modified with growth factors showed higher rates of smooth muscle cell proliferation. Notably, scaffolds bioconjugated with a concentration of 100 ng/mL PDGF-BB alone and a concentration of 500 ng/mL SDF-1α alone demonstrated the greatest degree of smooth muscle cell proliferation compared to other single-factor concentrations, which confirmed ideal concentrations reported by other studies. A novel finding was that the combination of these growth factors at the aforementioned concentrations showed synergy in the ability to increase the degree of SMC migration into the depth of the electrospun scaffold. CONCLUSIONS This series of experiments verifies that growth factors promote proliferation of SMCs on an electrospun scaffold, and that combining multiple factors can optimize migration of SMCs farther into the thickness of electrospun scaffolds in an in vitro setting. This study sought to utilize growth factors to promote the migration of smooth muscle cells (SMC) into an electrospun vascular scaffold. The hypothesis was that the use of growth factors would allow for acellular tissue-engineered vascular grafts that are capable of recruiting smooth muscle cellular proliferation and infiltration in situ. Scaffolds electrospun with poly(ε-caprolactone) (PCL)/collagen polymers were surface modified through the passive adsorption of platelet-derived growth factor with two beta subunits (PDGF-BB) and stromal derived growth factor 1α (SDF-1α) at various concentrations, individually and together. Single-factor studies investigated each factor at concentrations of 100 ng/mL, 250 ng/mL, and 500 ng/mL. Dual-factor studies investigated the combined effects of 100 ng/mL PDGF-BB and 500 ng/mL of SDF-1α. Compared to unmodified controls, scaffolds modified with growth factors showed higher rates of smooth muscle cell proliferation. Notably, scaffolds bioconjugated with a concentration of 100 ng/mL PDGF-BB alone and a concentration of 500 ng/mL SDF-1α alone demonstrated the greatest degree of smooth muscle cell proliferation compared to other single-factor concentrations, which confirmed ideal concentrations reported by other studies. A novel finding was that the combination of these growth factors at the aforementioned concentrations showed synergy in the ability to increase the degree of SMC migration into the depth of the electrospun scaffold. This series of experiments verifies that growth factors promote proliferation of SMCs on an electrospun scaffold, and that combining multiple factors can optimize migration of SMCs farther into the thickness of electrospun scaffolds in an in vitro setting.

Regulation of aromatase in cancer.

The regulation of aromatase, an enzyme involved in the biosynthesis of estrogen in normal and cancer cells, has been associated with growth factor signaling and immune response modulation. The tissue-specific regulatory roles of these factors are of particular importance as local aromatase expression is strongly linked to cancer development/progression and disease outcomes in patients. Therefore, aromatase has become a chemotherapeutic target and aromatase inhibitors (AIs) are used in the clinic for treating hormone-dependent cancers. Although AIs have shown promising results in the treatment of cancers, the emerging increase in AI-resistance necessitates the development of new and improved targeted therapies. This review discusses the role of tumor and stromal-derived growth factors and immune cell modulators in regulating aromatase. Current single-agent and combination therapies with or without AIs targeting growth factors and immune checkpoints are also discussed. This review highlights recent studies that show new connections between growth factors, mediators of immune response, and aromatase regulation.

ErbB3 Targeting Enhances the Effects of MEK Inhibitor in Wild-Type BRAF/NRAS Melanoma.

