Expression Of LIF Research Articles

Administration of dexamethasone disrupts endometrial receptivity by alteration of expression of miRNA 223, 200a, LIF, Muc1, SGK1, and ENaC via the ERK1/2-mTOR pathway.

Successful implantation of embryos requires endometrial receptivity. Glucocorticoids are one of the factors influencing the implantation window. In this study, 40 female BALB/c mice were used to study the impacts of dexamethasone administration on endometrial receptivity markers during implantation window. The mice mated and were randomly divided into four groups: control (vehicle), dexamethasone (100 μg/kg, IP), PP242 (30 mg/kg, IP), and dexamethasone + PP242 (Dex + PP242). On the Day 4th and 5th of gestation, mice received their respective treatments and were killed on the 5th day. To assess the expression of Muc1, leukemia inflammatory inhibitor (LIF), serum/glucocorticoid-inducible kinase 1 (SGK1), epithelial Na+ channel (ENaC), miRNA 200a, and miRNA 223-3p in the endometrium real-time polymerase chain reaction was performed. Furthermore, using Western blot analysis protein expressions of extracellular signal-regulated kinase 1/2 (ERK1/2), mammalian target of rapamycin (mTOR), and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) were evaluated. Periodic Acid-Schiff staining was used to examine the histomorphological changes of the uterus. According to the results dexamethasone declined the expression of LIF, whereas upregulated expression of Muc1, SGK1, ENaC mRNA, miRNA 200a, and miRNA 223-3p in the endometrium. In addition, PP242, an mTOR inhibitor, induced mRNA expression of Muc1, miRNA200a, and miRNa223-3p whereas it declined the expression of LIF. Moreover, activity of the ERK1/2-mTOR pathway in the endometrial cells was deterred by dexamethasone and PP242. Nonstop epithelium proliferation and elevated surface glycoproteins layer on epithelium of dexamethasone and/or PP242-received groups were divulged through histochemical analysis. According to the above mentioned results, uterine receptivity during implantation period was declined by dexamethasone, at least in part, through modulation of involved genes in endometrial receptivity and inhibition of the ERK1/2-mTOR pathway.

Immunophenotype and Immune-Modulatory Activities of Human Fetal Cartilage-Derived Progenitor Cells

We have previously reported human fetal cartilage progenitor cells (hFCPCs) as a novel source of therapeutic cells showing high proliferation and stem cell properties superior to those of adult mesenchymal stem cells (MSCs). In this study, we investigated the immunophenotype and immune-modulatory activities of hFCPCs. With institutional review board approval, hFCPCs were isolated from fetuses at 11–13 weeks of gestation. hFCPCs showed strong expression of HLA class I molecules but low or no expression of HLA class II and co-stimulatory molecules, which was not changed significantly after 4 days of IFN-γ treatment. In a mixed lymphocyte reaction (MLR), hFCPCs showed no allogeneic immune response to peripheral blood lymphocytes (PBLs) and suppressed concanavalin A (Con A)-mediated proliferation of PBLs in a dose-dependent manner. In addition, hFCPCs inhibited Con A-induced secretion of pro-inflammatory cytokines TNF-α and IFN-γ from PBLs but showed no significant decrease of secretion of IL-10, anti-inflammatory cytokine. Co-culture of hFCPCs with stimulated PBLs for 4 days resulted in a significant increase in CD4+CD25+FoxP3+ T regulatory cells (Tregs). hFCPCs expressed LIF, TGF-β1, TSG-6, and sHLA-G5 but did not express IDO and HGF. Stimulation of hFCPCs with TNF-α for 12 h showed slight induction in the expression of LIF, TSG-6, IDO, and HGF, whereas stimulation with IFN-γ did not affect expression of any of these factors. These results suggest that hFCPCs have low allogeneic immunogenicity and immune-modulatory activity in vitro, comparable to those of MSCs. However, compared with MSCs, hFCPCs were less responsive to TNF-α and IFN-γ, and the mechanisms underlying responses to these two cell types appeared distinct.

