Targeting LIF-mediated paracrine interaction for pancreatic cancer therapy and monitoring.

Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis largely owing to inefficient diagnosis and tenacious drug resistance. Activation of pancreatic stellate cells (PSCs) and consequent development of dense stroma are prominent features accounting for this aggressive biology1,2. The reciprocal interplay between PSCs and pancreatic cancer cells (PCCs) not only enhances tumour progression and metastasis but also sustains their own activation, facilitating a vicious cycle to exacerbate tumorigenesis and drug resistance3-7. Furthermore, PSC activation occurs very early during PDAC tumorigenesis8-10, and activated PSCs comprise a substantial fraction of the tumour mass, providing a rich source of readily detectable factors. Therefore, we hypothesized that the communication between PSCs and PCCs could be an exploitable target to develop effective strategies for PDAC therapy and diagnosis. Here, starting with a systematic proteomic investigation of secreted disease mediators and underlying molecular mechanisms, we reveal that leukaemia inhibitory factor (LIF) is a key paracrine factor from activated PSCs acting on cancer cells. Both pharmacologic LIF blockade and genetic Lifr deletion markedly slow tumour progression and augment the efficacy of chemotherapy to prolong survival of PDAC mouse models, mainly by modulating cancer cell differentiation and epithelial-mesenchymal transition status. Moreover, in both mouse models and human PDAC, aberrant production of LIF in the pancreas is restricted to pathological conditions and correlates with PDAC pathogenesis, and changes in the levels of circulating LIF correlate well with tumour response to therapy. Collectively, these findings reveal a function of LIF in PDAC tumorigenesis, and suggest its translational potential as an attractive therapeutic target and circulating marker. Our studies underscore how a better understanding of cell-cell communication within the tumour microenvironment can suggest novel strategies for cancer therapy.

Tumor-derived LIF promotes chemoresistance via activating tumor-associated macrophages in gastric cancers.

Chemotherapy is the preferred clinical treatment for advanced stage gastric cancer (GC) patients, of which efficacy could be markedly impaired due to the development of chemoresistance. Alternatively activated or M2-type tumor associated macrophages (TAMs) are recruited under chemotherapy and are highly implicated in the chemoresistance development, but underlying molecular mechanism for TAM activation is largely unknown. Here, we present that tumor-derived Leukemia inhibitory factor (LIF) induced by chemo drugs represses the chemo sensitivity of gastric tumor cells in a TAM-dependent manner. Mechanistically, cisplatin-induced HIF1α signaling activation directly drive the transcription of LIF, which promotes the resistance of gastric tumors to chemo drug. Further study revealed that tumor cell-derived LIF stimulates macrophages into tumor-supporting M2-type phenotype via activating STAT3 signaling pathway. Therapeutically, blocking LIF efficiently elevates chemo sensitivity of tumor cells and further represses the growth rates of tumors under chemotherapy. Therefore, our study reveals a novel insight in understanding the cross talking between tumor cells and immune cells and provides new therapeutic targets for gastric cancer.

Inhibition of hypothalamic leukemia inhibitory factor exacerbates diet-induced obesity phenotype.