MEK-ERK1/2 signaling is elevated in melanomas that are wild-type for both BRAF and NRAS (WT/WT), but patients are insensitive to MEK inhibitors. Stromal-derived growth factors may mediate resistance to targeted inhibitors, and optimizing the use of targeted inhibitors for patients with WT/WT melanoma is a clinical unmet need. Here, we studied adaptive responses to MEK inhibition in WT/WT cutaneous melanoma. The Cancer Genome Atlas data set and tumor microarray studies of WT/WT melanomas showed that high levels of neuregulin-1 (NRG1) were associated with stromal content and ErbB3 signaling. Of growth factors implicated in resistance to targeted inhibitors, NRG1 was effective at mediating resistance to MEK inhibitors in patient-derived WT/WT melanoma cells. Furthermore, ErbB3/ErbB2 signaling was adaptively upregulated following MEK inhibition. Patient-derived cancer-associated fibroblast studies demonstrated that stromal-derived NRG1 activated ErbB3/ErbB2 signaling and enhanced resistance to a MEK inhibitor. ErbB3- and ErbB2-neutralizing antibodies blocked the protective effects of NRG1 in vitro and cooperated with the MEK inhibitor to delay tumor growth in both cell line and patient-derived xenograft models. These results highlight tumor microenvironment regulation of targeted inhibitor resistance in WT/WT melanoma and provide a rationale for combining MEK inhibitors with anti-ErbB3/ErbB2 antibodies in patients with WT/WT cutaneous melanoma, for whom there are no effective targeted therapy options.Significance: This work suggests a mechanism by which NRG1 regulates the sensitivity of WT NRAS/BRAF melanomas to MEK inhibitors and provides a rationale for combining MEK inhibitors with anti-ErbB2/ErbB3 antibodies in these tumors. Cancer Res; 78(19); 5680-93. ©2018 AACR.

Nrf2 protects against diabetic dysfunction of endothelial progenitor cells via regulating cell senescence

Diabetes is associated with an increased risk of cardio-vascular disease. A decrease in the number and functionality of endothelial progenitor cells (EPCs) leads to reduced endothelial repair and the development of cardiovascular disease. The aim of the present study was to explore the effect and underlying mechanisms of nuclear factor erythroid 2-related factor 2 (Nrf2) on EPC dysfunction caused by diabetic mellitus. The biological functions of EPCs in streptozotocin-induced diabetic mice were evaluated, including migration, proliferation, angiogenesis and the secretion of vascular endothelial growth factor (VEGF), stromal-derived growth factor (SDF) and nitric oxide (NO). Oxidative stress levels in diabetic EPCs were also assessed by detecting intracellular reactive oxygen species (ROS), superoxide dismutase (SOD) and malondialdehyde (MDA). EPC senescence was evaluated by measuring p16 and b-gal expression and observing the senescence-associated secretory phenotype. In addition, the function of EPCs and level of oxidative stress were assessed following Nrf2 silencing or activation. Nrf2 silencing resulted in a decrease of EPC biological functions, accelerated cell senescence and increased oxidative stress, as indicated by ROS and MDA upregulation accompanied with decreased SOD activity. Furthermore, Nrf2 silencing inhibited migration, proliferation and secretion in EPCs, while it increased oxidative stress and cell senescence. Nrf2 activation protected diabetic EPCs against the effects of oxidative stress and cell senescence, ameliorating the biological dysfunction of EPCs derived from mice with diabetes. In conclusion, Nrf2 overexpression protected against oxidative stress-induced functional damage in EPCs derived from diabetic mice by regulating cell senescence.

The HGF/c-MET Pathway Is a Driver and Biomarker of VEGFR-inhibitor Resistance and Vascular Remodeling in Non-Small Cell Lung Cancer.

Purpose: Resistance to VEGFR inhibitors is a major obstacle in the treatment of non-small cell lung cancer (NSCLC). We investigated the cellular mechanisms mediating resistance of NSCLCs to VEGFR tyrosine kinase inhibitors.Experimental Design: We generated murine models of human NSCLC and performed targeted inhibition studies with the VEGFR TKIs cediranib and vandetanib. We used species-specific hybridization of microarrays to compare cancer (human) and stromal (mouse) cell transcriptomes of TKI-sensitive and -resistant tumors. We measured tumor microvascular density and vessel tortuosity to characterize the effects of therapy on the tumor vascular bed. Circulating cytokine and angiogenic factor levels in patients enrolled in VEGFR TKI trials were correlated with clinical outcomes.Results: Murine xenograft models of human lung adenocarcinoma were initially sensitive to VEGFR TKIs, but developed resistance to treatment. Species-specific microarray analysis identified increased expression of stromal-derived hepatocyte growth factor (HGF) as a candidate mediator of TKI resistance and its receptor, c-MET, was activated in cancer cells and tumor-associated stroma. A transient increase in hypoxia-regulated molecules in the initial response phase was followed by adaptive changes resulting in a more tortuous vasculature. Forced HGF expression in cancer cells reduced tumor sensitivity to VEGFR TKIs and produced tumors with tortuous blood vessels. Dual VEGFR/c-MET signaling inhibition delayed the onset of the resistant phenotype and prevented the vascular morphology alterations. In patients with cancer receiving VEGFR TKIs, high pretreatment HGF plasma levels correlated with poorer survival.Conclusions: HGF/c-MET pathway mediates VEGFR inhibitor resistance and vascular remodeling in NSCLC. Clin Cancer Res; 23(18); 5489-501. ©2017 AACR.

