CSF-1 Expression Research Articles

Gata3 regulates the pathogenicity of Tbet-expressing Th17 cells in EAE

Abstract Tbet+RORgt+ Th17 cells are thought to be key players in the pathology of experimental autoimmune encephalomyelitis (EAE) and GM-CSF production has been proposed to be one mechanism through which Th17 are encephalitic. However, the mechanisms through which Csf2 expression is regulated are unclear. Based on prior work highlighting a regulatory role for Gata3 in NKp46+ ILC3s, we hypothesized that Gata3 might play an analogous role in Tbet+ Th17 cells during EAE. Profiling Gata3 expression in MOG-immunized mice revealed that Th17 cells dynamically express Gata3 during their differentiation and maintain low levels thereafter. Therefore to determine how Gata3 might affect the pathogenicity of Tbet+ Th17 cells, we utilized tamoxifen inducible Gata3 knockout (Cre-ERT2 Gata3Fl/Fl) mice as donors in a transfer EAE model. In this model, a post-immunization deletion of Gata3 resulted in a failure to generate EAE symptoms due to a five-fold reduction in GM-CSF production, despite a normal Th17 and Tbet+ Th17 cell responses. To assess how Gata3 might affect the expression of Csf2 in a cell-intrinsic manner, we isolated Cd45.1/Cd45.2 and Cre-ERT2 Gata3Fl/Fl CD4+ effector T cells from the CNS of tamoxifen or vehicle treated co-transfer recipient mice for RNAseq. Interestingly, Gata3-deficient CD4+ T cells displayed stark reductions in Bhlhe40, Egr2, and Csf2 expression in a cell-intrinsic manner, which was confirmed in follow-up experiments. Taken together, our data suggests that Gata3 promotes the pathogenicity of Tbet+ Th17 cells in EAE, possibly via regulation of Bhlhe40 and GM-CSF expression. This research was supported by the Intramural Research Program of NIAID, NIH.

Roles of acyl-CoA synthetase long-chain family member 5 and colony stimulating factor 2 in inhibition of palmitic or stearic acids in lung cancer cell proliferation and metabolism.

Lung cancer is a heterogeneous and complex disease with the highest incidence and mortality rate. The present study aims at defining the lung cancer phenome specificity of lipidomic profiles, screening target lipid-dependent transcriptional alternations, identifying target lipid-associated target genes, and exploring molecular mechanisms. Lung cancer-specific and lung cancer subtype-specific target lipids palmitic acid (C16:0) and stearic acid (C18:0) were found as target lipids by integrating clinical phenomics, lipidomics, and transcriptomics and exhibited antiproliferative effects in sensitive cells. The metabolism-associated gene ACSL5 or inflammation-associated gene CCL3 was identified in lung adenocarcinoma or small lung cancer cells, respectively. C16:0 or C18:0 could upregulate ACSL5 or CSF2 expression in a time- and dose-dependent pattern, and the deletion of both genes led to the insensitivity of cells. Target lipids increased the expression of PDK4 gene in different patterns and inhibited cell proliferation through alterations of intracellular energy. Thus, our data provide a new strategy to investigate the trans-points between clinical and phenomics and lipidomics and target lipid-associated molecular mechanisms to benefit from the discovery of new therapies.

Potent BRD4 inhibitor suppresses cancer cell-macrophage interaction

Small molecule inhibitor of the bromodomain and extraterminal domain (BET) family proteins is a promising option for cancer treatment. However, current BET inhibitors are limited by their potency or oral bioavailability. Here we report the discovery and characterization of NHWD-870, a BET inhibitor that is more potent than three major clinical stage BET inhibitors BMS-986158, OTX-015, and GSK-525762. NHWD-870 causes tumor shrinkage or significantly suppresses tumor growth in nine xenograft or syngeneic models. In addition to its ability to downregulate c-MYC and directly inhibit tumor cell proliferation, NHWD-870 blocks the proliferation of tumor associated macrophages (TAMs) through multiple mechanisms, partly by reducing the expression and secretion of macrophage colony-stimulating factor CSF1 by tumor cells. NHWD-870 inhibits CSF1 expression through suppressing BRD4 and its target HIF1α. Taken together, these results reveal a mechanism by which BRD4 inhibition suppresses tumor growth, and support further development of NHWD-870 to treat solid tumors.

