Preferential, enhanced breast cancer cell migration on biomimetic electrospun nanofiber ‘cell highways’

BACKGROUND: Aggressive metastatic breast cancer cells seemingly evade surgical resection and current therapies, leading to colonization in distant organs and tissues and poor patient prognosis. Therefore, high-throughput in vitro tools allowing rapid, accurate, and novel anti-metastatic drug screening are grossly overdue. Conversely, aligned nanofiber constitutes a prominent component of the late-stage breast tumor margin extracellular matrix. This parallel suggests that the use of a synthetic ECM in the form of a nanoscale model could provide a convenient means of testing the migration potentials of cancer cells to achieve a long-term goal of providing clinicians an in vitro platform technology to test the efficacy of novel experimental anti-metastatic compounds. METHODS: Electrospinning produces highly aligned, cell-adhesive nanofiber matrices by applying a strong electric field to a polymer-containing solution. The resulting fibrous microstructure and morphology closely resembles in vivo tumor microenvironments suggesting their use in analysis of migratory potentials of metastatic cancer cells. Additionally, a novel interface with a gel-based delivery system creates CXCL12 chemotactic gradients to enhance CXCR4-expressing cell migration. RESULTS: Cellular dispersions of MCF-10A normal mammary epithelial cells or human breast cancer cells (MCF-7 and MDA-MB-231) seeded on randomly-oriented nanofiber exhibited no significant differences in total or net distance traveled as a result of the underlying topography. Cells traveled ~2-5 fold greater distances on aligned fiber. Highly-sensitive MDA-MB-231 cells displayed an 82% increase in net distance traversed in the presence of a CXCL12 gradient. In contrast, MCF-7 cells exhibited only 31% increase and MCF-10A cells showed no statistical difference versus control or vehicle conditions. MCF-10A cells displayed little sensitivity to CXCL12 gradients, while MCF-7 cells displayed early sensitivity when CXCL12 concentrations were higher. MDA-MB-231 cells displayed low relative expression levels of CXCR4, but high sensitivity resulting in 55-fold increase at late time points due to CXCL12 gradient dissipation. CONCLUSIONS: This model could create clinical impact as an in vitro diagnostic tool for rapid assessment of tumor needle biopsies to confirm metastatic tumors, their invasiveness, and allow high-throughput drug screening providing rapid development of personalized therapies. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/1471-2407-14-825) contains supplementary material, which is available to authorized users

M-CSF Signals through the MAPK/ERK Pathway via Sp1 to Induce VEGF Production and Induces Angiogenesis In Vivo

BACKGROUND: M-CSF recruits mononuclear phagocytes which regulate processes such as angiogenesis and metastases in tumors. VEGF is a potent activator of angiogenesis as it promotes endothelial cell proliferation and new blood vessel formation. Previously, we reported that in vitro M-CSF induces the expression of biologically-active VEGF from human monocytes. METHODOLOGY AND RESULTS: In this study, we demonstrate the molecular mechanism of M-CSF-induced VEGF production. Using a construct containing the VEGF promoter linked to a luciferase reporter, we found that a mutation reducing HIF binding to the VEGF promoter had no significant effect on luciferase production induced by M-CSF stimulation. Further analysis revealed that M-CSF induced VEGF through the MAPK/ERK signaling pathway via the transcription factor, Sp1. Thus, inhibition of either ERK or Sp1 suppressed M-CSF-induced VEGF at the mRNA and protein level. M-CSF also induced the nuclear localization of Sp1, which was blocked by ERK inhibition. Finally, mutating the Sp1 binding sites within the VEGF promoter or inhibiting ERK decreased VEGF promoter activity in M-CSF-treated human monocytes. To evaluate the biological significance of M-CSF induced VEGF production, we used an in vivo angiogenesis model to illustrate the ability of M-CSF to recruit mononuclear phagocytes, increase VEGF levels, and enhance angiogenesis. Importantly, the addition of a neutralizing VEGF antibody abolished M-CSF-induced blood vessel formation. CONCLUSION: These data delineate an ERK- and Sp1-dependent mechanism of M-CSF induced VEGF production and demonstrate for the first time the ability of M-CSF to induce angiogenesis via VEGF in vivo