Transplantation of stromal-derived factor 1α and basic fibroblast growth factor primed insulin-producing cells reverses hyperglycaemia in diabetic rats

The defective insulin production is associated with severely reduced islet cell mass leading to diabetes. Growth factors preconditioned stem cells have arisen as an effective therapy to treat many diseases including diabetes. The current study was designed to assess the effect of pretreatment of ASCs derived IPCs with combination of stromal cell derived factor 1 alpha (SDF1α) and basic fibroblast growth factor (bFGF) in improving glucose tolerance in streptozotocin induced diabetic rats. The results showed maximally significant reduction in hyperglycaemia and fibrosis, while up-regulation of survival and pancreas-specific genes, insulin levels and homing of transplanted cells in SDF-1α + bFGF IPCs transplanted rats as compared with other groups. Moreover, increased expression of insulin, glucagon and Glut-2 in pancreas of the SDF-1α + bFGF IPCs transplanted group indicated more regeneration of pancreas. Hence, the use of IPCs preconditioned with SDF-1α + bFGF would be more effective for treating diabetes.

Differential response of human cardiac stem cells and bone marrow mesenchymal stem cells to hypoxia-reoxygenation injury.

Cardiosphere-derived cells (CDCs) and bone marrow mesenchymal stem cells (MSCs) are popularly used in stem cell therapy for myocardial regeneration. The cell type that survives and maintains stem cell characteristics in the adverse microenvironment following ischemia-reperfusion injury is presumed to be ideal for transplantation. The study was therefore aimed at identifying the cell type with relatively greater resistance to ischemia-reperfusion injury. CDCs were isolated from the right atrial appendage and MSCs from bone marrow of patients who underwent coronary artery bypass graft surgery. Ischemia-reperfusion injury was simulated in vitro by subjecting the cells to hypoxia (0.5% O2) followed by reintroduction of oxygen (HR injury). Greater resistance of CDCs to HR injury was apparent from the decreased expression of senescence markers and lower proportion of apoptotic cells (one-sixth of that in MSCs). HR injury retarded cell cycle progression in MSCs. Consequent to HR injury, cell migration and secretion of stromal-derived growth factor were stimulated, significantly in CDCs. The differentiation to myocyte lineage and angiogenesis assessed by tube formation ability was better for CDCs. Release of vascular endothelial growth factor was relatively more in CDCs and was further stimulated by HR injury. Differentiation to osteogenic and angiogenic lineage was stimulated by HR injury in MSCs. Compared to MSCs, CDCs appear to be the cell of choice for promoting myocardial regeneration by virtue of its survival capacity in the event of ischemic insult along with higher proliferation rate, migration efficiency, release of growth factors with paracrine effects and differentiation to cardiac lineage.

In vitro formation of neuroclusters in microfluidic devices and cell migration as a function of stromal-derived growth factor 1 gradients

ABSTRACTCentral nervous system (CNS) cells cultured in vitro as neuroclusters are useful models of tissue regeneration and disease progression. However, the role of cluster formation and collective migration of these neuroclusters to external stimuli has been largely unstudied in vitro. Here, 3 distinct CNS cell types, medulloblastoma (MB), medulloblastoma-derived glial progenitor cells (MGPC), and retinal progenitor cells (RPC), were examined with respect to cluster formation and migration in response to Stromal-Derived Growth Factor (SDF-1). A microfluidic platform was used to distinguish collective migration of neuroclusters from that of individual cells in response to controlled concentration profiles of SDF-1. Cell lines were also compared with respect to expression of CXCR4, the receptor for SDF-1, and the gap junction protein Connexin 43 (Cx43). All cell types spontaneously formed clusters and expressed both CXCR4 and Cx43. RPC clusters exhibited collective chemotactic migration (i.e. movement as clusters) along SDF-1 concentration gradients. MGPCs clusters did not exhibit adhesion-based migration, and migration of MB clusters was inconsistent. This study demonstrates how controlled microenvironments can be used to examine the formation and collective migration of CNS-derived neuroclusters in varied cell populations.