MiR-423-5p may regulate ovarian response to ovulation induction via CSF1

BackgroundWe have previously shown that hsa-miR-423-5p expression in ovarian granulosa cells is decreased in high ovarian response populations. The objective of the present study was to find the target gene and mechanism for miR-423-5p involved in ovarian response regulation.Methods(a) TargetScan was used to predict the target gene of hsa-miR-423-5p. (b) A model for hsa-miR-423-5p overexpression or inhibition was constructed by transfecting KGN cells with lentivirus. CSF1 mRNA and protein expression and luciferase activity were measured. (c) The cell cycles of control and lentivirus treated KGN cells were analyzed. Western blot was used to measure the expression of CDKN1A in KGN cells. (d) The concentration of E2 in KGN cell culture medium were measured.Results(a) TargetScan revealed that the 3′ un-translated region of CSF1 matched 11 bases at the 5′ end of miR-423-5p, making it a likely target gene. (b) Overexpression or inhibition of miR-423-5p were associated with respective decreases or increases in CSF1 expression (both mRNA and protein) (p < 0.05) and luciferase activity (p < 0.05). (c) When miR-423-5p expression increased, the number of G0/G1 phase cells and the expression of CDKN1A protein increased while estradiol concentrations in the cell culture solution decreased (p < 0.05). However, when miR-423-5p expression decreased, the number of S phase cells increased and E2 concentrations increased while the expression of CDKN1A protein decreased (p < 0.05).ConclusionsColony stimulating factor 1 is a target gene of miR-423-5p and that it may regulate ovarian response to ovulation induction by affecting granulosa cells proliferation and estrogen secretion.

PLX3397 treatment inhibits constitutive CSF1R-induced oncogenic ERK signaling, reduces tumor growth, and metastatic burden in osteosarcoma

Osteosarcoma (OSA) is a heterogeneous and aggressive solid tumor of the bone. We recently identified the colony stimulating factor 1 receptor (Csf1r) gene as a novel driver of osteosarcomagenesis in mice using the Sleeping Beauty (SB) transposon mutagenesis system. Here, we report that a CSF1R-CSF1 autocrine/paracrine signaling mechanism is constitutively activated in a subset of human OSA cases and is critical for promoting tumor growth and contributes to metastasis. We examined CSF1R and CSF1 expression in OSAs. We utilized gain-of-function and loss-of-function studies (GOF/LOF) to evaluate properties of cellular transformation, downstream signaling, and mechanisms of CSF1R-CSF1 action. Genetic perturbation of CSF1R in immortalized osteoblasts and human OSA cell lines significantly altered oncogenic properties, which were dependent on the CSF1R-CSF1 autocrine/paracrine signaling. These functional alterations were associated with changes in the known CSF1R downstream ERK effector pathway and mitotic cell cycle arrest. We evaluated the recently FDA-approved CSF1R inhibitor Pexidartinib (PLX3397) in OSA cell lines in vitro and in vivo in cell line and patient-derived xenografts. Pharmacological inhibition of CSF1R signaling recapitulated the in vitro genetic alterations. Moreover, in orthotopic OSA cell line and subcutaneous patient-derived xenograft (PDX)-injected mouse models, PLX3397 treatment significantly inhibited local OSA tumor growth and lessened metastatic burden. In summary, CSF1R is utilized by OSA cells to promote tumorigenesis and may represent a new molecular target for therapy.

Cranial irradiation acutely and persistently impairs injury-induced microglial proliferation

Microglia, the resident immune cells of the central nervous system (CNS), play multiple roles in maintaining CNS homeostasis and mediating tissue repair, including proliferating in response to brain injury and disease. Cranial irradiation (CI), used for the treatment of brain tumors, has a long-lasting anti-proliferative effect on a number of cell types in the brain, including oligodendrocyte progenitor and neural progenitor cells; however, the effect of CI on CNS-resident microglial proliferation is not well characterized. Using a sterile cortical needle stab injury model in mice, we found that the ability of CNS-resident microglia to proliferate in response to injury was impaired by prior CI, in a dose-dependent manner, and was nearly abolished by a 20 ​Gy dose. Similarly, in a metastatic tumor model, prior CI (20 ​Gy) reduced microglial proliferation in response to tumor growth. The effect of irradiation was long-lasting; 20 ​Gy CI 6 months prior to stab injury significantly impaired microglial proliferation. We also investigated how stab and/or irradiation impacted levels of P2Y12R, CD68, CSF1, IL-34 and CSF1R, factors involved in the brain’s normal response to injury. P2Y12R, CD68, CSF1, and IL-34 expression were altered by stab similarly in irradiated mice and controls; however, CSF1R was differentially affected. qRT-PCR and flow cytometry analyses demonstrated that CI reduced overall Csf1r mRNA levels and microglial specific CSF1R protein expression, respectively. Interestingly, Csf1r mRNA levels increased after injury in unirradiated controls; however, Csf1r levels were persistently decreased in irradiated mice, and did not increase in response to stab. Together, our data demonstrate that CI leads to a significant and lasting impairment of microglial proliferation, possibly through a CSF1R-mediated mechanism.