BACKGROUND: The consumption of large amounts of dietary fats can trigger an inflammatory response in the hypothalamus and contribute to the dysfunctional control of caloric intake and energy expenditure commonly present in obesity. The objective of this study was to identify chemokine-related transcripts that could be involved in the early stages of diet-induced hypothalamic inflammation. METHODS: We used immunoblot, PCR array, real-time PCR, immunofluorescence staining, glucose and insulin tolerance tests, and determination of general metabolic parameters to evaluate markers of inflammation, body mass variation, and glucose tolerance in mice fed a high-fat diet. RESULTS: Using a real-time PCR array, we identified leukemia inhibitory factor as a chemokine/cytokine undergoing a rapid increase in the hypothalamus of obesity-resistant and a rapid decrease in the hypothalamus of obesity-prone mice fed a high-fat diet for 1 day. We hypothesized that the increased hypothalamic expression of leukemia inhibitory factor could contribute to the protective phenotype of obesity-resistant mice. To test this hypothesis, we immunoneutralized hypothalamic leukemia inhibitory factor and evaluated inflammatory and metabolic parameters. The immunoneutralization of leukemia inhibitory factor in the hypothalamus of obesity-resistant mice resulted in increased body mass gain and increased adiposity. Body mass gain was mostly due to increased caloric intake and reduced spontaneous physical activity. This modification in the phenotype was accompanied by increased expression of inflammatory cytokines in the hypothalamus. In addition, the inhibition of hypothalamic leukemia inhibitory factor was accompanied by glucose intolerance and insulin resistance. CONCLUSION: Hypothalamic expression of leukemia inhibitory factor may protect mice from the development of diet-induced obesity; the inhibition of this protein in the hypothalamus transforms obesity-resistant into obesity-prone mice.

A Key Role for Leukemia Inhibitory Factor in C26 Cancer Cachexia.

Cachexia is an exacerbating event in many types of cancer that is strongly associated with a poor prognosis. We have identified cytokine, signaling, and transcription factors that are required for cachexia in the mouse C26 colon carcinoma model of cancer. C2C12 myotubes treated with conditioned medium from C26 cancer cells induced atrophy and activated a STAT-dependent reporter gene but not reporter genes dependent on SMAD, FOXO, C/EBP, NF-κB, or AP-1. Of the gp130 family members IL-11, IL-6, oncostatin M (OSM), and leukemia inhibitory factor (LIF), only OSM and LIF were sufficient to activate the STAT reporter in myotubes. LIF was elevated in C26 conditioned medium (CM), but IL-6, OSM, TNFα, and myostatin were not. A LIF-blocking antibody abolished C26 CM-induced STAT reporter activation, STAT3 phosphorylation, and myotube atrophy but blocking antibodies to IL-6 or OSM did not. JAK2 inhibitors also blocked C26 CM-induced STAT reporter activation, STAT3 phosphorylation, and atrophy in myotubes. LIF at levels found in the C26 CM was sufficient for STAT reporter activation and atrophy in myotubes. In vivo, an increase in serum LIF preceded the increase in IL-6 in mice with C26 tumors. Overexpression of a dominant negative Stat3Cß-EGFP gene in myotubes and in mouse muscle blocked the atrophy caused by C26 CM or C26 tumors, respectively. Taken together, these data support an important role of LIF-JAK2-STAT3 in C26 cachexia and point to a therapeutic approach for at least some types of cancer cachexia.

Primate-specific microRNA-637 inhibits tumorigenesis in hepatocellular carcinoma by disrupting signal transducer and activator of transcription 3 signaling.

UNLABELLED: MiR-637 (microRNA-637) is a primate-specific miRNA belonging to the small noncoding RNA family, which represses gene regulation at the post-transcriptional expression level. Although it was discovered approximately 5 years ago, its biomedical significance and regulatory mechanism remain obscure. Our preliminary data showed that miR-637 was significantly suppressed in four HCC cell lines and, also, in most of the hepatocellular carcinoma (HCC) specimens, thereby suggesting that miR-637 would be a tumor suppressor in HCC. Simultaneously, the enforced overexpression of miR-637 dramatically inhibited cell growth and induced the apoptosis of HCC cells. The transcription factor, signal transducer and activator of transcription 3 (Stat3), is constitutively activated in multiple tumors, and aberrant Stat3 activation is linked to the promotion of growth and desensitization of apoptosis. Our study showed that Stat3 tyrosine 705 phosphorylation and several Stat3-regulated antiapoptotic genes were down-regulated in miR-637 mimics-transfected and Lv-miR637-infected HCC cells. In addition, miR-637 overexpression negatively regulated Stat3 phosphorylation by suppressing autocrine leukemia inhibitory factor (LIF) expression and exogenous LIF-triggered Stat3 activation and rescued cell growth in these cells. A nude mice model also demonstrated the above-described results, which were obtained from the cell model. Furthermore, we found that LIF was highly expressed in a large proportion of HCC specimens, and its expression was inversely associated with miR-637 expression. CONCLUSION: Our data indicate that miR-637 acted as a tumor suppressor in HCC, and the suppressive effect was mediated, at least in part, by the disruption of Stat3 activation.

