Leukemia Inhibitory Factor Research Articles

Overcoming poor ovarian response in assisted reproductive technology programs in patients of late reproductive age

Objective: to increase the effectiveness of in vitro fertilization in patients of late reproductive age by optimizing prepregnancy preparation for the IVF program.Materials and methods: The study included 67 patients of late reproductive age with a history of unsuccessful IVF attempts, divided into two groups at the second stage of the study, depending on the volume of prepregnancy preparation: group 1 (33 patients), group 2 (34 patients). The control group (50 patients) were healthy women of reproductive age. Immunohistochemical examination of the endometrium and microbiological examination of the uterine cavity, hormonal status of peripheral blood, study of the concentration of melatonin in peripheral blood and follicular fluid, antioxidant status of follicular fluid, expression of leukemia inhibitory factor (LIF) in the cervical secretion were performed.Results: in patients with age-related dysregulation of the hypothalamic-pituitary-ovarian axis and impaired fertile function, a systemic and local (in follicles) decrease in melatonin levels was revealed. The studied effects of melatonin suggested that its use in patients of late reproductive age with reduced follicular reserve and a "poor" response to ovarian stimulation may increase the effectiveness of in vitro fertilization (IVF).Conclusions: The inclusion in the pre-gravidar preparation of a complex of water-soluble polypeptide fractions of the epiphysis, stimulating the production of endogenous melatonin, made it possible to increase the effectiveness of IVF in patients by an average of 3.1 times according to the criterion of "clinical pregnancy" and 4.1 times according to the criterion of "live birth".

Multiple cancer cell types release LIF and Gal3 to hijack neural signals

Neural signals can significantly influence cancer prognosis. However, how cancer cells may proactively modulate the nervous system to benefit their own survival is incompletely understood. In this study, we report an overlapping pattern of brain responses, including that in the paraventricular nucleus of the hypothalamus, in multiple mouse models of peripheral cancers. A multi-omic screening then identifies leukemia inhibitory factor (LIF) and galectin-3 (Gal3) as the key cytokines released by these cancer cell types to trigger brain activation. Importantly, increased plasma levels of these two cytokines are observed in patients with different cancers. We further demonstrate that pharmacologic or genetic blockage of cancer cell-derived LIF or Gal3 signaling abolishes the brain responses and strongly inhibits tumor growth. In addition, ablation of peripheral sympathetic actions can similarly restore antitumor immunity. These results have elucidated a novel, shared mechanism of multiple cancer cell types hijacking the nervous system to promote tumor progression.

Targeting metastasis-initiating cancer stem cells in gastric cancer with leukaemia inhibitory factor

Gastric cancer’s (GC) bad prognosis is usually associated with metastatic spread. Invasive cancer stem cells (CSC) are considered to be the seed of GC metastasis and not all CSCs are able to initiate metastasis. Targeting these aggressive metastasis-initiating CSC (MIC) is thus vital. Leukaemia inhibitory factor (LIF) is hereby used to target Hippo pathway oncogenic members, found to be induced in GC and associated with CSC features. LIF-treated GC cell lines, patient-derived xenograft (PDX) cells and/or CSC tumourspheres underwent transcriptomics, laser microdissection-associated proteomics, 2D and 3D invasion assays and in vivo xenograft in mice blood circulation. LIFR expression was analysed on tissue microarrays from GC patients and in silico from public databases. LIF-treated cells, especially CSC, presented decreased epithelial to mesenchymal transition (EMT) phenotype and invasion capacity in vitro, and lower metastasis initiation ability in vivo. These effects involved both the Hippo and Jak/Stat pathways. Finally, GC’s high LIFR expression was associated with better clinical outcomes in patients. LIF treatment could thus represent a targeted anti-CSC strategy to fight against metastatic GC, and LIFR detection in primary tumours could constitute a potential new prognosis marker in this disease.

Green Synthesis of Pyrrole Derivatives Catalyzed by Molecular Sieves: DFT, ADMT, and Molecular Docking Investigations

AbstractA new synthetic methodology for the preparation by a different alternative method of five aryl pyrrole derivatives is produced from the corresponding ketoximes, giving diverse five‐membered N‐heterocycles in satisfactory yields. For the first time in literature, the synthetic catalyst molecular sieves were described. The simplicity and high response yield of this procedure are its standout qualities. Additionally, spectroscopic techniques (FT‐IR, 1H NMR, 13C NMR, elemental analysis) were used to confirm the structures of all synthesized compounds. FT‐IR and NMR computations with B3LYP/6‐311G** level by utilizing the G09 W package were performed to compare with observed data and evaluate the structural confirmation of novel pyrrole derivatives. Then, the lipophilic characterization and water solubility were computed to enlighten the physicochemical attitude. The ADMT properties were calculated to elucidate the possible bioavailability tendencies of the compounds and the adverse behaviour in terms of both medicinal and environmental. FMO analyses were also conducted to indicate/show the reactivity tendencies and possible regions. The molecular docking analyses of the optimized molecules were performed against Bovine Serum Albumin and Leukemia Inhibitory Factor. In conclusion, this study underscores the crucial role of green chemistry principles in developing novel synthetic methodologies, using environmentally benign catalysts like zeolite 3 Å.

Transcriptome profiling reveals superovulation with the gonadotropin-releasing hormone agonist trigger impaired embryo implantation in mice.