Modeling the inhibition of breast cancer growth by GM-CSF

M-CSF is overexpressed in breast cancer and is known to stimulate macrophages to produce VEGF resulting in angiogenesis. It has recently been shown that the growth factor GM-CSF injected into murine breast tumors slowed tumor growth by secreting soluble VEGF receptor-1 (sVEGFR-1) that binds and inactivates VEGF. This study presents a mathematical model that includes all the components above, as well as MCP-1, tumor cells, and oxygen. The model simulations are representative of the in vivo data through predictions of tumor growth using different protocol strategies for GM-CSF for the purpose of predicting higher degrees of treatment success. For example, our model predicts that once a week dosing of GM-CSF would be less effective than daily, twice a week, or three times a week treatment because of the presence of essential factors required for the anti-tumor effect of GM-CSF. (C) 2012 Elsevier Ltd. All rights reserved

Abstract PR03: Macrophage phenotype drives tumor program via epigenetic machinery carried in secreted microvesicles

Abstract Macrophage phenotypes are reported to regulate tumor progression, angiogenesis, and metastasis in breast cancer by producing soluble factors modulating these programs. Macrophages also communicate via secreted microvesicles (MVs) which are taken-up by neighboring epithelium. MVs contain mRNAs coding the epigenetic regulating machinery, DNA methyltranferases (DNMTs) and histone deacetylases (HDACs), which augment or silence expression via promoter CpG island methylation. Tie2-expressing monocytes (TEMs) is a subset of monocytes reported to augment tumor angiogenesis and metastasis. Recently, we found that increased levels of colony stimulating factor-1 (CSF1) can expand the TEM population in circulation, enabling an influx into breast tumors. Interestingly, we also found that expansion of TEMs by hypoxia was regulated by HIF-1α; and not HIF-2α, but only once they enter the tumor proper. We hypothesized that MVs secreted from M1 and M2 macrophages or TEMs contain epigenetic regulatory machinery which regulate CpG island methylation and gene expression of tumor suppressor genes (TSGs) and genes driving epithelial-to-mesenchymal transition (EMT). We differentiated M1, M2, and TEMs in vitro from CD14+ monocytes isolated from peripheral blood. These cell populations were confirmed using flow cytometry for CD68 and CD80 for M1, CD163 for M2, and CD14/Tie2 for TEMs, as well as M1 (IL-6 and TNFα) and M2 (IL-10 and mannose receptor-1) gene expression profiles. After, we collected MVs using high speed centrifugation techniques characterized by flow cytometry and isolated their nucleic acid content. Using qRT-PCR, we found differential presence of mRNAs for DNMTs and HDACs between M1, M2, and TEM MVs. We cultured these MVs with MCF-10A normal mammary epithelial cells or BEAS-2B normal lung epithelial cells (target cells) for 24 hours and demonstrated MV uptake using Syto RNASelect (RNA) and DiIC16(3) (lipid membrane) and confocal microscopy. After, we isolated RNA and DNA from the target cells and analyzed DNMTs, HDACs, and EMT mRNA expression as well as methyl-specific PCR for CpG island methylation in the promoters of EMT genes. We found that MVs from M1 macrophages increased DNMTs mRNA expression compared to MVs produced from M2 and quiescent macrophages (M0) as well as untreated target cells. To the contrary, HDACs mRNA expression in these target cells cultured with M1-derived MVs was abrogated compared to target cells cultured with MVs from M2 and M0 macrophages and untreated target cells. As a result of the differential MV-carrying DNMTs and HDACs mRNA transferred to the target cells, we found significant differences in CpG island promoter methylation and resultant gene expression in a signature of EMT genes, including TWIST, WNT5A, VIM, FOXC2, KRT19, STAT3, SNAI1 BMP1, TGFb, DSP, AKT1, NUDT13, and ZEB1. The regulation of EMT and tumor suppressor gene promoter methylation and gene expression in MDA-MB-231 human breast cancer cells, as well as the disparate regulation of methylation and gene expression patterns on these target cells as well endothelial cells (HUVEC) by MVs collected from CD14+/Tie2+ TEMs is ongoing. Our current and ongoing work, we establish that M1 and M2 macrophages, and TEMs, secrete MVs containing distinct epigenetic profiles which are taken-up by target cells to regulate promoter methylation and gene expression of TSGs and genes driving EMT. This program of macrophage function may be important in the progression of solid tumors via inhibition of TSGs and activation of a signature of EMT genes in normal epithelial cells as well as to direct the endothelium to support tumor progression. This abstract is also presented as Poster A31. Citation Format: Duaa Dakhlallah, Ivory Patterson, Amy C. Gross, Randall Evans, Tim D. Eubank. Macrophage phenotype drives tumor program via epigenetic machinery carried in secreted microvesicles. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr PR03. doi:10.1158/1538-7445.CHTME14-PR03</jats:p