Birth control vaccine targeting leukemia inhibitory factor.

The population explosion and unintended pregnancies resulting in elective abortions continue to impose major public health issues. This calls for a better method of contraception. Immunocontraception has been proposed as a valuable alternative that can fulfill most, if not all, of the properties of an ideal contraceptive. There are several targets that are being explored for contraceptive vaccine development. Leukemia inhibitory factor (LIF), a member of interleukin-6 family, is required for embryo development and successful blastocyst implantation in several mammalian species. The present study was conducted to examine if LIF can be a target for the development of a birth control vaccine. Three sequences from LIF and two sequences from LIF-receptor (LIF-R) that span the regions involved in ligand-receptor binding were delineated, and peptides were synthesized based upon these sequences. Antibodies raised against these five peptides reduced LIF bioactivity in an in vitro culture assay using BA/F3 mLIF-R-mpg130 cells. Vaccines were prepared by conjugating these peptides to various carrier proteins. Immunization of female mice with these peptide vaccines induced a long-lasting, circulating as well as local antibody response in various parts of the genital tract, and resulted in a significant (P ≤ 0.05) inhibition in fertility in all the three trials; the LIF-R peptide vaccines proved to be a better vaccine target. The data indicate that LIF/LIF-R is an excellent target for the development of a birth control vaccine. This is the first study, to our knowledge, that examined LIF/LIF-R as a target for immunocontraception. The findings of this study can be easily translated to humans since LIF/LIF-R is also important for implantation and pregnancy in women.

Understanding the first steps in embryonic stem cell exit from the pluripotent state.

BACKGROUND: We are interested in understanding how a given cell type, in response to external cues from its environment, makes the decision to differentiate. In the case of mouse embryonic stem cells (mESCs), the key external factor that maintains their undifferentiated state is the cytokine leukemia inhibitory factor (LIF). LIF removal causes mESCs to exit their pluripotent state and differentiate into more restricted precursors. Although LIF is known to activate multiple different phosphorylation cascades, the mechanisms by which its removal leads to mESC differentiation are not well understood. STUDY DESIGN AND METHODS: In order to identify the molecular events that occur upon LIF removal, we developed a set of novel experimental approaches that allowed identification and quantification of global phosphorylation changes that occur when mESCs are deprived of LIF. These included growth of mESCs on permeable membranes and development of a robust and sensitive phospho-proteomics platform to quantify early signaling events. RESULTS: In addition to the well-characterized tyrosine 705 phosphorylation of STAT3, LIF removal results in the rapid phosphorylation of multiple other proteins known to regulate the mESC self-renewal on both tyrosine, serine, and threonine residues. We hypothesize that these unique posttranslational modifications help drive the exit of mESCs from the pluripotent state. CONCLUSIONS: Our data set the stage for future studies investigating the functional role of these phosphorylation events in mESCs. These studies were greatly facilitated by the National Blood Foundation, whose support in the crucial initiation phase of these studies was invaluable.

Embryo implantation is blocked by intraperitoneal injection with anti-LIF antibody in mice.