Microglial Homeostasis Requires Balanced CSF-1/CSF-2 Receptor Signaling

CSF-1R haploinsufficiency causes adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP). Previous studies in the Csf1r+/- mouse model of ALSP hypothesized a central role ofelevated cerebral Csf2 expression. Here, we showthat monoallelic deletion of Csf2 rescues most behavioral deficits and histopathological changes in Csf1r+/- mice by preventing microgliosis and eliminating most microglial transcriptomic alterations, including those indicative of oxidative stress and demyelination. We also show elevation of Csf2 transcripts and of several CSF-2 downstream targets in the brains of ALSP patients, demonstrating that the mechanisms identified in the mouse model are functional in humans. Our data provide insights into the mechanisms underlying ALSP. Because increased CSF2 levels and decreased microglial Csf1r expression have also been reported in Alzheimer's disease and multiple sclerosis, we suggest that the unbalanced CSF-1R/CSF-2 signaling we describe in the present study may contribute to the pathogenesis of other neurodegenerative conditions.

BHLHE40 Promotes TH2 Cell-Mediated Antihelminth Immunity and Reveals Cooperative CSF2RB Family Cytokines.

The transcription factor BHLHE40 is an emerging regulator of the immune system. Recent studies suggest that BHLHE40 regulates type 2 immunity, but this has not been demonstrated in vivo. We found that BHLHE40 is required in T cells for a protective TH2 cell response in mice infected with the helminth Heligmosomoides polygyrus bakeri H. polygyrus elicited changes in gene and cytokine expression by lamina propria CD4+ T cells, many of which were BHLHE40 dependent, including production of the common β (CSF2RB) chain family cytokines GM-CSF and IL-5. In contrast to deficiency in GM-CSF or IL-5 alone, loss of both GM-CSF and IL-5 signaling impaired protection against H. polygyrus Overall, we show that BHLHE40 regulates the TH2 cell transcriptional program during helminth infection to support normal expression of Csf2, Il5, and other genes required for protection and reveal unexpected redundancy of common β chain-dependent cytokines previously thought to possess substantially divergent functions.

Targeting IL-17A/glucocorticoid synergy to CSF3 expression in neutrophilic airway diseases.

IL-17A plays a critical role in the pathogenesis of steroid-resistant neutrophilic airway inflammation, which is a hallmark of severe asthma and chronic obstructive pulmonary disease (COPD). Through RNA sequencing analysis of transcriptomes of human airway smooth muscle cells treated with IL-17A, dexamethasone (DEX, a synthetic glucocorticoid drug), alone or in combination, we identified a group of genes that are synergistically induced by IL-17A and DEX, including the neutrophil-promoting cytokine CSF3. In type-17 (Th17/IL-17Ahi) preclinical models of neutrophilic severe asthma (acute and chronic) and COPD, although DEX treatment was able to reduce the expression of neutrophil-mobilizing CXCL1 and CXCL2 in lung tissue, CSF3 expression was upregulated by DEX treatment. We found that DEX treatment alone failed to alleviate neutrophilic airway inflammation and pathology, and even exacerbated the disease phenotype when CSF3 was highly induced. Disruption of the IL-17A/DEX synergy by IL-17A inhibition with anti-IL-17A mAb or cyanidin-3-glucoside (C3G, a small-molecule IL-17A blocker) or depletion of CSF3 effectively rendered DEX sensitivity in type-17 preclinical models of neutrophilic airway diseases. Our study elucidates what we believe is a novel mechanism of steroid resistance in type-17 neutrophilic airway inflammation and offers an effective steroid-sparing therapeutic strategy (combined low-dose DEX and C3G) for treating neutrophilic airway diseases.

Association of CXCR2+ granulocytic myeloid derived suppressor cells with the development of cholangiocarcinoma: A possible target for intervention.