IL1-Induced JAK/STAT Signaling Is Antagonized by TGFβ to Shape CAF Heterogeneity in Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is poorly responsive to therapies and histologically contains a paucity of neoplastic cells embedded within a dense desmoplastic stroma. Within the stroma, cancer-associated fibroblasts (CAF) secrete tropic factors and extracellular matrix components, and have been implicated in PDAC progression and chemotherapy resistance. We recently identified two distinct CAF subtypes characterized by either myofibroblastic or inflammatory phenotypes; however, the mechanisms underlying their diversity and their roles in PDAC remain unknown. Here, we use organoid and mouse models to identify TGFβ and IL1 as tumor-secreted ligands that promote CAF heterogeneity. We show that IL1 induces LIF expression and downstream JAK/STAT activation to generate inflammatory CAFs and demonstrate that TGFβ antagonizes this process by downregulating IL1R1 expression and promoting differentiation into myofibroblasts. Our results provide a mechanism through which distinct fibroblast niches are established in the PDAC microenvironment and illuminate strategies to selectively target CAFs that support tumor growth. SIGNIFICANCE: Understanding the mechanisms that determine CAF heterogeneity in PDAC is a prerequisite for the rational development of approaches that selectively target tumor-promoting CAFs. Here, we identify an IL1-induced signaling cascade that leads to JAK/STAT activation and promotes an inflammatory CAF state, suggesting multiple strategies to target these cells in vivo. See related commentary by Ling and Chiao, p. 173. This article is highlighted in the In This Issue feature, p. 151.

Evaluation of Diabetes Effects on the Expression of Leukemia Inhibitory Factor and Vascular Endothelial Growth Factor A Genes and Proteins at the Time of Endometrial Receptivity after Superovulation in Rat Model.

Background:Diabetes, a major metabolic disorder, seems to affect the fertility rates of women in various ways. Due to the uncertainty of the effects of diabetes along with superovulation treatment on the infertility, we investigate the effects of ovulation induction treatment as therapeutic approach on the expression of leukemia inhibitory factor (LIF) and vascular endothelial growth factor A (VEGFA) as two main factors which are involved in the implantation in the streptozotocin (STZ)-induced type 1 diabetic rats.Materials and Methods:Type 1 diabetes was induced by injections of STZ in Wistar rats. The animals were kept in diabetic conditions for 4 weeks, while some were treated with insulin for treatment. After treatment, the ovulation was induced by human menopausal gonadotropin (hMG) and human chorionic gonadotropin (hCG). The rats were then sacrificed and the expression of LIF and VEGFA was checked by immunohistochemistry staining method, and the relative expression of LIF and VEGFA was measured by quantitative reverse transcription polymerase chain reaction (RT-PCR) and Western blotting methods.Results:It was observed that diabetes and insulin treatment for diabetes altered the expression of Lif and VEGFA in both mRNA and protein levels. However, superovulation treatment seems to ameliorate this alternation for both factors.Conclusion:According to our results, diabetes and insulin therapy could alter the expression of Lif and VEGFA genes and proteins that are effective in endometrial receptivity and implantation process. It seems in diabetic cases, the effect of hCG and hMG therapy by itself could regulate the level of expression and presence of these two genes and proteins.

MicroRNA-146a Promotes Growth of Acute Leukemia Cells by Downregulating Ciliary Neurotrophic Factor Receptor and Activating JAK2/STAT3 Signaling.

PurposeAcute leukemia (AL) is classified as acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). This study aimed to investigate the effect of miR-146a on childhood AL and its underlying molecular mechanisms.Materials and MethodsBone marrow samples were obtained from 39 AL children and 10 non-cancer controls. The expressions of miR-146a and ciliary neurotrophic factor receptor (CNTFR) were detected by quantitative real-time polymerase chain reaction (qRT-PCR) in ALL and AML pediatric patients, as well as ALL (Jurkat) and AML (HL-60) cells. Correlations between miR-146a and clinical indicators were explored. A targeting relationship between miR-146a and CNTFR was detected by dual luciferase reporter gene assay. Cell proliferation, apoptosis, migration, and invasion of Jurkat and HL-60 cells were measured by MTT assay, flow cytometry, and transwell assay, respectively. LIF expression was detected by qRT-PCR in Jurkat and HL-60 cells. The expression of p-JAK2, JAK2, p-STAT3, and STAT3 in HL-60 cells was measured by Western blot.ResultsmiR-146a was increased in ALL and AML pediatric patients, while CNTFR was decreased. miR-146a expression was associated with immunophenotype, karyotype, fusion gene, and SIL-TAL1. CNTFR was a target gene of miR-146a. miR-146a could promote cell proliferation, migration, and invasion, as well as inhibit cell apoptosis in Jurkat and HL-60 cells by downregulating CNTFR. Meanwhile, miR-146a inhibited the expression of LIF and activated JAK2/STAT3 pathway by downregulating CNTFR.ConclusionmiR-146a could promote the proliferation, migration, and invasion and inhibit the apoptosis of AL Jurkat and HL-60 cells by downregulating CNTFR and activating the JAK2/STAT3 pathway.

Heterogeneity of Mesenchymal Stromal Cells in Myelodysplastic Syndrome-with Multilineage Dysplasia (MDS-MLD).