Abstract A50: Targeting CXCR4 and CXCR7 to mobilize and kill prostate tumor cells

Abstract Prostate cancer bone metastasis is a lethal incurable disease. Prostate cancer cells home to bone, where they are termed disseminated tumor cells (DTCs) and may reside dormant for years and/or grow into overt metastatic lesions. The bone homing process is largely thought to occur via local signaling of stromal-derived growth factor 1 (SDF1), secreted by osteoblasts within the bone marrow, to C-X-C chemokine receptor 4 (CXCR4), expressed on prostate cancer cells. Hematopoietic stem cells (HSCs) are also recruited to bone via SDF1/CXCR4 signaling. Specifically, HSCs reside in the osteoblastic endosteal niche, where they remain mostly quiescent. We have previously shown that prostate tumor cells occupy this HSC niche, and that the niche affords DTCs a chemoprotective status. Similar to HSCs, prostate DTCs can be mobilized from the niche by inhibiting the SDF1/CXCR4 signaling axis via small molecule CXCR4 inhibitors such as AMD3100 (trade name Plerixafor). Combining CXCR4 antagonism with the microtubule inhibitor docetaxel in mice injected subcutaneously with prostate cancer cells causes greater tumor regression than either compound alone. It is thought that mobilization of DTCs from the bone (we refer to mobilized DTCs as mobDTCs) causes them to proliferate in circulation, causing them to become susceptible to chemotherapy. We are using this strategy in a first-in-man clinical trial; we will treat bone metastatic prostate cancer patients with a potent next-generation CXCR4 antagonist coupled with standard docetaxel treatment. SDF1 also binds to the receptor CXCR7, which is overexpressed on prostate cancer cells as well. It is unknown whether CXCR7 will play a compensatory role when CXCR4 is antagonized. Therefore, it is crucial that we determine individual and overlapping roles of CXCR4 and CXCR7 in prostate cancer cells, particularly during bone metastasis. We hypothesized that CXCR7 can compensate for loss of CXCR4 function. We first assessed CXCR4 and CXCR7 expression across a panel of prostate cancer cell lines. We then stably overexpressed CXCR4 and CXCR7 in prostate cancer PC3 cells and determined classic cellular properties necessary for metastasis in vitro, such as proliferation, migration, and invasion. PC3 cells overexpressing either CXCR4 or CXCR7 had higher proliferation rates relative to PC3 parental cells. In addition, they were more migratory and invasive than parental cells. We are currently working to delete CXCR4 and/or CXCR7 from PC3 cells using the CRISPR-Cas9 system. We will perform in vivo xenograft metastasis experiments using prostate cancer cells with CXCR4 and/or CXCR7 overexpressed or knocked out. We hypothesize that loss of both receptors will be required to prevent bone metastasis. Future work will include small molecule inhibition of both CXCR4 and CXCR7 to mobilize prostate DTCs from bone. Citation Format: Kenneth C. Valkenburg, Sounak Roy, Kenneth J. Pienta. Targeting CXCR4 and CXCR7 to mobilize and kill prostate tumor cells. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr A50.

Mesenchymal Stem Cell Exosomes Induce Proliferation and Migration of Normal and Chronic Wound Fibroblasts, and Enhance Angiogenesis In Vitro.