565 Background: Cholangiocarcinoma (CCA) is the second most common primary liver malignancy, with increasing incidence. Currently, surgical resection offers the only chance for cure, however the prognosis remains poor in part due to high rates of unresectability, recurrence, and poor response to conventional therapy. Thus, new systemic therapies represent an unmet medical need. Few preclinical models exist for identifying and testing new targeted or immune based therapies. Here we present our findings of the immune infiltrate in human CCA tumor microenvironment (TME) and a spontaneous murine model that faithfully recapitulates human disease. Methods: Histology and immunohistochemistry (IHC) staining was performed on human CCA and adjacent normal liver. Mice with targeted hepatic Kras activation and loss of p53 (KPPC) spontaneously develop CCA. KPPC hepatic tumors and normal livers from littermate controls underwent histological and gene expression studies. Flow cytometric analysis was performed on bone marrow, spleen, peripheral blood, CCA tumors and normal littermate livers. Results: Digital IHC quantification of archival human CCA specimens demonstrated elevated levels of CD15+CXCR2+ granulocytic myeloid derived suppressor cells (G-MDSC) compared to adjacent normal liver (p = &lt; 0.05). In addition, the CXCR2 ligand, CXCL5, was significantly elevated in CCA tumors compared to adjacent normal liver. In KPPC mice, flow cytometric analysis of hepatic tumors showed an abundance of CD45+ leukocytes comprised of immunosuppressive G-MDSC vs normal littermate controls (p = 0.0007) which recapitulates human disease. qRT-PCR demonstrated significantly increased expression of G-csf, Csf1, Cxcl1, Cxcl2, and Cxcl5 (p = &lt; 0.0001) in CCA KPPC tumors compared to normal livers. Accordingly, granulocytes in KPPC mice were elevated in both the bone marrow and blood compared to normal littermate controls. Conclusions: These data suggest CCA co-opts the ELR+ cytokine/CXCR2 axes to mobilize and recruit immunosuppressive G-MDSC to the TME. Targeted therapy against tumor infiltrating neutrophils can be tested in this pre-clinical model to inform clinical translation.

Expression of colony-stimulating factor 1 and interleukin-34 in gingival tissue and gingival fibroblasts from periodontitis patients and controls.

Colony-stimulating factor 1 (CSF-1) and interleukin (IL)-34 are important for the functions of myeloid lineage cells and are involved in several chronic inflammatory conditions associated with tissue degeneration. The aim of this study is to evaluate the expression of CSF-1 and IL-34 in gingival tissue and gingival fibroblasts (GF) from patients with periodontitis and controls. Gingival biopsies were obtained from 19 periodontitis patients and 15 controls. Expression of CSF-1 and IL-34 in gingival tissue was assessed by western blot and localization by immunohistochemistry. Expression of CSF1 and IL34 mRNA in GF was analyzed by real-time polymerase chain reaction and protein expression visualized by immunofluorescence stainings. CSF-1 and IL-34 secretion from GF was evaluated in response to tumor necrosis factor-alpha (TNF-α), IL-1β, Escherichia coli lipopolysaccharide (Ec-LPS) and Porphyromonas gingivalis lipopolysaccharide (Pg-LPS) stimulation, using enzyme-linked immunosorbent assays. CSF-1 was increased in gingival tissue from periodontitis patients compared with controls (P<0.05) whereas IL-34 expression was similar. In GF from a non-periodontitis donor, stimulation with either TNF-α, IL-1β, Ec-LPS, or Pg-LPS, increased the secretion of CSF-1 (P<0.05) and Ec-LPS stimulation increased IL-34 (P<0.05). CSF-1 and IL-34 were expressed and secreted constitutively from GF, with comparable levels in GF from periodontitis patients and controls. Inflammatory stimuli increased the secretion of CSF-1 and IL-34 with comparable levels measured from GF from periodontitis patients and controls (P<0.05). The expression of CSF-1 and IL-34 in gingival tissue and fibroblasts suggests involvement in myeloid cell functions during periodontal inflammation.

Characterization of a p53/miR-34a/CSF1R/STAT3 Feedback Loop in Colorectal Cancer.