Leukemia inhibitory factor (LIF) is essential for embryo implantation in mice and plays an important role in other mammals including humans. Intraperitoneal (i.p.) injections with anti-LIF antibody (7.5 µg/g body weight, 3 times) between D3 (D1 = day of vaginal plug detection) and D4 effectively blocked embryo implantation; complete inhibition was achieved in C57BL/6J mice, and implantation was dramatically reduced in ICR mice (reduced to 27%). Normal rabbit IgG used as the control did not disturb embryo implantation. Anti-LIF antibody was localized not only in the stroma, but also in the luminal epithelium and the glandular lumen after i.p. injections. Growth-arrested blastocysts were recovered from the uterus without any implantation sites in both strains. Blastocysts made contact with the LE on the antimesometrial side; however, uterine stromal cells did not undergo secondary decidual reaction, and the uterine lumen was open, even at D7. Several regions of decidualization in ICR mice treated with anti-LIF antibody were smaller than those of the control, and development of blastocysts was delayed. The expression of LIF-regulated genes, such as immune-responsive gene-1 and insulin-like growth factor binding protein-3, was significantly decreased in C57BL/6J mice treated with anti-LIF antibody compared with the control, but not in ICR mice. The present study demonstrated that simple ip injections of an antibody are sufficient to block one of the important factors involved in embryo implantation in mice, and this method should also be easily applicable to the investigation of other factors involved in implantation.

An engineered ligand trap inhibits leukemia inhibitory factor as pancreatic cancer treatment strategy.

Leukemia inhibitory factor (LIF), a cytokine secreted by stromal myofibroblasts and tumor cells, has recently been highlighted to promote tumor progression in pancreatic and other cancers through KRAS-driven cell signaling. We engineered a high affinity soluble human LIF receptor (LIFR) decoy that sequesters human LIF and inhibits its signaling as a therapeutic strategy. This engineered 'ligand trap', fused to an antibody Fc-domain, has ~50-fold increased affinity (~20 pM) and improved LIF inhibition compared to wild-type LIFR-Fc, potently blocks LIF-mediated effects in pancreatic cancer cells, and slows the growth of pancreatic cancer xenograft tumors. These results, and the lack of apparent toxicity observed in animal models, further highlights ligand traps as a promising therapeutic strategy for cancer treatment.

Blocking LIF action in the uterus by using a PEGylated antagonist prevents implantation: a nonhormonal contraceptive strategy.

Blastocyst implantation is a critical stage in the establishment of pregnancy. Leukemia inhibitory factor (LIF) is essential for mouse blastocyst implantation and also plays a role in human pregnancy. We examined the effect of a potent LIF antagonist (LA) on mouse implantation. In mice, LIF expression peaks on day 3.5 of pregnancy (D3.5) (D0.5 = day of mating plug detection) in the uterine glandular epithelium. LA (7 mg/kg per day) administered from D2.5 to D4.5 via four hourly i.p. injections plus continuous administration via miniosmotic pump resulted in complete implantation failure. To improve its pharmacokinetic properties, we conjugated LA to polyethylene glycol (PEG), achieving a significant increase in serum levels. PEGylated LA (PEGLA) (37.5 mg/kg per day) administered via three i.p. injections between D2.5 and D3.5 also resulted in complete implantation failure. PEGLA immunolocalized to the uterine luminal epithelium at the time of blastocyst implantation. Both LA and PEGLA reduced phosphorylation of the downstream signaling molecule STAT3 in luminal epithelial cells on D3.5. The effects of PEGLA were found to be endometrial, with no embryo-lethal effects observed. These data demonstrate that administration of a PEGylated LIF antagonist is an effective method of targeting LIF signaling in the endometrium and a promising novel approach in the development of nonhormonal contraceptives for women.

Role of a LIF antagonist in LIF and OSM induced MMP-1, MMP-3, and TIMP-1 expression by primary articular chondrocytes.