Although chronic wounds are common and continue to be a major cause of morbidity and mortality, treatments for these conditions are lacking and often ineffective. A large body of evidence exists demonstrating the therapeutic potential of mesenchymal stem cells (MSCs) for repair and regeneration of damaged tissue, including acceleration of cutaneous wound healing. However, the exact mechanisms of wound healing mediated by MSCs are unclear. In this study, we examined the role of MSC exosomes in wound healing. We found that MSC exosomes ranged from 30 to 100-nm in diameter and internalization of MSC exosomes resulted in a dose-dependent enhancement of proliferation and migration of fibroblasts derived from normal donors and chronic wound patients. Uptake of MSC exosomes by human umbilical vein endothelial cells also resulted in dose-dependent increases of tube formation by endothelial cells. MSC exosomes were found to activate several signaling pathways important in wound healing (Akt, ERK, and STAT3) and induce the expression of a number of growth factors [hepatocyte growth factor (HGF), insulin-like growth factor-1 (IGF1), nerve growth factor (NGF), and stromal-derived growth factor-1 (SDF1)]. These findings represent a promising opportunity to gain insight into how MSCs may mediate wound healing.

O096 : Matrix metalloproteinase-10 contributes to hepatocellular carcinoma development in a novel crosstalk with stromal derived growth factor 1/C-X-C chemokine receptor 4 axis

O096 : Matrix metalloproteinase-10 contributes to hepatocellular carcinoma development in a novel crosstalk with stromal derived growth factor 1/C-X-C chemokine receptor 4 axis

Novel Evidence That, in Addition to Proteolytic Enzymes, Lipolytic Enzymes Are Involved in Mobilization of Hematopoietic Stem/Progenitor Cells (HSPCs) - an Important Pro-Mobilizing Role Identified for Hematopoietic-Specific Phospholipase C (PLCβ2)

Background. Hematopoietic stem/progenitor cells (HSPCs) express the chemokine receptor CXCR4 and the very late antigen 4 receptor (VLA-4, also known as alpha4beta1 integrin) on their cell surface and are retained in bone marrow (BM) niches by interaction of these receptors with their respective ligands, α-chemokine stromal-derived growth factor 1 (SDF-1) and vascular adhesion molecule 1 (VCAM-1, also known as CD106), which are expressed by cells in the BM microenvironment (e.g., osteoblasts and fibroblasts). Mobilization studies employing small molecule antagonists of CXCR4 or VLA-4 indicate the importance of both axes in retention of HSPCs in the BM microenvironment. Furthermore, it has been postulated that a crucial role in the mobilization process plays activation of a proteolytic microenvironment (J Clin Invest. 2003;111:187-96) and complement cascade (ComC) (Blood 2004;103:2071-8) in the BM microenvironment. However, to our surprise no attention has been paid so far to the role of lipolytic enzymes. Phospholipase C (PLC) is an enzyme released by neutrophils that cleaves the phosphoglycerol bond in glycosylphospahtidylinositol (GPI anchor), a glycolipid that is attached to the C-termini of several important proteins during posttranslational modification. GPI-linked proteins are thought to be preferentially located in lipid rafts. The most important GPI-anchored proteins include VCAM-1, complement inhibitors CD55 and CD59, and uPAR. We have reported that for retention of HSPCs in BM, both CXCR4 and VLA-4 have to be incorporated into membrane lipid rafts (Blood 2005;105:40-48).Hypothesis. Based on our previous observation that the HSPCs of paroxysmal nocturnal hemoglobinuria patients show defective retention in BM due to lack of functional GPI anchor (Leukemia 2012; 26:1722—5) we have hypothesized that PLC in normal BM may facilitate mobilization of HSPCs by perturbing expression of GPI anchor, which is so crucial for proper retention of HSPCs in BM.Materials and Methods. PLC activity has been measured by ELISA in the BM of mobilized mice as well in conditioned media from neutrophils exposed to several pro-mobilizing factors (G-CSF, AMD3100, C3a, C5a, desArgC5a, and S1P). We also tested the effect of PLC on the expression of VCAM-1 on BM-derived stroma and CD55 and CD59 antigens on BM mononuclear cells. The effect of PLC on incorporation of CXCR4 and VLA-4 into membrane lipid rafts has been studied by confocal microscopy employing murine Sca-1+ and human CD34+ cells. Here, to test our hypothesis, mobilization studies using AMD3100 and G-CSF have been performed in PLCβ2-KO (PLCβ2–/–) mice and their wild type (WT) littermates.Results. Our data indicate that the PLC level increases in BM during mobilization and is released from neutrophils in response to several pro-mobilization factors (G-CSF, AMD3100, C3a, C5a, desArgC5a, and S1P). PLC efficiently cleaves VCAM-1 expressed on BM stromal cells and thus perturbs the VCAM-1–VLA4 interaction as well as removes CD55 and CD59 from BM mononuclear cells, which enhances the pro-mobilizing effects of the ComC. PLC also inhibits lipid raft formation on HSPCs and by this means impairs the normal BM-retention function of CXCR4 and VLA-4. Finally, what is most important, we observed a mobilization defect in PLCβ2–/– mice, as evaluated by the number of mobilized leucocytes, SKL cells, and CFU-GM.Conclusions. We have established for the first time that, in addition to proteolytic enzymes, lipolytic enzymes, including LPC, are upregulated in the BM microenvironment, and that PLC promotes mobilization of HSPCs by perturbing the BM-retention function of GPI-anchored proteins. These data support an important role for GPI anchor-dependent proteins in the retention of HSPCs in BM niches. DisclosuresNo relevant conflicts of interest to declare.