Background & AimsThe miR-34a gene is a direct target of p53 and is commonly silenced in colorectal cancer (CRC). Here we identified the receptor tyrosine kinase CSF1R as a direct miR-34a target and characterized CSF1R as an effector of p53/miR-34a-mediated CRC suppression.MethodsAnalyses of TCGA-COAD and three other CRC cohorts for association of mRNA expression and signatures with patient survival and molecular subtypes. Bioinformatics identification and experimental validation of miRNA and transcription factor targets. Functional analysis of factors/pathways in the regulation of epithelial-mesenchymal transition (EMT), invasion, migration, acquired chemo-resistance and metastasis. Analyses of protein expression and CpG methylation within primary human colon cancer samples.ResultsIn primary CRCs increased CSF1R, CSF1 and IL34 expression was associated with poor patient survival and a mesenchymal-like subtype. CSF1R displayed an inverse correlation with miR-34a expression. This was explained by direct inhibition of CSF1R by miR-34a. Furthermore, p53 repressed CSF1R via inducing miR-34a, whereas SNAIL induced CSF1R both directly and indirectly via repressing miR-34a in a coherent feed-forward loop. Activation of CSF1R induced EMT, migration, invasion and metastasis of CRC cells via STAT3-mediated down-regulation of miR-34a. 5-FU resistance of CRC cells was mediated by CpG-methylation of miR-34a and the resulting elevated expression of CSF1R. In primary CRCs elevated expression of CSF1R was detected at the tumor invasion front and was associated with CpG methylation of the miR-34a promoter as well as distant metastasis.ConclusionsThe reciprocal inhibition between miR-34a and CSF1R and its loss in tumor cells may be relevant for therapeutic and prognostic approaches towards CRC management.

Identification of miR-210-5p in human placentae from pregnancies complicated by preeclampsia and intrauterine growth restriction, and its potential role in the pregnancy complications.

Preeclampsia (PE) and intrauterine growth restriction (IUGR) are pregnancy complications resulting from abnormal placental development. As epigenetic regulators, microRNAs can regulate placental development and contribute to the disease pathophysiology by influencing the expression of genes involved in placental development or disease. Our previous study revealed an increase in miR-210-5p expression in placentae from patients with early-onset pregnancy complications and identified candidate gene targets for miR-210-5p. The purpose of this study was to: (i) validate candidate gene targets predicted for miR-210-5p from microRNA-RNA expression data, and (ii) overexpress miR-210-5p in a trophoblast cell line (HTR-8/SVneo) to assess impact on trophoblast cell functions. Integration of the miRNA and RNA sequencing expression data revealed 8 candidate gene targets for miR-210-5p in patients with PE only or PE+IUGR. Luciferase reporter assays identified two gene targets for miR-210-5p, CSF1 and ITGAM. Real-time PCR confirmed the decreased expression of CSF1 and ITGAM in patients with PE+IUGR. Immunohistochemistry of placentae from late second trimester identified CSF1 and ITGAM in intermediate trophoblast cells in the decidua. Expression levels of CSF1 and ITGAM were reduced in HTR-8/SVneo cells following increased miR-210-5p expression. Concomitantly, HTR-8/SVneo cells demonstrate an average 45% reduction in cell migration. These findings suggest that miR-210-5p may contribute to dysfunction of intermediate trophoblasts and potentially contribute to the disease process of these pregnancy complications.

1,3-Dicaffeoylquinic acid targeting 14-3-3 tau suppresses human breast cancer cell proliferation and metastasis through IL6/JAK2/PI3K pathway

14-3-3τ plays a critical role in tumorigenesis and metastasis of breast cancer and can be used as new drug target protein. Dicaffeoylquinic acids (DCQAs), natural products, have antioxidant, antimicrobial, and anti-inflammatory activities. In this study, the anticancer effects of DCQAs on breast cancer cells MCF-7, MDA-MB-231 cell lines and mechanism in triple negative breast cancer (TNBC) were investigated. First, we screened for DCQAs that could bind to 14-3-3τ and had a significant inhibitory effect on breast cancer cells. MTT, colony formation, transwell migration, and flow cytometric assays revealed that 1,3-DCQA was the best one of 14-3-3τ binding protein from DCQAs against breast cancer cell proliferation and metastasis but safe for normal cells. Through molecular docking simulation, overexpression and knockdown assays, we confirmed that 14-3-3τ was one of 1,3-DCQA target protein. Eukaryotic transcriptome sequencing and western blot analysis demonstrated that 1,3-DCQA binds to 14-3-3τ to prevent breast cancer proliferation and metastasis via Jak/PI3K/Akt and Raf/ERK pathway, which promote IL6 and CSF3 expression raised by CREB (CREBBP, CREB5) and induced cell apoptosis via Bad/Bax/caspase 9 signaling pathway. Our results provided evidence that 1,3-DCQA can be used as a novel lead compound against breast cancer by inhibition of 14-3-3 protein.