Bone marrow niche constituents have been implicated in the genesis of clonal hematopoietic dysfunction in myelodysplastic syndromes (MDS), though the exact role of stroma in the pathogenesis of MDS remains to be defined. We have evaluated the characteristics of mesenchymal stromal cells in a cohort of patients with MDS with multilineage dysplasia (MDS-MLD). MSCs were cultured from bone marrow aspirates of MDS-MLD patients and controls with healthy bone marrow. Phenotypic characterization, cell cycle, and apoptosis were analyzed by flow cytometry. Targeted gene expression analysis was done using a reverse-transcription polymerase chain reaction (Q-PCR). MSCs derived from MDS patients (MDS-MSCs) showed normal morphology, phenotype, karyotype and differentiation potential towards adipogenic and osteogenic lineages. However, these MDS-MSCs showed significantly altered cell cycle status and displayed a shift towards increased apoptosis compared to control MSCs (C-MSCs). The gene expression profile of niche responsive/regulatory cytokines showed a trend towards lower expression VEGF, SCF, and ANGPT with no changes in expression of CXCL12A and LIF compared to C-MSCs. The expression levels of Notch signaling components like Notch ligands (JAGGED-1 and DELTA-LIKE-1), receptors (NOTCH1, NOTCH3) and downstream gene (HES1) showed an aberrant expression pattern in MDS-MSCs compared to C-MSCs. Similarly, Q-PCR analysis of Wnt signaling inhibitory ligands (DKK-1 and DKK-2) in MDS-MSCs showed a three-fold increase in mRNA expression of DKK1 and a two-fold increase in DKK2 compared to C-MSCs. These data suggested that MDS-MSCs have an altered proliferation characteristic as well as a dysregulated cytokine secretion and signaling profile. These changes could contribute to the pathogenesis of MDS.

150 Effect of bovine seminal plasma and sperm on endometrial gene expression

In cattle, most pregnancy losses are sustained before implantation. Many factors are involved in implantation failure, but in mice, pigs and humans there is increased evidence of a role for the maternal immune system and its regulation by seminal plasma (SP). However, there is little evidence for a role of SP in bovine fertility, where dilution or removal of SP before AI is routine. Therefore, the aim of this work was to determine the effect of bull SP or sperm on endometrial gene expression. To this end, 6 heifers were oestrous synchronised and slaughtered 12h after the onset of oestrus. Five endometrial explants from the horn ipsilateral to the preovulatory follicle were obtained from each animal. Explants were incubated with (1) RPMI medium (control); (2) epididymal sperm (106 epididymal sperm mL−1); (3) complete ejaculate (106 ejaculated sperm mL−1+25% SP); (4) ejaculated sperm alone (106 ejaculated sperm mL−1); and (5) SP alone (25% SP). Epididymal sperm were collected and pooled from the cauda epididymis of 3 bulls slaughtered in a commercial abattoir. In addition, complete ejaculates were obtained from 3 other bulls using an artificial vagina. After pooling the samples, a small volume was washed through a density gradient to obtain the ejaculated sperm, and the rest of the ejaculate was filtered to obtain sperm-free SP. The RPMI media was used to dilute sperm and SP to the working concentrations. After 6h of incubation, explants were snap frozen. The RNA quality was assessed with an Agilent 2100 bioanalyzer (Agilent, Santa Clara, CA, USA) before RT-qPCR analysis. Interestingly, SP had a dramatic effect on endometrium RNA integrity, as evidenced by a lower RNA integrity number in explants exposed to a complete ejaculate (2.4±0.14) or SP (2.4±0.06), in comparison with the control, epididymal sperm, or ejaculated sperm treatments (6.7±0.43, 6.9±0.32, 6.7±0.30, respectively; P<0.05). Due to the low RNA quality, those treatments were excluded from further analysis. However, this finding is currently being explored, along with the possibility of this effect being inherent to species that ejaculate intravaginally. We then compared the ability of ejaculated sperm (which have been exposed to SP) and epididymal sperm (which have never had contact with SP) to regulate the endometrial expression of IL1A, IL1B, IL8, IL6, PTGES2, TNFA, and LIF. Although IL6, IL1A and LIF increased in all animals when exposed to either ejaculated or epididymal sperm, there was no effect of treatment. In conclusion, these data did not support the notion that exposure of sperm to SP is important for the immune regulation of the bovine uterus. In addition, the negative effect of SP on the endometrium, together with the fact that bulls are intravaginal ejaculators, suggests that any putative immunoregulatory role of this fluid in the cattle uterus is indirect. Further analysis of the effect of SP on the vagina and cervix will help elucidate whether this response is present in this species and whether it can propagate to more distal regions of the reproductive tract. This work was supported by Science Foundation Ireland (13/IA/1983, 16/IA/4474).