Obesity-mediated regulation of HGF/c-Met is associated with reduced basal-like breast cancer latency in parous mice.

It is widely thought that pregnancy reduces breast cancer risk, but this lacks consideration of breast cancer subtypes. While a full term pregnancy reduces risk for estrogen receptor positive (ER+) and luminal breast cancers, parity is associated with increased risk of basal-like breast cancer (BBC) subtype. Basal-like subtypes represent less than 10% of breast cancers and are highly aggressive, affecting primarily young, African American women. Our previous work demonstrated that high fat diet-induced obesity in nulliparous mice significantly blunted latency in C3(1)-TAg mice, a model of BBC, potentially through the hepatocyte growth factor (HGF)/c-Met oncogenic pathway. Experimental studies have examined parity and obesity individually, but to date, the joint effects of parity and obesity have not been studied. We investigated the role of obesity in parous mice on BBC. Parity alone dramatically blunted tumor latency compared to nulliparous controls with no effects on tumor number or growth, while obesity had only a minor role in further reducing latency. Obesity-associated metabolic mediators and hormones such as insulin, estrogen, and progesterone were not significantly regulated by obesity. Plasma IL-6 was also significantly elevated by obesity in parous mice. We have previously reported a potential role for stromal-derived hepatocyte growth factor (HGF) via its cognate receptor c-Met in the etiology of obesity-induced BBC tumor onset and in both human and murine primary coculture models of BBC-aggressiveness. Obesity-associated c-Met concentrations were 2.5-fold greater in normal mammary glands of parous mice. Taken together, our studies demonstrate that, parity in C3(1)-TAg mice dramatically reduced BBC latency compared to nulliparous mice. In parous mice, c-Met is regulated by obesity in unaffected mammary gland and is associated with tumor onset. C3(1)-TAg mice recapitulate epidemiologic findings such that parity drives increased BBC risk and potential microenvironmental alterations in c-Met signaling may play a role in etiology.

Abstract 1658: Enhanced shedding of extracellular vesicles from amoeboid prostate cancer cells: Potential effects on the tumor microenvironment

Abstract The gene encoding the cytoskeletal regulator DIAPH3 is lost at high frequency in metastatic prostate cancer, and silencing of DIAPH3 evokes a transition to an amoeboid phenotype in multiple tumor cell backgrounds. This amoeboid transformation is accompanied by increases in plasma membrane deformations (blebbing), cell migration, invasion, and metastasis. DIAPH3 silencing also promotes the shedding of atypically large (>1μm) extracellular vesicles (EV) containing bioactive cargo. Whether loss of DIAPH3 also stimulates the release of bioactive nano-sized EV (exosomes) is not established. Here we examined the mechanism of release and potential biological functions of EV shed from DIAPH3-silenced and other prostate cancer cells. We observed that stimulation of LNCaP cells with the prostate stroma-derived growth factor heparin-binding EGF-like growth factor (HB-EGF), combined with p38MAPK inhibition caused exosome shedding, a process mediated by ERK1/2 hyperactivation. DIAPH3 silencing in DU145 cells also increased rates of exosome production. EV isolated from DIAPH3-silenced cells activated AKT1 and androgen signaling, increased proliferation of recipient tumor cells, and suppressed proliferation of human macrophages and peripheral blood mononuclear cells (PBMC). DU145-derived EV contained miR-125a, which suppressed AKT1 expression and proliferation in recipient human peripheral blood mononuclear cells and macrophages. Our findings suggest that EV across a wide size range (∼70nm to >1um) are produced by amoeboid prostate cancer cells as a result of DIAPH3 loss and/or growth factor stimulation. These EV may condition the tumor microenvironment through multiple mechanisms, including promoting the growth of cancer cells and suppression of tumor-infiltrating immune cells. Citation Format: Jayoung Kim, Samantha Morley, Minh Le, Denis Bedoret, Dale Umetsu, Dolores Di Vizio, Michael Freeman. Enhanced shedding of extracellular vesicles from amoeboid prostate cancer cells: Potential effects on the tumor microenvironment. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1658. doi:10.1158/1538-7445.AM2014-1658