Spatial Mapping of Myelopoiesis Reveals the Bone Marrow Niche for Monocyte Dendritic Cell Progenitors

Monocytes, macrophages and dendritic cells are indispensable for innate immunity. Myelopoiesis takes place in the bone marrow (BM) through differentiation of a common myeloid progenitor into monocyte dendritic cell progenitors (MDP) or granulocyte monocyte progenitors (GMP). MDP differentiate into myeloid progenitors (MoP) or common dendritic progenitors (cDP) that give rise to monocytes or dendritic cells (DC) repectively. GMP can also generate neutrophil-like monocytes via an intermediate monocyte progenitor [MP; Immunity. 2017 47(5):890] and neutrophils via a granulocyte progenitor (GP). CSF1 (M-CSF) is the key cytokine that regulates monopoiesis, CSF1 loss causes profound defects in monocyte, dendritic cell, macrophage and osteoclast generation. Classical studies using Csf1-/- chimeric mice demonstrated that CSF1 is produced by BM stromal cells. However the identity of these CSF1-producing cells is unknown. A major limitation is the lack of immunofluorescence protocols to map progenitor interaction with candidate niches. We have found that CD11b-Ly6C-CD117+CD115+ cells are MDP; Lin-Ly6C-CD117+CD16/32+CD115- cells are GMP; CD11b-Ly6C+CD117+CD115+ are MP/MoP; CD11b-Ly6C+CD117+CD115- are GP; CD11b+CD115+Ly6Chi and CD11b+CD115+Ly6Clo cells are classical and non-classical monocytes; and MHCIIhi reticular cells are DC. We used the markers above to map the 3D position of every myeloid cell in the sternum and assessed the relationships between myeloid progenitors, their offspring and candidate niches in situ with single cell resolution. To test whether the interactions observed were specific we obtained the X, Y and Z coordinates for every hematopoietic cell in the sternum (detected using αCD45 and αTer119). We then used these coordinates to randomly place each type of myeloid cell, at the same frequencies found in vivo, through the BM to generate a random distribution for each myeloid cell type. HSC localize to sinusoidal, arteriolar and endosteal niches. However, myeloid progenitors are exclusively perisinusoidal (mean MDP distance to sinusoids, arterioles, and endosteum observed 5, 134, and 105μm vs 9, 86, and 69µm in the random simulation). Myeloid progenitors rarely localize with HSC indicating that progenitors abandon the HSC niche upon differentiation. Strikingly, we found that granulopoiesis, monopoiesis and DCpoiesis occur in distinct sinusoidal locations and that MDP are tightly associated with sinusoids, dendritic cells and Ly6Clo monocytes (2.0 DC and 4.4 Ly6Clo monocytes observed within 50µm of an MDP vs 0.9 DC and 1.8 Ly6Clo monocytes in the random simulation p=0.02 and p&amp;lt;0.0001) but not with MoP/MP or Ly6Chi classical monocytes. The results above suggest that the stromal cells that provide the signals that regulate MDP will localize to the sinusoids. Analyses of Csf1 expression in two recently published scRNAseq studies of BM stroma showed that perivascular stromal cells and osteoblastic cells are the major CSF1 sources with sinusoidal endothelial cells expressing much lower levels. Csf1 deletion in perivascular cells using LepR-cre mice and in osteoblastic cells using Ocn-cre mice did not impact Ly6Chi classical or Ly6Clo non-classical monocytes in peripheral blood. We also did not find any defects in BM MDP, GMP, MoP numbers or colony forming activity or in monocyte or dendritic cell numbers. In sharp contrast we found that conditional Csf1 deletion in endothelial cells using Cdh5-cre mice led to a 3.9-fold defect in Ly6Clo non-classical monocytes in the blood (1.89 vs 0.47 x105/ml in the +/- controls vs Cdh5-cre:Csf1-/Δ mice; p=0.03). In the BM these mice showed a 1.4 reduction in MDP numbers (0.72 vs 0.5x104/femur; p=0.04) further compounded by a 2.7-fold loss in MDP-derived CFU-M (22 vs 8 colonies/100 cells; p=0.009) indicating a dramatic reduction in MDP function. This in turn led to a 2.3-fold reduction in Ly6Clo non classical monocytes (9.5 vs 4.1x104/femur; p=0.01) and a 1.2-fold reduction in cDC (2.7 vs 2.1 x104cDC/femur p=0.005). In summary we have imaged for the first time myeloid progenitors; mapped their differentiation into mature myeloid cells; quantified their interaction with candidate niche cells; showed that sinusoids are the exclusive site of monocyte and dendritic cell production; and demonstrated that endothelial cells are a niche that regulates MDP numbers and function via CSF1. Disclosures No relevant conflicts of interest to declare.

