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.

Leukemia inhibitory factor protects against liver steatosis in nonalcoholic fatty liver disease patients and obese mice.

Nonalcoholic fatty liver disease (NAFLD) is one of the most common chronic liver diseases worldwide. However, the molecular mechanisms that promote dysregulation of hepatic triglyceride metabolism and lead to NAFLD are poorly understood, and effective treatments are limited. Leukemia inhibitory factor (LIF) is a member of the interleukin-6 cytokine family and has been shown to regulate a variety of physiological processes, although its role in hepatic triglyceride metabolism remains unknown. In the present study, we measured circulating LIF levels by ELISA in 214 patients with biopsy-diagnosed NAFLD as well as 314 normal control patients. We further investigated the potential role and mechanism of LIF on hepatic lipid metabolism in obese mice. We found that circulating LIF levels correlated with the severity of liver steatosis. Patients with ballooning, fibrosis, lobular inflammation, and abnormally elevated liver injury markers alanine transaminase and aspartate aminotransferase also had higher levels of serum LIF than control patients. Furthermore, animal studies showed that white adipose tissue-derived LIF could ameliorate liver steatosis through activation of hepatic LIF receptor signaling pathways. Together, our results suggested that targeting LIF-LIF receptor signaling might be a promising strategy for treating NAFLD.

Leukemia inhibitory factor is a novel biomarker to predict lymph node and distant metastasis in pancreatic cancer.

Pancreatic cancer has a low survival rate, and most patients have lymph node metastasis and distant metastasis at the time of diagnosis. Despite efforts to improve overall survival (OS) and recurrence free survival (RFS), the prognosis of pancreatic cancer remains poor, underscoring the importance of identifying new biomarkers to predict metastasis in patients with pancreatic cancer. Leukemia inhibitory factor (LIF) is overexpressed in many types of cancer and is involved in the development of various malignancies including pancreatic cancer. However, the role of LIF as a biomarker to predict metastasis in pancreatic cancer remains unclear. In this study, univariate and multivariate Cox regression analyses identified LIF expression in pancreatic tumor tissues as an independent risk factor related to worse OS and RFS. LIF overexpression was related to poor clinicopathological features such as lymph node metastasis and Pathological stage (pTNM) stage. Serum LIF levels were higher in pancreatic cancer patients than in healthy controls. The area under the receiver operating characteristic curve indicated that serum LIF is more effective than other biomarkers (CA199 and CEA) for predicting lymph node and distant metastasis.

The expression and significance of leukemia inhibitory factor, interleukin-6 and vascular endothelial growth factor in Chinese patients with endometriosis.

OBJECTIVE: To observe the levels of leukemia inhibitory factor (LIF), interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF) in blood, peritoneal fluid, ectopic endometrial tissue, and ectopic endometrial stromal cells of patients with endometriosis, and to compare expression of IL-6, LIF and VEGF expression between endometriotic and non-endometriotic patients underwent laparoscopic surgery. METHODS: Thirty-one patients who underwent laparoscopic surgery for endometriosis in our hospital from January 2018 to January 2020 were included in the observation group, and 32 patients who underwent laparoscopic surgery for uterine fibroids, ovarian serous cystadenoma, and ovarian teratomas, were included in the control group. The levels of LIF, IL-6 and VEGF in the blood and peritoneal fluid of the two groups of patients were detected. The levels of LIF, IL-6 and VEGF in ectopic endometrial tissue and self-paired eutopic endometrial tissue, ectopic endometrial stromal cells and self-paired eutopic endometrial stromal cells of patients in the observation group were detected. After treating the primary cultured ectopic endometrial stromal cells with LIF and IL-6 alone or in combination, the changes of VEGF mRNA of ectopic endometrial stromal cells and the VEGF levels in the supernatant were observed. RESULTS: The levels of LIF, IL-6 and VEGF in the blood and peritoneal fluid of the observation group were all higher than those of the control group (P < 0.05), and the levels of LIF, IL-6 and VEGF in the peritoneal fluid of the observation group were significantly higher than those in the blood (P < 0.05). In the observation group, the expression levels of LIF-mRNA and IL-6 mRNA in the ectopic endometrial tissue were higher than those in the self-paired eutopic endometrial tissues (P < 0.05), while the expression level of VEGF mRNA in the ectopic endometrial tissues was lower than that in the self-paired eutopic endometrial tissues (P < 0.05). Besides, the mRNA expression levels of LIF, IL-6 and VEGF in ectopic endometrial stromal cells of the observation group were all higher than those in the self-paired eutopic endometrial stromal cells (P < 0.05). After stimulating ectopic endometrial stromal cells with LIF, IL-6 and LIF + IL-6, respectively, the VEGF levels in the supernatant were all significantly higher than that in the blank control group (P < 0.05). CONCLUSION: The LIF, IL-6 and VEGF levels in blood and peritoneal fluid were increased in patients with endometriosis, and increased LIF and IL-6 in ectopic endometriosis stromal cells can play a synergistic role in increasing the VEGF levels, which may be involved in the occurrence and development of endometriosis.