Leukemia inhibitory factor promotes extracellular matrix synthesis in degenerative nucleus pulposus cells via MAPK-ERK1/2 signaling pathway

Extracellular matrix (ECM) anabolism and catabolism imbalance is key feature of chondrocyte and intervertebral disc nucleus pulposus (NP) cell degeneration. The role of LIF as a multifunctional cytokine in the ECM metabolism of chondrocytes is controversial, but no relevant research in the ECM metabolism of NP cells. This study aimed to explore the biofunction and related mechanisms of LIF in the degenerative NP cells. We obtained an increase in the expression of LIF in the human degenerated NP specimens. The addition of recombinant human leukemia inhibitory factor (rhLIF) to the degenerated NP cells cultured in vitro was found to stimulate the synthesis of ECM, and rhLIF could activate the ERK1/2 signaling pathway. However, coculture with PD98059, a signal inhibitor of ERK1/2, blocked the effect of rhLIF on the synthesis of ECM. To furtherly clarify the role of LIF, we carried out animal experiments and found that rhLIF treatment could successfully delay the degree of degeneration of the intervertebral disc in a rabbit model; but with the addition of PD98059, the function of rhLIF for degeneration protection disappeared. In summary, this study demonstrates that LIF plays a role in promoting ECM synthesis in the degenerated NP cells as a protective role in intervertebral disc degeneration (IDD), which is related to the activation of ERK1/2 signaling pathway.

Empowering the immune fate of bone marrow mesenchymal stromal cells: gene and protein changes.

Bone marrow mesenchymal stromal cells (BM-MSCs) are referred as a promising immunotherapeutic cell product. New approaches using empowered MSCs should be developed as for the treatment or prevention of different immunological diseases. Such preconditioning by new licensing stimuli will empower the immune fate of BM-MSCs and, therefore, promote a better and more efficient biological. Here, our main goal was to establish the immunological profile of BM-MSCs following inflammatory priming and in particular their capacity to adjust their immune-related proteome and transcriptome. To run this study, we have used BM-MSC cell cultures, a pro-inflammatory cytokine cocktail priming, flow cytometry analysis, qPCR and ELISA techniques. Different expression levels of several immunological mediators such as COX-1, COX-2, LIF, HGF, Gal-1, HO-1, IL-11, IL-8, IL-6 and TGF-β were constitutively observed in BM-MSCs. Inflammation priming substantially but differentially modulated the gene and protein expression profiles of these mediators. Thus, expressions of COX-2, LIF, HGF, IL-11, IL-8 and IL-6 were highly increased/induced and those of COX-1, Gal-1, and TGF-β were reduced. Collectively, we demonstrated that BM-MSCs are endowed with a specific and modular regulatory machinery which is potentially involved in immunomodulation. Moreover, BM-MSCs are highly sensitive to inflammation and respond to such signal by properly adjusting their gene and protein expression of regulatory factors. Using such preconditioning may empower the immune fate of MSCs and, therefore, enhance their value for cell-based immunotherapy.

Therapeutic Effects of VEGF Gene-Transfected BMSCs Transplantation on Thin Endometrium in the Rat Model.

Objective Bone mesenchymal stem cells (BMSCs) transplantation has a therapeutic effect on the thin endometrium in animal researches and clinical trials. The present study aims at assessing whether transplantation of VEGF-transfected BMSCs (VEGF-BMSCs) have a better therapeutic effect on endometrial regeneration and endometrial receptivity compared with BMSCs therapy alone. Methods Sprague-Dawley (SD) rats were used in the study. Thin endometrium model was established with 95% ethanol injection into uterine. VEGF-BMSCs or BMSCs was transplanted via tail vein IV injection. Endometrial thickness, morphology, and pinopodes were assessed by hematoxylin and eosin (HE) staining and scanning electron microscope (SEM). The proteins and mRNAs expressions of markers for endometrial cells and endometrial receptivity were measured after treatment. The fertility testing was done to assess the embryo implantation efficiency. Results VEGF-BMSCs transplantation significantly increased endometrial thickness compared with the BMSCs group and the control group. There was no significant difference in endometrial thickness between VEGF-BMSCs group and sham operation group. Importantly, in protein level, expressions of cytokeratin, vitamin, VEGF, LIF, and integrin ανβ 3 in VEGF-BMSC group were increased dramatically compared with those of the control group and BMSC group both 4 days and 8 days after stem cells transplantation. Accordingly, mRNA expression of LIF and integrin α ν β 3 was significantly upregulated compared with those of the control group and BMSC group both 4 and 8 days after treatment. The pinopodes were developed better in the VEGF-BMSCs group and the sham operation group compared with BMSCs group and the control group. The number of embryo implantation is largest in the sham operation group, followed by VEGF-BMSCs group, BMSCs group, and the control group. Conclusions Transplantation of VEGF gene-transfected BMSCs may be a better therapeutic treatment for thin endometrium than stem cell therapy alone.