SCIDOT-26. THE ROLE OF TUMOR-DERIVED GRANULOCYTE MACROPHAGE COLONY STIMULATING FACTOR (GM-CSF/CSF2) IN REGULATION OF MICROGLIA-DEPENDENT INVASION IN GLIOMAS

Abstract Brain resident immune cells (microglia) and peripheral macrophages accumulate in malignant gliomas and constitute for 30–50% of the tumor mass. These immune cells are polarized by factors released by glioma and become the pro-invasive, immunosuppressive cells that support tumor progression. We have previously found that tumor-derived granulocyte macrophage colony stimulating factor (GM-CSF/Csf-2) is a crucial factor controlling accumulation of microglia and macrophages in murine gliomas. The analysis of TCGA dataset revealed overexpression of the CSF2 gene (encoding GM-CSF) in a set of mesenchymal glioblastomas (most aggressive WHO grade IV gliomas) and its association with high immune gene expression. To study the role of GM-CSF in microglia-stimulated glioma invasion, we used a co-culture system, which mimics microglia interactions with tumor cells. We silenced the expression of CSF2 in glioma cells and found reduced microglia-dependent invasion of glioma cells. To translate those results into clinically relevant setting, we designed and tested humanized short peptides interfering with binding of GM-CSF to its receptor. Selected peptide effectively inhibited binding of GM-CSF to its receptor as demonstrated with different methods. We selected the non-cytotoxic peptides that potently blocked microglia-dependent glioma invasion in cell co-cultures. Blocking GM-CSF-receptor signaling pathway with a neutralizing antibody against a GM-CSF receptor also inhibited microglia-dependent invasion of glioma cells. Altogether, our results demonstrate that glioma-derived GM-CSF supports pro-tumorigenic polarization of microglia turning them into cells that facilitate glioma growth and shape the immune microenvironment. The study was supported by grant 2014/15/B/NZ3/04704 from The National Science Centre, Poland.

TMIC-64. THE ROLE OF TUMOR-DERIVED GRANULOCYTE MACROPHAGE COLONY STIMULATING FACTOR (GM-CSF/CSF2) IN REGULATION OF MICROGLIA-DEPENDENT INVASION IN GLIOMAS

Abstract Brain resident immune cells (microglia) and peripheral macrophages accumulate in malignant gliomas and constitute for 30–50% of the tumor mass. These immune cells are polarized by factors released by glioma and become the pro-invasive, immunosuppressive cells that support tumor progression. We have previously found that tumor-derived granulocyte macrophage colony stimulating factor (GM-CSF/CSF2) is a crucial factor controlling accumulation of microglia and macrophages in murine gliomas. The analysis of TCGA dataset revealed overexpression of the CSF2 gene (encoding GM-CSF) in a set of mesenchymal glioblastomas and its association with high immune gene expression. To study the role of GM-CSF in microglia-stimulated glioma invasion, we used a co-culture system, which mimics microglia interactions with tumor cells. We silenced the expression of CSF2 in glioma cells and found reduced microglia-dependent invasion of glioma cells. To translate those results into clinically relevant setting, we designed and tested humanized short peptides interfering with binding of GM-CSF to its receptor. Selected peptide effectively inhibited binding of GM-CSF to its receptor as demonstrated with different methods. We selected the non-cytotoxic peptides that potently blocked microglia-dependent glioma invasion in cell co-cultures. Blocking GM-CSF-receptor signaling pathway with a neutralizing antibody against a GM-CSF receptor also inhibited microglia-dependent invasion of glioma cells. Altogether, our results demonstrate that glioma-derived GM-CSF supports pro-tumorigenic polarization of microglia turning them into cells that facilitate glioma growth and shape the immune microenvironment. This work was supported by statutory budget of Nencki Institute of Experimental Biology and grant PBS3/B7/19/2015 from The National Centre for Research and Development, Poland.

CSF1-associated decrease in endometrial macrophages may contribute to Asherman's syndrome.