Cartilage degradation is mediated by matrix metalloproteinases (MMPs) and their inhibitors, tissue metalloproteinases (TIMPs), which are transcriptionally regulated by a variety of growth factors and cytokines. The levels of various MMPs as well as TIMPs have been shown to increase in response to certain cytokines. These include leukaemia inhibitory factor (LIF) and Oncostatin M (OSM), both of which have been detected in the synovial fluids of patients with rheumatoid arthritis (RA). However, the role of LIF and OSM in the regulation of various MMPs and TIMPs is still incompletely understood. The aims of this study were to examine the effects of LIF and OSM on MMP-1, MMP-3, and TIMP-1 production. In addition, the capacity of the LIF antagonist, MH35-BD, to block LIF and OSM induced MMP expression was examined. Primary chondrocytes, isolated from porcine metacarpophalangeal cartilage, were cultured in the presence and absence of LIF and OSM, with and without a predetermined concentration of the LIF antagonist. We analysed the levels of MMP-1, MMP-3 and TIMP-1 expression using qRT-PCR, Northern blot, and ELISA assays. The results indicate that LIF and OSM increase the expression of MMP-1, MMP-3, and TIMP-1 several fold. Furthermore their expression is reduced to basal levels in the presence of the LIF antagonist MH35-BD.

Determining the LIF-sensitive period for implantation using a LIF-receptor antagonist.

Uteri of Lif null mice do not support embryo implantation. Since deletion of some genes often prevents the survival of null mice to adulthood, we have used a proven inhibitor of leukaemia inhibitory factor (LIF) signalling to identify the precise window of time during which LIF is required in vivo, and assessed the cellular expression of several LIF-associated targets. On day 4 of pregnancy, mice were injected with hLIF-05 (inhibitor) into the uterine lumen, with corresponding volumes of PBS (vehicle) injected into the contralateral horn. On days 5 and 6, the number of implantation sites was recorded and the uteri processed for immunohistochemistry. Blockade of LIF on day 4 reduced embryo implantation by 50% (P

LIF regulates CXCL9 in tumor-associated macrophages and prevents CD8+ T cell tumor-infiltration impairing anti-PD1 therapy.

Cancer response to immunotherapy depends on the infiltration of CD8+ T cells and the presence of tumor-associated macrophages within tumors. Still, little is known about the determinants of these factors. We show that LIF assumes a crucial role in the regulation of CD8+ T cell tumor infiltration, while promoting the presence of protumoral tumor-associated macrophages. We observe that the blockade of LIF in tumors expressing high levels of LIF decreases CD206, CD163 and CCL2 and induces CXCL9 expression in tumor-associated macrophages. The blockade of LIF releases the epigenetic silencing of CXCL9 triggering CD8+ T cell tumor infiltration. The combination of LIF neutralizing antibodies with the inhibition of the PD1 immune checkpoint promotes tumor regression, immunological memory and an increase in overall survival.

Blockade of leukemia inhibitory factor as a therapeutic approach to KRAS driven pancreatic cancer.

KRAS mutations are present in over 90% of pancreatic ductal adenocarcinomas (PDAC), and drive their poor outcomes and failure to respond to targeted therapies. Here we show that Leukemia Inhibitory Factor (LIF) expression is induced specifically by oncogenic KRAS in PDAC and that LIF depletion by genetic means or by neutralizing antibodies prevents engraftment in pancreatic xenograft models. Moreover, LIF-neutralizing antibodies synergize with gemcitabine to eradicate established pancreatic tumors in a syngeneic, KrasG12D-driven, PDAC mouse model. The related cytokine IL-6 cannot substitute for LIF, suggesting that LIF mediates KRAS-driven malignancies through a non-STAT-signaling pathway. Unlike IL-6, LIF inhibits the activity of the Hippo-signaling pathway in PDACs. Depletion of YAP inhibits the function of LIF in human PDAC cells. Our data suggest a crucial role of LIF in KRAS-driven pancreatic cancer and that blockade of LIF by neutralizing antibodies represents an attractive approach to improving therapeutic outcomes.

Leukemia inhibitory factor functions as a growth factor in pancreas carcinoma cells: Involvement of regulation of LIF and its receptor expression.