Decreased expression of LIF mRNA in patients withmyoma uteri.

The aim of this study was to evaluate endometrial receptivity by measuring HOXA-10, HOXA-11, and LIF gene expressions in women with polycystic ovary syndrome. A total of 48 women were included in this clinical study. Thepatients were allocated to two groups: study group consisted of 28 patients with myoma uteri and control group consisted of 20 patients without myoma uteri. Endometrial sampling was performed during the proliferative phase. The biopsies obtained from the patients with myoma uteri were taken from the place where the fibroids were localized. HOXA-10, HOXA-11, and LIF expressions were measured in the endometrial sampling material. Demographic data of the patients such as age, obstetric and gynecologic history, medical conditions, medications, surgical history, last menstrual period were recorded. Also, the number, size, localization, and type of the myoma were registered. The mean age of the patients was 42.07 and 38.17, respectively. HOXA-11 levels in the study and control groups were 0.004 ± 0.001 and 0.010 ± 0.001, respectively ( P < 0.90). Paradoxically, HOXA-10 levels were found to be higher in the study group than the control group, but the difference was not statistically significant ( P < 0.25). LIF levels were significantly lower in the study group ( P < 0.05). Our results showed that myoma uteri might lead to a decrease in implantation rate by diminishing LIF gene expressions. However, there were no differences between the two groups in terms of HOXA-10 and HOXA-11 levels.

C-Fos is necessary for HGF-mediated gene regulation and cell migration in Schwann cells

We previously reported that the expression of hepatocyte growth factor (HGF) was highly induced after peripheral nerve damage, and that c-Fos is one of many cellular genes whose expressions are affected by the increased level of HGF[1]. c-Fos is an important component of AP-1 heterodimer, but its role has not been clearly understood in the context of HGF and Schwann cells (SCs). In this study, we investigated the relationship between HGF and c-Fos. First, it was confirmed that the c-Fos was increased in SCs after nerve injury, while this effect abrogated by PHA-665752, an inhibitor of c-met receptor. When primary SCs were treated with recombinant HGF protein, c-Fos expression was regulated in a typical quick, transient fashion at both RNA and proteins levels. HGF-mediated induction of c-Fos expression was highly suppressed by specific inhibitors of ERK and CREB, respectively. The knock down of c-Fos expression by siRNA almost completely blocked various HGF-mediated effects in SCs, such as induction of gene expression of GDNF, LIF, and c-Myc, and migration of SCs, indicating that c-Fos might play a key role in HGF effects. Taken together, our results suggested that c-Fos plays a key role(s) in HGF-mediated effects on neurotrophic genes and cell migration.

The Difference of Integrin ανβ3 Expression, Leukemia Inhibitory Factors and Superoxide Dismutase Serum Concentration in the Provision of Kebar Extract (Biophytum petersianum Klotczh), Metformin, and Their Combination to Mouse models of Endometriosis

Background: In endometriosis there is a decrease in endometrial receptivity. Kebar and Metformin grasses are expected to improve the condition of endometrial receptivityObjective: To find out whether there is any effect of giving Kebar grass extract and metformin on endometrial receptivity Methods: The study was designed by using a simple experimental randomization. It was conducted in Obstetrics Gynecology Department - Faculty of Medicine Diponegoro University and Faculty of Veterinary Medicine Airlangga University. Sample calculation was employed. The study was done within five groups including control group with normal mice, group of endometriosis mouse models given placebo, group of endometriosis mouse models given Kebar grass extract, group of endometriosis mouse models given metformin, and group of endometriosis mouse models given the combination of Kebar grass – metformin extract. The examination materials of ανβ3 integrin and Leukemia Inhibitory Factors were taken from the uteri of the mice and measured by the Rammele Scale Index (Immuno Reactive Score), while the Superoxide Dismutase examination using ELISA was derived from mice serum. These examinations were performed by two veterinariansResults: The expression of ανβ3 integrin was significantly higher in the group of mouse models given Kebar grass extract as well as the combination of Kebar and metformin grass extracts (p &lt;0.05) rather than in the group of endometriosis mouse models with no treatment, whereas LIF expression was significantly higher in the group given Kebar grass extract, metformin and combination of both extracts (p &lt;0.05). Serum SOD levels remained the same.Conclusion: The expressions of ανβ3 Integrin and LIF are higher due to the provision of Kebar grass extract as well as the combination of Kebar grass and metformin extracts