Asherman's syndrome (AS) is characterized by endometrial fibrosis leading to intrauterine adhesions and symptoms like hypomenorrhea, infertility, and recurrent pregnancy loss. Macrophages are key regulators of inflammation, tissue repair, regeneration, and fibrosis. However, the role of macrophages in AS remains unclear. Endometrial biopsies of AS patients and controls were collected during the late proliferating phase of menstrual cycle. Fibrosis and proliferation markers were detected by Masson's trichrome staining and immunohistochemistry. Macrophages were examined by immunostaining and flow cytometry. The expression levels of CCL2, CSF1, CSF1R, and GM-CSF were detected by quantitative real-time polymerase chain reaction (q-PCR) and immunohistochemistry. A well-differentiated endometrial cell line Ishikawa (IK) was used for in vitro studies. Macrophages differentiating from THP-1 monocytic cells were polarized by IL-4/IL-13. Their culture supernatants (M(IL-4/13)-S) were applied to H2 O2 or bleomycin-damaged IK cells. In AS patients, endometrial stroma was replaced by fibrous tissue and cell proliferation was reduced. Macrophages in endometrial tissue were mainly alternative activated macrophages and their number was significantly decreased in AS patients. The CSF1 expression level was reduced in AS patients. M(IL-4/13)-S promoted the growth and migration of IK cells and inhibited H2 O2 -induced apoptosis. M(IL-4/13)-S protected IK cells from bleomycin-induced fibrosis. Macrophages are critical cells involved in the process of endometrial repair and fibrosis. The decreased amount of endometrial macrophages may be attributed to the reduced expression level of CSF1. Manipulation of macrophage activation/function may provide a novel therapeutic target for AS.

MiR-532-5p acts as a tumor suppressor and inhibits glioma cell proliferation by targeting CSF1.

Recent studies have discovered a class of micro-RNAs (miRNAs), which are dysregulated in various tumors and associated with carcinogenesis. In our research, we aim to uncover the molecular functions of miR-532-5p in glioma development. Real Time-quantitative Polymerase Chain Reaction (RT-qPCR) was performed to detect miR-532-5p expression in 48 glioma samples and 4 glioma cell lines. The Pearson's Chi-square test was used to determine the association of miR-532-5p expression with several clinicopathological indexes in glioma patients. Besides, cell proliferation assay, colony formation assay, and Ethynyl deoxyuridine (EdU) incorporation assay were performed to explore in vitro effects of miR-532-5p on glioma cells. Furthermore, the interaction between miR-532-5p and CSF1 in glioma was studied by performing Western blot assay and Dual-Luciferase Reporter Gene Assay. Downregulated miR-532-5p expression was observed in glioma tissues compared with adjacent normal samples. MiR-532-5p expression was associated with the KPS score and tumor grading in glioma patients. Moreover, cell proliferation of glioma was inhibited after overexpression of miR-532-5p in vitro. Furthermore, CSF1 was a target of miR-532-5p in glioma. After overexpression of miR-532-5p, CSF1 was downregulated at mRNA and protein levels in vitro Besides, the expression of CSF1 in glioma tissues was negatively related to that of miR-532-5p. Malignant phenotypes of glioma cells were remarkably suppressed through the overexpression of miR-532-5p. MiR-532-5p/CSF1 axis was identified as a new therapeutic intervention for the treatment of glioma.

Circular RNA circ_0067934 functions as an oncogene in glioma by targeting CSF1.

The importance of circular RNAs in malignant tumors increases the attention of researchers. The role of circ_0067934 in glioma remains unclear. Our study aims to uncover how circ_0067934 functions in glioma development. Real Time-quantitative Polymerase Chain Reaction (RT-qPCR) was utilized to determine the level of circ_0067934 in glioma tissues. Circ_0067934 was knocked down in glioma cells. Cell migrated and invaded ability was detected through functional assay in vitro and in vivo. Further mechanism assays were performed to explore the potential targets of circ_0067934. The circ_0067934 was highly expressed in glioma tissues compared with adjacent samples. The expression of circ_0067934 was upregulated in glioma cell lines. The cell migrated and invaded ability of glioma cells was inhibited after circ_0067934 was knocked down. Besides, CSF1 expression was decreased via knockdown of circ_0067934. Furthermore, tumor metastasis was inhibited after circ_0067934 was knocked down in nude mice. The circ_0067934 could suppress cell migration and invasion of glioma by upregulating CSF1.