Leukemia inhibitory factor (LIF) is a pleiotrophic cytokine, which plays an important role in inducing cancer cachexia. We have previously reported that LIF promotes cell proliferation in some human carcinoma cells through c-fos, jun-B and cyclin-E expression. In the present study, we analyzed the regulation of LIF and its receptor (LIFR) expression in pancreatic carcinoma cells. Seven pancreatic carcinoma cells expressed constitutively LIF and its heterodimer receptor (LIFR and gp130) mRNA in RPMI-1640 medium without FBS. The amount of LIF immunoreactive protein was 132.5+/-52 pg/10(6) cells in culture supernatants without FBS. Pro-inflammatory cytokines, such as TNF-alpha, IL-1beta, IL-6, IL-8, or LIF, enhanced the expression of LIF mRNA in Hs-700T and Hs-766T cells. Addition of LIF significantly induced cell proliferation of Hs700T in 13 days LIF dose-dependently. However, anti-LIF IgG failed to suppress cell proliferation in Hs-700T cells. LIF acted as a paracrine growth factor in Hs-700T cells, which expressed low amount of LIF without stimuli. Cellular signal transductions by LIF was down-regulated by inhibitors of protein kinase C (PKC), protein tyrosine kinase (PTK), and Ca/Calmodulin. LIF induced phosphorylation of STAT3. Moreover, exogenous LIF upregulated the expression of LIFR mRNA. Antisense LIFR oligonucleotide significantly suppressed cell growth in the presence of LIF in Hs-700T cells. These results suggest that cytokine network might alter the expression and responsiveness to LIF in tumor microenvironment.

Monoclonal anti-leukemia inhibitory factor antibody inhibits blastocyst implantation in the rhesus monkey.

INTRODUCTION: Leukemia inhibitory factor (LIF) appears important in the process of blastocyst implantation in primates. In the present study, we investigated the effect on pregnancy outcome of the administration of a monoclonal antibody (mAb) to recombinant human (rh) LIF (anti-LIF mAb) into the uterine cavity of mated female rhesus monkeys during the peri-implantation period. METHODS: Two milligrams of either mouse mAb to rhLIF or isotype-matched immunoglobulin (Ig) was administered into the uterine cavity on estimated Day 5 or Day 8 after ovulation either through the vagina (n=33) or through the oviduct (n=29) of successfully mated females. RESULTS: There was a significant (p<.04) decline in pregnancy outcome in groups treated with anti-LIF mAb (8 pregnancies from 32 animals) compared with control animals (24 pregnancies from 30 animals). There was, however, no significant difference in pregnancy inhibition between a group of animals subjected to vaginal route treatment and a group of animals subjected to oviductal route treatment, as well as between a group subjected to anti-LIF mAb on Day 5 after ovulation and a group subjected to anti-LIF mAb on Day 8 after ovulation. No significant change was seen in the number of viable pregnancy in animals treated with 6 mg of anti-LIF mAb (5 pregnancies from 16 animals) compared with animals treated with 2 mg of anti-LIF mAb (8 pregnancies from 32 animals). Serum profiles of estradiol, progesterone, monkey chorionic gonadotropin and mouse IgG did not show any difference among different treatment subgroups during the luteal phase. However, among animals treated with anti-LIF mAb, the mean area under the curve for serum mouse IgG in pregnant animals (234+/-55 microg/mL day) was significantly (p<.01) less than that of nonpregnant animals (1325+/-97 microg/mL day). CONCLUSION: The results of the present study put forward the proof of concept that LIF plays a critical role in the process of blastocyst implantation in the rhesus monkey.

Leukemia Inhibitory Factor Is Necessary for Ovulation in Female Rhesus Macaques.