Differential expression of leukemia inhibitory factor and insulin like growth factor-1 between normal pregnancies, partial hydatidiform moles and complete hydatidiform moles

IntroductionLeukemia inhibitory factor (LIF) and insulin like growth factor-1 (IGF-1) are two of the most important growth factors mediating trophoblast actions. We hypothesized that the localization and expression patterns of LIF and IGF-1 in partial and complete hydatidiform moles (HM) compared with normal first trimester placentas may provide an understanding of the proliferative processes in HMs. MethodsThe study population included curettage material of women diagnosed as complete or partial HM as a result of histopathological and immunohistochemical examination (complete HM group, n = 8; partial HM group, n = 8) and women undergoing dilatation&curettage for unwanted pregnancies (control group, n = 8). Expression of LIF and IGF-1 among placental cell groups was evaluated immunohistochemically and given a score depending on immunostaining intensity. ResultsIn normal chorionic villi strong expression of LIF and IGF-1 was present. Both LIF and IGF-1 expressions were weaker in the chorionic villi of complete HMs. In complete mole decidua there was a significant decrease in glandular and endothelial IGF-1 expression along with a decrease in decidual cell LIF expression compared to normal first trimester decidua. LIF expression in extravillous trophoblasts was stronger in complete molar placentas compared to normal placentas. DiscussionLIF and IGF-1 are important regulators of trophoblast proliferation and invasion. Differential expression of LIF and IGF-1 in molar trophoblasts and chorionic villi might have a role in regulation of trophoblasts in complete moles. Decreased expression of glandular IGF-1 and decidual LIF might be related to the decidual changes during trophoblastic proliferation and invasion of decidua in complete HMs.

Abstract 1751: MSC-1 is a first-in-class humanized monoclonal antibody that modulates the tumor microenvironment by inhibiting a novel cancer immunotherapy target, LIF

Abstract Leukemia Inhibitory Factor (LIF) is a member of the IL-6 family of cytokines and is involved in many physiological and pathological processes including the promotion of an immunosuppressive environment to support embryo implantation, down-regulation of autoimmune processes and the regulation of stem cell homeostasis and differentiation. In cancer, LIF is hypothesized to have a complex role tumor development and progression, creating an immunosuppressive tumor microenvironment as well as promoting the activity of cancer initiating cells (CICs). LIF is highly expressed in a subset of tumors across multiple tumor types, e.g. glioblastoma multiforme (GBM), non-small cell lung cancer (NSCLC), colon, ovarian, prostate, and pancreatic cancer, and correlates with poor prognosis. Given the pleiotropic role LIF is hypothesized to play in cancer, inhibition of LIF represents an exciting new therapeutic concept sitting at the intersection of two key therapeutic approaches in oncology: blockade of tumor evasion of the immune system and blockade of tumor growth via inhibition of CICs. We have identified and developed MSC-1, a first-in-class humanized IgG1 monoclonal antibody that is a potent and selective inhibitor of LIF. MSC-1 cross reacts with mouse and non-human primate LIF and inhibits LIF signaling by blocking the recruitment of gp130 to the LIF-LIFR-gp130 signaling complex. The efficacy of MSC-1 was evaluated in multiple mouse tumor models and the mechanism of action investigated. LIF inhibition with MSC-1 or shRNAs reduced tumor growth in multiple syngeneic tumor models (NSCLC, ovarian and colon), and clear target engagement was shown for MSC-1. Investigations into the mechanism of action identified that inhibition of LIF with MSC-1 reprogrammed the tumor microenvironment by decreasing immunosuppressive M2 macrophages and increasing the number of intratumoral NK cells and total/activated T cells. MSC-1 also decreased immunosuppressive M2 macrophages in an orthotopic GBM xenograft model and human GBM organotypic tumor slices in an ex vivo model. Similarly, immunosuppressive macrophage genes were decreased when monocytes were co-cultured with supernatants from a GBM cell line in which LIF expression had been knocked-down. Given the effects of MSC-1 on intratumoral immune cells, we hypothesized that MSC-1 could be effectively combined with checkpoint inhibitors and we are currently evaluating MSC-1/checkpoint inhibitor combination therapy. Taken together, these findings form the basis of a robust therapeutic hypothesis, whereby MSC-1 treatment will lead to clinical activity in multiple cancer indications. A Phase I dose-escalation and expansion study of MSC-1 is planned to initiate early 2018 in patients with advanced solid tumors that will incorporate target engagement and PD biomarkers, as well as safety and efficacy endpoints. Citation Format: Angus Sinclair, Robin Hallett, Patricia Giblin, Isabel Huber-Ruano, Judit Anido, Naimish Pandya, Kimberly Hoffman, Ada Sala, Monica Pascual, Vanessa Chigancas, Swetha Raman, Johan Fransson, Jean-Philippe Julien, Robert Wasserman, Jeanne Magram, Joan Seoane. MSC-1 is a first-in-class humanized monoclonal antibody that modulates the tumor microenvironment by inhibiting a novel cancer immunotherapy target, LIF [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1751.