Matrix metalloproteinase-1-mediated mesenchymal stem cell tumor tropism is dependent on crosstalk with stromal derived growth factor 1/C-X-C chemokine receptor 4 axis.

Human bone marrow-derived mesenchymal stem cells (MSCs) have the unique ability to home toward injuries or tumor sites. We have previously shown that the tumor-tropic property is dependent on the intrinsic expression and activity of the matrix remodeling gene, matrix metalloproteinase 1 (MMP-1). Herein, crosstalk between MMP-1/protease activated receptor 1 (PAR-1) and the G-protein coupled receptor stromal-derived growth factor 1 (SDF-1)/C-X-C chemokine receptor 4 (CXCR-4) in facilitating cell migration was investigated. Gain-of-function and RNA interference (RNAi) technology were used to evaluate the interplay between the key players. The downstream effect on the tumor-tropic migration of MSCs was investigated using modified Boyden chamber assay. Neutralizing PAR-1 activation using monoclonal antibody and targeted knockdown of MMP-1 using RNAi resulted in decreased expression of SDF-1, which was not observed in control-RNAi-transfected cells. Overexpression of CXCR-4 failed to promote MSC migration; the percentage of migrated cells toward tumor cell conditioned medium was similar to the vector-transduced and the CXCR-4-transduced MSCs. Furthermore, inhibition of SDF-1/CXCR-4 signaling using AMD3100 reduced MSC migration through the deregulation of MMP-1 promoter activities, protein expression, and metalloproteinase activity. Collectively, our results showed that MMP-1-mediated MSC tumor tropism is dependent on crosstalk with the SDF-1/CXCR-4 axis.

Blocking SDF-1α/CXCR4 Downregulates PDGF-B and Inhibits Bone Marrow–Derived Pericyte Differentiation and Tumor Vascular Expansion in Ewing Tumors

Bone marrow cells (BMC) are critical to the expansion of the tumor vessel network that supports Ewing sarcoma growth. BMCs migrate to the tumor and differentiate into endothelial cells and pericytes. We recently demonstrated that stromal-derived growth factor 1α (SDF-1α) regulates platelet-derived growth factor B (PDGF-B) and that this pathway plays a critical role in bone marrow-derived pericyte differentiation in vitro. We investigated the role of SDF-1α/PDGF-B in the tumor microenvironment in vivo in promoting bone marrow-derived pericyte differentiation in Ewing tumors. The CXCR4 antagonist AMD 3100 was used to disrupt the SDF-1α/CXCR4 axis in vivo in two xenograft Ewing tumor models. BMCs from GFP(+) transgenic mice were transplanted into lethally irradiated nude mice to track BMC migration to the tumor site. Following BMC engraftment, tumor-bearing mice received daily subcutaneous injections of either PBS or AMD 3100 for 3 weeks. Tumors were resected and tumor sections were analyzed by immunohistochemistry. AMD 3100 inhibited BMC differentiation into desmin(+) and NG2(+) pericytes, affected the morphology of the tumor vasculature, decreased perfusion, and increased tumor cell apoptosis. We observed smaller vessels with tiny lumens and a decrease in the microvessel density. AMD 3100 also inhibited PDGF-B protein expression in vitro and in vivo. SDF-1α in the tumor microenvironment plays a critical role in promoting pericyte formation and Ewing sarcoma tumor neovascularization by regulating PDGF-B expression. Interfering with this pathway affects tumor vascular morphology and expansion.