Although the requirement of pituitary-derived LH for ovulation is well documented, the intrafollicular paracrine and autocrine processes elicited by LH necessary for follicle rupture are not fully understood. Evaluating a published rhesus macaque periovulatory transcriptome database revealed that mRNA encoding leukemia inhibitory factor (LIF) and its downstream signaling effectors are up-regulated in the follicle after animals receive an ovulatory stimulus (human chorionic gonadotropin [hCG]). Follicular LIF mRNA and protein levels are below the limit of detection before the administration of hCG but increase significantly 12 hours thereafter. Downstream LIF receptor (LIFR) signaling components including IL-6 signal transducer, the receptor associated Janus kinase 1, and the transcription factor signal transducer and activator of transcription 3 also exhibit increased expression in the rhesus macaque follicle 12 hours after administration of an ovulatory hCG bolus. A laparoscopic ovarian evaluation 72 hours after the injection of a LIF antagonist (soluble LIFR) into the rhesus macaque preovulatory follicle and hCG administration revealed blocking LIF action prevented ovulation (typically occurs 36-44 h after hCG). Moreover, ovaries removed 52 hours after both hCG and intrafollicular soluble LIFR administration confirmed ovulation was blocked as evidenced by the presence of an intact follicle and a trapped cumulus-oocyte complex. These findings give new insight into the role of LIF in the primate ovary and could lead to the development of new approaches for the control of fertility.

Blocking Endogenous Leukemia Inhibitory Factor During Placental Development in Mice Leads to Abnormal Placentation and Pregnancy Loss.

The placenta forms the interface between the maternal and fetal circulation and is critical for the establishment of a healthy pregnancy. Specialized trophoblast cells derived from the embryonic trophectoderm play a pivotal role in the establishment of the placenta. Leukemia inhibitory factor (LIF) is one of the predominant cytokines present in the placenta during early pregnancy. LIF has been shown to regulate trophoblast adhesion and invasion in vitro, however its precise role in vivo is unknown. We hypothesized that LIF would be required for normal placental development in mice. LIF and LIFRα were immunolocalized to placental trophoblasts and fetal vessels in mouse implantation sites during mid-gestation. Temporally blocking LIF action during specific periods of placental development via intraperitoneal administration of our specific LIFRα antagonist, PEGLA, resulted in abnormal placental trophoblast and vascular morphology and reduced activated STAT3 but not ERK. Numerous genes regulating angiogenesis and oxidative stress were altered in the placenta in response to LIF inhibition. Pregnancy viability was also significantly compromised in PEGLA treated mice. Our data suggest that LIF plays an important role in placentation in vivo and the maintenance of healthy pregnancy.

Leukemia Inhibitory Factor (LIF) Inhibition during Mid-Gestation Impairs Trophoblast Invasion and Spiral Artery Remodelling during Pregnancy in Mice.

The placenta forms the interface between the maternal and fetal circulation and is critical for the establishment of a healthy pregnancy. Trophoblast cell proliferation, migration and invasion into the endometrium are fundamental events in the initiation of placentation. Leukemia inhibitory factor (LIF) has been shown to promote trophoblast invasion in vitro, however its precise role in trophoblast invasion in vivo is unknown. We hypothesized that LIF would be required for normal trophoblast invasion and spiral artery remodeling in mice. Both LIF and its receptor (LIFRα) co-localized with cytokeratin-positive invasive endovascular extravillous trophoblasts (EVT) in mouse implantation sites during mid-gestation. Temporally blocking LIF action during specific periods of placental development via administration of our unique LIFRα antagonist, PEGLA, resulted in abnormal trophoblast invasion and impaired spiral artery remodeling compared to PEG control. PEGLA-treated mouse decidual vessels were characterized by retention of α-smooth muscle actin (αSMA)-positive vascular smooth muscle cells (VSMCs), while PEG control decidual vessels were remodelled by cytokeratin-positive trophoblasts. LIF blockade did not alter F4/80-positive decidual macrophage numbers between treatment groups, but resulted in down-regulation of decidual transcript levels of monocyte chemoattractant protein-1 (MCP-1) and interleukin-10 (IL-10), which are important immune cell activation factors that promote spiral artery remodeling during pregnancy. Our data suggest that LIF plays an important role in trophoblast invasion in vivo and may facilitate trophoblast-decidual-immune cell cross talk to enable adequate spiral artery remodeling.