Abstract 5875: Development of LIFR inhibitor EC359 as a novel therapeutic for ovarian cancer

Abstract Background: Ovarian cancer (OCa) is the deadliest of all gynecologic cancers. OCa patients initially respond to standard combinations of surgical and cytotoxic therapy; however, ~80% will develop recurrence and inevitably succumb to chemotherapy-resistant disease. OCa stem cells are implicated in the tumor initiation and therapy resistance. LIFR signaling plays a critical role in OCa progression and stemness. Further, high circulating LIF levels correlate with tumor recurrence and chemoresistance. The autocrine loop involving LIF, LIFR and STAT3 axis drives sustained fibroblast production of inflammatory mediators. This represents a significant problem and a critical need exists for development of novel therapies targeting the LIFR axis for treating OCa. Methods: We have rationally designed and synthesized a small organic molecule (EC359) that emulates the LIF-LIFR binding site and functions as a LIFR inhibitor from a library of compounds. In silico docking studies were used to identify the putative interaction of the EC359 and LIF/LIFR complex. Binding of EC359 to LIFR was confirmed using surface plasmon resonance (SPR) and IP assays. Mechanistic studies were conducted using Western, RT-qPCR, and RNA-Seq analysis. Xenograft models were used for preclinical evaluation and toxicity. The efficacy of EC359 was tested using Patient-Derived eXplants (PDeX). Results: Global analysis of online databases revealed negative correlation of OCa survival with LIFR expression. Molecular docking studies showed EC359 interacts at the LIF-LIFR binding interface. SPR studies confirmed interaction of EC359 to LIFR. Western analysis of eight cells that represent four subtypes of OCa confirmed higher expression of LIF and LIFR. EC359 reduced the growth of eight OCa cells with high potency (IC50 10-50 nM) and promoted apoptosis. EC359 treatment reduced stemness of OCa cells. EC359 activity is dependent on the level of expression of LIFR with little activity on cells that do not express LIFR. EC359 significantly reduced the viability of carboplatin- and taxol-resistant OCa cells. Mechanistic studies showed EC359 interacts with LIFR and block its interaction with LIF. EC359 treatment reduced the STAT3 phosphorylation, mTOR and downstream survival signaling cascades. RNA sequencing revealed unique pathways blocked by EC359. Treatment of xenograft tumors with EC359 significantly reduced the tumor volume compared to control. Further, using PDeX of OCa, we demonstrated that EC359 has potential to reduce the proliferation. Pharmacologically, EC359 exhibited high oral bioavailability and long half-life with a wide therapeutic window. Conclusions: EC359 is a novel agent that targets LIF-LIFR axis and has activity against chemotherapy-resistant and primary OCa tumors. EC359 has the distinct pharmacologic advantages of oral bioavailability, in vivo stability, and is associated with minimal systemic side effects. Citation Format: Suryavathi Viswanadhapalli, Hareesh B Nair, Bindu Santhamma, Gangadhara R Sareddy, Yiliao Luo, Xinlei Pan, Edward R Kost, Ramachandran Murali, Rajeshwar Rao Tekmal, Klaus J Nickisch, Ratna K Vadlamudi. Development of LIFR inhibitor EC359 as a novel therapeutic for ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5875.

Oviductal glycoprotein 1 (OVGP1) is expressed by endometrial epithelium that regulates receptivity and trophoblast adhesion

To study the regulation and functions of oviductal glycoprotein 1 (OVGP1) in endometrial epithelial cells. Expression of OVGP1 in mouse endometrium during pregnancy and in the endometrial epithelial cell line (Ishikawa) was studied by immunofluorescence, Western blotting, and RT-PCR. Regulation of OVGP1 in response to ovarian steroids and human chorionic gonadotropin (hCG) was studied by real-time RT-PCR. OVGP1 expression was knockdown in Ishikawa cells by shRNA, and expression of receptivity associated genes was studied by real-time RT-PCR. Adhesion of trophoblast cell line (JAr) was studied by in vitro adhesion assays. OVGP1 was localized exclusively in the luminal epithelial cells of mouse endometrium at the time of embryo implantation. Along with estrogen and progesterone, hCG induced the expression of OVGP1 in Ishikawa cells. Knockdown of OVGP1 in Ishikawa cells reduced mRNA expression of ITGAV, ITGB3, ITGA5, HOXA10, LIF, and IL15; it increased the expression of HOXA11, MMP9, TIMP1, and TIMP3. Supernatants derived from OVGP1 knockdown Ishikawa cells reduced the adhesiveness of JAr cells in vitro. Expression of OVGP1 mRNA was found to be significantly lowered in the endometrium of women with recurrent implantation failure. OVGP1 is specifically induced in the luminal epithelium at the time of embryo implantation where it regulates receptivity-related genes and aids in trophoblast adhesion.