Probiotic Lactobacillus reuteri Attenuates the Stressor-Enhanced Severity of Citrobacter rodentium Infection

ABSTRACT Stressor exposure has been shown to enhance host susceptibility and the severity of a plethora of illnesses, including gastrointestinal disease. In mice, susceptibility to Citrobacter rodentium has been shown to be dependent on host genetics as well as the composition of the intestinal microbiota, but the effects of stressor exposure on this gastrointestinal pathogen have not been elucidated fully. Previously, our lab showed that exposure to the prolonged-restraint stressor prior to a challenge with C. rodentium alters the intestinal microbiota community structure, including a reduction of beneficial genera such as Lactobacillus , which may contribute to stressor-enhanced C. rodentium -induced infectious colitis. To test the effects of stressor exposure on C. rodentium infection, we exposed resistant mice to a prolonged-restraint stressor concurrent with pathogen challenge. Exposure to prolonged restraint significantly enhanced C. rodentium -induced infectious colitis in resistant mice, as measured by increases in colonic histopathology, colonic inflammatory mediator gene production, and pathogen translocation from the colon to the spleen. It was further tested if the beneficial bacterium Lactobacillus reuteri could reduce the stressor-enhanced susceptibility to C. rodentium -enhanced infectious colitis. While L. reuteri treatment did not reduce all aspects of stressor-enhanced infectious colitis, it did significantly reduce pathogen translocation from the colon to the spleen. Taken together, these data demonstrate the deleterious effects that prolonged stressor exposure can have at the onset of a gastrointestinal infection by its ability to render a resistant mouse highly susceptible to C. rodentium . Probiotic treatment ameliorated the systemic manifestations of stress on colonic infection. </jats:p

GM-CSF Induces Expression of Soluble VEGF Receptor-1 from Human Monocytes and Inhibits Angiogenesis in Mice

AbstractGM-CSF promotes homeostasis of myeloid cells. We report that GM-CSF upregulates mRNA and protein production of the soluble form of membrane bound VEGF receptor-1 (sVEGFR-1) in human monocytes. This sVEGFR-1 was biologically active, as cell-free supernatants from GM-CSF-stimulated monocytes blocked detection of endogenously expressed VEGF and inhibited endothelial cell migration and tube formation, even in the presence of exogenous rhVEGF. VEGF activity was recovered by neutralizing sVEGFR-1. To determine whether these events were important in vivo, Matrigel plugs were incubated with rhVEGF, rhGM-CSF, or rhGM-CSF/rhVEGF and injected into mice. Plugs containing GM-CSF or GM-CSF/VEGF had less endothelial cell invasion than plugs containing rhVEGF and were similar to plugs incubated with PBS alone. Neutralizing antibodies specific for sVEGFR-1 injected in these plugs reversed the effects of GM-CSF or GM-CSF/VEGF, while an isogenic antibody did not. Thus, GM-CSF and monocytes play a vital role in angiogenesis through the regulation of VEGF and sVEGFR-1