Enhanced shedding of extracellular vesicles from amoeboid prostate cancer cells

The gene encoding the cytoskeletal regulator DIAPH3 is lost at high frequency in metastatic prostate cancer, and DIAPH3 silencing evokes a transition to an amoeboid tumor phenotype in multiple cell backgrounds. This amoeboid transformation is accompanied by increased tumor cell migration, invasion, and metastasis. DIAPH3 silencing also promotes the formation of atypically large (>1 μm) membrane blebs that can be shed as extracellular vesicles (EV) containing bioactive cargo. Whether loss of DIAPH3 also stimulates the release of nano-sized EV (e.g., exosomes) is not established. Here we examined the mechanism of release and potential biological functions of EV shed from DIAPH3-silenced and other prostate cancer cells. We observed that stimulation of LNCaP cells with the prostate stroma-derived growth factor heparin-binding EGF-like growth factor (HB-EGF), combined with p38MAPK inhibition caused EV shedding, a process mediated by ERK1/2 hyperactivation. DIAPH3 silencing in DU145 cells also increased rates of EV production. EV isolated from DIAPH3-silenced cells activated AKT1 and androgen signaling, increased proliferation of recipient tumor cells, and suppressed proliferation of human macrophages and peripheral blood mononuclear cells. DU145 EV contained miR-125a, which suppressed AKT1 expression and proliferation in recipient human peripheral blood mononuclear cells and macrophages. Our findings suggest that EV produced as a result of DIAPH3 loss or growth factor stimulation may condition the tumor microenvironment through multiple mechanisms, including the proliferation of cancer cells and suppression of tumor-infiltrating immune cells.

Alterations of connexin 43 expression and SDF-1α secretion of bone marrow mesenchymal stem cells co-cultured with myeloma cells

To construct a co- culture system of mesenchymal stem cells (MSC) and multiple myeloma (MM) cells and investigate the alterations of connexin 43 (CX43) expression and stromal derived growth factor (SDF)- 1α secretion of MSC. CX43 expression and SDF- 1α secretion of MM cell lines (RPMI8226) and human primary MM cells were analyzed by western blot and immunofluorescence. Western blot, RT- PCR and immunofluorescence were employed to detect the alterations of CX43 expression and distribution in MSC directly and indirectly co-cultured with myeloma cells. Lucifer yellow dye spread was utilized to evaluate gap junctional intercellular communication (GJIC) between co- cultured MSC. Transwell was applied to study the transmigration of RPMI8266 induced by MSC under the condition of 18α- glycyrrhetinic acid (18α-GA). The level of SDF- 1α was detected by EILSA. RPMI8266, U266 and one-third primary MM cells expressed CX43 at low or moderate levels. CX43 wasn't expressed in XG- 4 and XG- 7 cells but highly expressed in MSC. The expressions of CX43 mRNA of MSC were up- regulated after directly and indirectly co- cultured with RPMI8226, 1.36 and 2.10 times that of MSC cultured alone respectively. Western blot analysis showed that CX43 protein expression of MSC was also up-regulated, mainly distributed in cytoplasm. Lucifer yellow dye spread showed that GJIC was up-regulated in MSC. SDF-1α concentration in supernatant of MSC directly and indirectly co-cultured with RPMI8226 were (373.02±10.11)pg/ml and (309.71±10.71)pg/ml respectively, which were higher than that of MSC cultured alone (237.84±9.23)pg/ml (P<0.01), and could be inhibited by 18α-GA [(237.84±9.23)pg/ml and (94.31±6.44)pg/ml] respectively (P<0.01). 18α-GA could inhibit the transmigration of RPMI8226 induced by MSC, decrease from (8.00±0.67)% to (4.82±0.19)%. CX43 expression of MSC was up-regulated after directly and indirectly co-cultured with MM cells, which could improve the level of SDF-1α secretion of MSC. GJ inhibitor could downregulate SDF-1α secretion of MSC and inhibit the transmigration of MM cells induced by MSC.