Has the time come to include low-level laser photobiomodulation as an adjuvant therapy in the treatment of impaired endometrial receptivity?

Low-level laser therapy (LLLT) has been used in photobiomodulation to promote healing, regenerate, and restore tissue function. Women with persistent thin endometrium were assumed to encounter diminished activity in the regenerative and functional capacity of their endometrium. The aim of this study was to examine the ability of LLLT in 635nm wavelength to enhance the proliferation and gene expression of in vitro cultured endometrial cells. Single (SE) versus multiple exposures (ME) to LLLT were examined in the study groups and compared to controls. A fluence dose of 4.27J/cm2 for 16min was given once in the SE or divided in three equal sessions in days 2, 4, and 6 of the culture in ME. Cellular response was evaluated by measuring viable cell numbers and surface area. Pattern of MUC1, ITGA5, ITGB3, LIF, and PTEN gene expression was assessed using the qRT-PCR. Greater numbers of cells were found in both study groups (P < 0.001) as compared to controls. The surface area of cells at the end of culture phase was highly significant (P < 0.001) in ME when compared to SE and controls. A statistically significant difference was found in terms of gene expression in both irradiated groups (P < 0.001) as compared to controls, although greater difference in PTEN tumour suppressor gene (Phosphatase and tensin homolog) expression was toward ME. The introduction of LLLT to the armamentarium of infertility will serve as a new adjuvant therapy in this field. The current study proofed that LLLT was able to increase the proliferative and functional capacity of cultured endometrial cells.

Müller Cell Biological Processes Associated with Leukemia Inhibitory Factor Expression.

Müller cells provide support to photoreceptors under many conditions of stress and degeneration. Leukemia inhibitory factor is known to be expressed in Müller cells, which is necessary to promote photoreceptor survival in stress. We hypothesize that Müller cells that express LIF are undergoing other biological processes or functions which may benefit photoreceptors in disease. In this study, we analyze an existing single Müller cell microarray dataset to determine which processes are upregulated in Müller cells that express LIF, by correlating LIF expression to the expression of other genes using a robust correlation method. Some enriched processes include divalent inorganic cation homeostasis, negative regulation of stem cell proliferation, and gamma-glutamyl transferase activity.

A phytoestrogen supplement prevents the altered gene expression associated with pregnancy implantation induced by IL-1β in endometrial epithelial cells

Phytoestrogens stimulate expression of the uterine estrogen receptor and regulate uterine functions in reproductive tissues. However, comprehensive understanding of the beneficial impacts of phytoestrogens on uterine biology at the molecular level remains unexplored. Interleukin-1β (IL-1β) expression is increased in the inflamed decidua and is associated with first trimester pregnancy loss. AglyMax-Sup has the same composition as that of the phytoestrogen supplement AglyMax but with added vitamins and other components. Expression of genes associated with implantation may be enhanced by AglyMax-Sup compared with AglyMax. We tested the hypothesis that AglyMax-Sup has greater effects on implantation compared with AglyMax, using RT-PCR and Western blotting in the endometrial epithelial cell line. Furthermore, we investigated the protective effect of AglyMax-Sup on IL-1βinduced changes in estrogen-responsive gene expression in endometrial epithelial cells. The purpose of this study was to compare the effects of the phytoestrogen supplement AglyMax-Sup with those of AglyMax on estrogen-responsive gene expression. AglyMax and AglyMax-Sup significantly (p<0.05) induced gene expression of glycodelin-A, HoxA10, IL-11, LIF, MEG-E8 and TGFβ1. AglyMax-Sup induced high levels of these genes compared with the levels induced by AglyMax. The enhanced expression of LIF, IL-11, integrin αV, and HOXA10 induced by AglyMax-Sup was abolished by the ER antagonist fulvestrant and the ERK inhibitor PD98059. Meanwhile, IL-1β inhibited progesterone plus estrogen-induced TGFβ1, glycodelin-A, HOXA10, and integrin αV expression. IL-1β-induced suppression of these expression was reversed by AglyMax-Sup. These results indicate that expression of genes associated with implantation may be increased by AglyMax-Sup compared with AglyMax. AglyMax-Sup might abrogate IL-1β-mediated changes that can affect embryo implantation via the MAPK pathway.