Leukaemia inhibitory factor in serum and follicular fluid of women with polycystic ovary syndrome and its correlation with IVF outcome.

Polycystic ovary syndrome (PCOS) is characterized by hyperandrogenism, ovarian dysfunction and polycystic ovarian morphology. Leukaemia inhibitory factor (LIF) affects many reproductive activities, including follicular development, embryo implantation and growth. The aim of this study was to evaluate LIF concentrations in serum and follicular fluid of women with PCOS and controls who underwent IVF with embryo transfer (IVF-ET). Serum and follicular fluid LIF concentrations were lower in women with PCOS compared with controls. Oestradiol concentrations in follicular fluid were higher in PCOS subjects compared with controls. LIF concentrations in serum (r = 0.6263, P < 0.05) and follicular fluid (r = 0.7093, P < 0.05) were negatively correlated with oestradiol concentration in the PCOS group. LIF concentrations in follicular fluid showed no difference between women who conceived and women who did not in both PCOS and control groups. However, LIF concentrations in embryo culture medium were higher in women who conceived following IVF compared with women who did not, in combined PCOS and control groups. The findings indicate that low LIF concentrations in serum and follicular fluid may contribute to disordered folliculogenesis in PCOS. LIF concentrations in embryo culture medium may predict the outcome of IVF treatment.

Expression of Heme Oxygenase-1 and Leukemia Inhibitory Factor in Maternal Plasma and Placental Tissue in a Lipopolysaccharide-Induced Late Pregnancy Preterm Birth Mouse Model.

OBJECTIVE: To investigate the expression of heme oxygenase-1 (HO-1) and leukemia inhibitory factor (LIF) in maternal plasma and placental tissue in intrauterine infection-induced preterm birth. STUDY DESIGN: Using a mouse model of intrauterine infection in preterm birth, we used magnetic beads to extract normal pregnant mouse spleen Treg cells, injecting them into lipopolysaccharide (LPS)-treated pregnant mice. The subjects were divided into 4 groups: control group, LPS group, LPS+PBS group, and LPS+Treg group. RT-PCR and Western blot were used to evaluate HO-1, LIF mRNA, and protein levels in the placenta. ELISA was used to detect these parameters in the peripheral blood of pregnant mice. RESULTS: The expression of HO-1 and LIF in the placenta of the LPS group was significantly decreased when compared to that of the control group (p<0.05). Serum HO-1 and LIF levels were higher than in the control group (p<0.05). In the Treg cell-treated group placental tissue HO-1 and LIF expression were significantly higher than in the LPS group (p<0.05), and serum HO-1 and LIF expression were significantly lower than in the LPS group (p<0.05). CONCLUSION: HO-1 and LIF participate with Treg cells in the maternal-fetal interface, producing a unique immune microenvironment.

Anti-allergic effects of So-Cheong-Ryong-Tang in ovalbumin-induced allergic rhinitis model.

Allergic rhinitis (AR) is an allergic inflammation of the nasal airways. The Korean herbal medicine, So-Cheong-Ryong-Tang (SCRT) has been typically used for the treatment of AR for hundreds of years. In the present study, we investigated whether SCRT suppresses the progression of AR in animal model. AR was induced by ovalbumin (OVA). Treatment with SCRT was assessed to study the effect of SCRT on AR in mice. Histological analysis, multiplex cytokine assay, blood analysis, cell viability assay, RT-PCR and Elisa assay were performed to verify inhibitory effect of SCRT on AR. SCRT reduced infiltration of inflammatory cells into nasal cavity. SCRT reduced infiltration of mast cells into nasal mucosa. SCRT reduced the levels of cytokines (IL-4 and LIF) in the serum. SCRT reduced the levels of leukocytes in the blood. SCRT decreased cell viability of HMC-1 cells and splenocyte. SCRT suppressed IL-4 level in HMC-1 cells and splenocyte cells in a dose-dependent manner. SCRT suppressed IL-6 level and TNF-α level in splenocyte. SCRT suppresses the progression of AR induced by OVA. SCRT might be a useful drug for the treatment of AR.

Immunologic Network and Response to Intramyocardial CD34+ Stem Cell Therapy in Patients With Dilated Cardiomyopathy.

BACKGROUND: Although stem cell therapy (SCT) is emerging as a potential treatment for patients with dilated cardiomyopathy (DCM), clinical response remains variable. Our objective was to determine whether baseline differences in circulating immunologic and nonimmunologic biomarkers may help to identify patients more likely to respond to intramyocardial injection of CD34(+)-based SCT. METHODS AND RESULTS: We enrolled from January 3, 2011 to March 5, 2012 37 patients with longstanding DCM (left ventricular ejection fraction [LVEF] <40%, New York Heart Association functional class III) who underwent peripheral CD34(+) stem cell mobilization with granulocyte colony-stimulating factor (G-CSF) and collection by means of apheresis. CD34(+) cells were labeled with (99m)Tc-hexamethylpropyleneamine oxime to allow assessment of stem cell retention at 18 hours. Response to SCT was predefined as an increase in LVEF of ≥5% at 3 months. The majority (84%) of patients were male with an overall mean LVEF of 27 ± 7% and a median N-terminal pro-B-type natriuretic peptide (NT-proBNP) level of 2,774 pg/mL. Nineteen patients (51%) were responders to SCT. There was no significant difference between responders and nonresponders regarding to age, sex, baseline LVEF, NT-proBNP levels, or 6-minute walking distance. With the use of a partial least squares (PLS) predictive model, we identified 9 baseline factors that were associated with both stem cell response and stem cell retention (mechanistic validation). Among the baseline factors positively associated with both clinical response and stem cell retention were G-CSF, SDF-1, LIF, MCP-1, and MCP-3. Among baseline factors negatively associated with both clinical response and retention were IL-12p70, FASL, ICAM-1, and GGT. A decrease in G-CSF at 3-month follow-up was also observed in responders compared with nonresponders (P = .02). CONCLUSIONS: If further validated, baseline immunologic and nonimmunologic biomarkers may help to identify patients with DCM who are more likely to respond to CD34(+)-based SCT.

Cytokine levels in the serum of healthy subjects.

Growing knowledge about the cytokine network response has led to a better comprehension of mechanisms of pathologies and to the development of new treatments with biological drugs, able to block specific molecules of the immune response. Indeed, when the cytokine production is deregulated, diseases often occur. The understanding of the physiological mechanism of the cytokine network would be useful to better comprehend pathological conditions. Moreover, since the immune system and response change their properties with development, differences in patients' age should be taken into account, both in physiological and in pathological conditions. In this study, we analyzed the profile of 48 cytokines and chemokines in the serum of healthy subjects, comparing adults (≥18 years) with young children and children (1-6 and 7-17 years). We found that a certain number of cytokines were not being produced in healthy subjects; others showed a constant serum level amongst the groups. Certain cytokines exhibited a downward or an upward trend with increasing age. The remaining cytokines were up- or downregulated in the group of the children with respect to the other groups. In conclusion, we drew some kinds of guidelines about the physiological production of cytokines and chemokines, underling the difference caused by aging.

Receptivity assessment of an ultrasonographic homogeneous endometrium in the late follicular phase of infertile women with natural cycles.

OBJECTIVE: The purpose of this study was to assess the receptivity of the homogeneous endometrium in the late follicular phase in infertile women with natural cycles. STUDY DESIGN: Twenty-eight infertile women with ultrasonographically homogeneous (group 1) or trilaminar (group 2) endometria in the late follicular phase underwent endometrial biopsies. Some molecular markers and development of pinopodes were evaluated. RESULTS: In the late follicular phase, the mean level of vascular endothelial growth factor was significantly lower in group 1 than in group 2 (0.96 ± 0.37 marks vs 1.39 ± 0.46 marks; P = .010). In the mid luteal phase, a decreased leukemia inhibitory factor and integrin alpha v beta 3 levels were found in group 1 (1.58 ± 0.99 marks vs 2.59 ± 0.61 marks; 1.85 ± 0.72 marks vs 2.60 ± 0.73 marks; 1.92 ± 0.91 marks vs 2.83 ± 0.57 marks; P = .003; P = .011; P = .004). The rate of fully developed pinopodes in the mid luteal phase was significantly decreased in group 1 (P = .018). CONCLUSION: An ultrasonographically homogeneous endometrium in the late follicular phase was associated with poor receptivity in infertile women with natural cycles.

Serum biomarkers for detecting ectopic pregnancy.

Unless an ectopic pregnancy is visible by ultrasound, diagnosis can be a challenge. Differentiating ectopic pregnancies from intrauterine pregnancies can be impossible without intervention or follow-up. This poses a clinical dilemma to the practitioner given the inherent danger to the mother of tubal rupture of an ectopic pregnancy versus the fear of intervening in the case of a desired pregnancy without certainty of diagnosis. Early diagnostic modalities are clearly lacking, and serum biomarkers are currently being investigated as a solution to need for a rapid and accurate test for ectopic pregnancy.

Intermittent hypoxia mobilizes bone marrow-derived very small embryonic-like stem cells and activates developmental transcriptional programs in mice.

BACKGROUND: obstructive sleep apnea is a prevalent disorder associated with cognitive dysfunction and cardiovascular and metabolic morbidity and is characterized by recurrent episodes of hypoxia during sleep. Bone marrow-derived very small embryonic-like (VSEL) pluripotent stem cells represent a recruitable pool that may play an important role in organ repair after injury. We hypothesized that exposure to intermittent hypoxia (IH) can mobilize VSELs from the bone marrow (BM) to peripheral blood (PB) in mice and can activate distinct transcriptional programs. METHODS: adult mice were exposed to IH or normoxia for 48 hours. VSELs were sorted from BM and PB using flow cytometry. Plasma levels of stem cell chemokines, stromal cell derived factor-1 (SDF-1), hepatocyte growth factor (HGF), and leukemia inhibitory factor (LIF) were measured. Transcriptional profiling of VSELs was performed, and differentially expressed genes were mapped to enriched functional categories and genetic networks. RESULTS: exposure to IH elicited migration of VSELs from BM to PB and elevations in plasma levels of chemokines. More than 1100 unique genes were differentially expressed in VSELs in response to IH. Gene Ontology and network analysis revealed the activation of organ-specific developmental programs among these genes. CONCLUSIONS: exposure to IH mobilizes VSELs from the BM to PB and activates distinct transcriptional programs in VSELs that are enriched in developmental pathways, including central nervous system development and angiogenesis. Thus, VSELs may serve as a reserve mobile pool of pluripotent stem cells that can be recruited into PB and may play an important role in promoting end-organ repair during IH.

Ferritin concentrations in synovial fluid are higher in osteoarthritis patients with HFE gene mutations (C282Y or H63D).

OBJECTIVES: In view of the clinical similarities between polyarticular osteoarthritis (POA) with metacarpophalangeal (MCP) joint involvement and the arthropathy that occurs in hereditary haemochromatosis (HH), it was hypothesized that osteochondral damage in both disorders may be due to localized iron overload. Accordingly, it was predicted that the concentration of ferritin in synovial fluid (SF) would be higher in OA patients with HFE gene mutations than in HFE wild-type (wt) OA patients. The aim of this study was to test this proposition. METHODS: Sequential patients with physician-diagnosed OA and, for comparison, diverse inflammatory diseases of the joints, who required diagnostic or therapeutic arthrocentesis, were studied. Participants underwent HFE genotyping. SF samples were assayed for ferritin and also for selected cytokines and matrix metalloproteinases (MMPs). RESULTS: Seventy-three patients with diverse rheumatic disorders were recruited. Of the 29 patients who had knee OA, 15 were wt and 14 were heterozygous for HFE mutations (C282Y or H63D). Mean SF ferritin concentrations in the wt and heterozygous OA groups were 273 and 655 ng/mL, respectively (p = 0.0146). CONCLUSIONS: A predicted difference in SF ferritin concentrations in patients with knee OA was confirmed. Concentrations of ferritin in the SF were found to be two- to threefold higher in knee OA patients with HFE gene mutations compared to wt patients. This finding is consistent with the possibility that, in OA patients with HFE gene mutations, localized iron overload may contribute either directly or indirectly to osteochondral damage, possibly in a similar way to that which occurs in the arthropathy that complicates HH.

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.

Evaluation of serum levels of interleukin-10, interleukin-11 and leukemia inhibitory factor in differentiation of eutopic and tubal ectopic pregnancies.

PURPOSE OF STUDY: To investigate whether serum levels of leukemia inhibitory factor (LIF), interleukin 10 (IL-10) and interleukin 11 (IL-11) are different in reference to the site of implantation. METHODS: Seventeen patients with laparoscopic diagnoses of tubal ectopic pregnancy (EP) and 19 patients with intrauterine pregnancy delivering healthy term neonates (IUP) were prospectively evaluated for LIF, IL-10 and IL-11 levels. The data were compared by using the Student's t-test, chi-square test, Kruskal-Wallis and the Mann-Whitney U test with Bonferroni's correction (p < 0.05) as appropriate. RESULTS: A statistically significant difference was observed in serum LIF levels between the EP and IUP groups (p = 0.002). Ranges of LIF were 15-300 and 70-1200 ng/ml for the IUP and EP groups, respectively. There were no significant differences between groups in terms of IL-10 and IL-11 levels. CONCLUSION: LIF, but not IL-10 or IL-11, levels may be increased in early tubal ectopic pregnancies when compared to normal intrauterine pregnancies.

Serum and cerebrospinal fluid cytokines in children with acute encephalopathy.

BACKGROUND: The pathogenesis of acute encephalopathy (AE) remains unclear, and a biomarker has not been identified. METHODS: Levels of 49 cytokines and chemokines, including osteopontin (OPN), were measured in serum and cerebrospinal fluid (CSF) of children with AE (n = 17) or febrile convulsion (FC; n = 8; control group). The AE group included acute necrotizing encephalopathy (n = 1), acute encephalopathy with biphasic seizures and late reduced diffusion (AESD; n = 3), clinically mild encephalitis/encephalopathy with a reversible splenial lesion (MERS; n = 4), and unclassified acute encephalopathy (UCAE; n = 9) that does not meet the criteria of syndrome classification. Five individuals with AE had neurological sequelae or death (poor prognosis), whereas 12 were alive without neurological sequelae (good prognosis). RESULTS: The CSF:serum ratios of OPN, CC chemokine ligand (CCL)4, and interleukin (IL)-10 were significantly higher in AE than in FC. The CSF levels of macrophage inhibitory factor (MIF) and leukemia inhibitory factor (LIF) were significantly higher in the poor-prognosis group than in the good-prognosis group. The CSF:serum ratios of OPN were significantly higher in AESD and in MERS than in FC. The CSF:serum ratios of MIF and OPN were higher in MERS than in UCAE or FC. CONCLUSION: Our results suggest that microglia-related cytokines and chemokines such as OPN, MIF, and LIF could be novel biomarkers of AE, in addition to the previously reported IL-10 and CCL4, and that MIF and LIF may be markers of poor prognosis.

Effect of a six-hour marathon ultra-race on the levels of IL-6, LIF, and SCF.

The effects of 6-h marathon ultra-race (long aerobic work below the lactate threshold level) on the levels of IL-6, leukemia inhibiting factor (LIF), and stem cell growth factor (SCF) were studied. The athletes participating in the study had different endurance levels evaluated by the distance covered over 6 h. The level of IL-6 sharply increased after exercise. The degree of IL-6 increase correlated with the length of the distance covered (r=0.83, p=0.042). The concentration of LIF after exercise inversely correlated with the distance covered (r=-0.75), but this correlation was statistically insignificant. The IL-6/LIF proportion exhibited the highest correlation with the result in the marathon ultra-race. This parameter most fully characterized athlete endurance (r=0.92, p=0.009). Hence, the relationship of LIF with physical endurance was demonstrated. Involvement of LIF in antibody production processes can be responsible for it.

Ontogenetic features of the expression of mRNA isoforms for leukemia-inhibitory factor in human fetal tissues and mononuclear cells.

The expression of leukemia-inhibitory factor mRNA in human fetal tissues and mononuclear cells was studied during ontogeny. The expression of mRNA isoforms for leukemia-inhibitory factor was tissue-specific at the stage of prenatal development. The transition from antenatal and neonatal development to the postnatal period was accompanied by a decrease in the expression of mRNA isoforms for leukemia-inhibitory factor in mononuclear cells.