Superovulation is a critical step in assisted reproductive technology, but the use of human chorionic gonadotropin (hCG) as a trigger for superovulation can result in ovarian hyperstimulation. Thus, the use of Gonadotropin-releasing hormone agonist (GnRHa) trigger has been increasingly adopted, although it has been associated with a higher rate of pregnancy failure compared to natural cycles. This study aimed to investigate the effect of GnRHa trigger on embryo implantation in a mouse model. Mice in the superovulation (PG) group were administered 7.5 IU of PMSG, followed by the injection of 3.5 μg of GnRHa (Leuprorelin) 48 h later, while mice in the control group (CTR) mated naturally. We compared the number of oocytes, blastocysts, and corpus luteum between the two groups and the implantation sites after the transfer of natural blastocysts. Ovaries, uterus, and serum 2 and 4 days after mating were collected for qRT-PCR, transcriptome sequencing, and hormone assays. The PG group had more oocytes, blastocysts, and corpus luteum after superovulation than the CTR group. However, the mRNA expression of leukemia inhibitory factor (Lif) and the number of implantation sites were reduced in the PG group. The ELISA assay revealed that superovulation increased ovarian estrogen secretion. The transcriptome analysis showed that superphysiological estrogen led to a response of the uterus to a high estrogen signal, resulting in abnormal endometrium and extracellular matrix remodeling and up-regulation of ion transport and inflammation-related genes. Our findings suggest that a combination of PMSG and GnRHa trigger impaired embryo implantation in mice, as the excessive uterine response to superphysiological estrogen levels can lead to the change of gene expression related to endometrial remodeling, abnormal expression of uterine ion transport genes and excessive immune-related genes.

PTHrP intracrine actions divergently influence breast cancer growth through p27 and LIFR

The role of parathyroid hormone (PTH)-related protein (PTHrP) in breast cancer remains controversial, with reports of PTHrP inhibiting or promoting primary tumor growth in preclinical studies. Here, we provide insight into these conflicting findings by assessing the role of specific biological domains of PTHrP in tumor progression through stable expression of PTHrP (-36-139aa) or truncated forms with deletion of the nuclear localization sequence (NLS) alone or in combination with the C-terminus. Although the full-length PTHrP molecule (-36-139aa) did not alter tumorigenesis, PTHrP lacking the NLS alone accelerated primary tumor growth by downregulating p27, while PTHrP lacking the NLS and C-terminus repressed tumor growth through p27 induction driven by the tumor suppressor leukemia inhibitory factor receptor (LIFR). Induction of p27 by PTHrP lacking the NLS and C-terminus persisted in bone disseminated cells, but did not prevent metastatic outgrowth, in contrast to the primary tumor site. These data suggest that the PTHrP NLS functions as a tumor suppressor, while the PTHrP C-terminus may act as an oncogenic switch to promote tumor progression through differential regulation of p27 signaling.

Immunosuppressive role of BDNF in therapy-induced neuroendocrine prostate cancer.

Prostate stromal cells play a crucial role in the promotion of tumor growth and immune evasion in the tumor microenvironment (TME) through intricate molecular alterations in their interaction with prostate cancer (PCa) cells. While the impact of these cells on establishing an immunosuppressive response and influencing PCa aggressiveness remains incompletely understood. Our study shows that the activation of the leukemia inhibitory factor (LIF)/LIF receptor (LIFR) pathway in both prostate tumor and stromal cells, following androgen deprivation therapy (ADT), leads to the development of an immunosuppressive TME. Activation of LIF/LIFR signaling in PCa cells induces neuroendocrine differentiation (NED) and upregulates immune checkpoint expression. Inhibition of LIF/LIFR attenuates these effects, underscoring the crucial role of LIF/LIFR in linking NED to immunosuppression. Prostate stromal cells expressing LIFR contribute to NED and immunosuppressive marker abundance in PCa cells, while LIFR knockdown in prostate stromal cells reverses these effects. ADT-driven LIF/LIFR signaling induces brain-derived neurotrophic factor (BDNF) expression, which, in turn, promotes NED, aggressiveness, and immune evasion in PCa cells. Clinical analyses demonstrate elevated BDNF levels in metastatic castration-resistant PCa (CRPC) and a positive correlation with programmed death-ligand 1 (PDL1) and immunosuppressive signatures. This study shows that the crosstalk between PCa cells and prostate stromal cells enhances LIF/LIFR signaling, contributing to an immunosuppressive TME and NED in PCa cells through the upregulation of BDNF.

An in vitro three-dimensional (3D) testicular organoid culture system for efficient gonocyte maintenance and propagation using frozen/thawed neonatal bovine testicular tissues

Fertility preservation in prepubertal boys with cancer requires the cryopreservation of immature testicular tissues (ITTs) prior to gonadotoxic treatment. However, the limited number of germ cells in small human ITT biopsies necessitates the development of an in vitro culture system for germ cell expansion using frozen-thawed ITTs. Here, we generated testicular organoids for the in vitro maintenance and expansion of gonocytes from frozen-thawed two-week-old neonatal bovine ITTs. We investigated the effects of different cell-seeding densities, culture serums, seeding methods, and gonadotropin supplementations, on the maintenance and proliferation of enriched gonocytes. Our results demonstrated that enriched gonocytes and testicular cells from frozen-thawed neonatal ITTs could self-assemble into spheroid organoids in three days in an appropriate Matrigel-based culture environment. For the optimal formation of prepubertal testicular organoids, a seeding density of 1 × 106 cells/well is recommended over other densities. This strategy results in organoids with a mean diameter of 60.53 ± 12.12 μm; the mean number of organoids was 5.57 ± 1.60/105 μm2 on day 11. The viability of organoids was maintained at 79.75 ± 2.99% after being frozen and thawed. Supplementing the culture medium with glial cell-derived neurotrophic factor, fibroblast growth factor 2, and leukemia inhibitory factor, increased the proportion of KI67-positive proliferating cells in organoids, elevated the expression of C-KIT but reduced the expression of GFRα1 at day 28 when compared to those without hormone supplements (p< 0.05). In addition, supplementing the culture medium with follicle-stimulating hormone and testosterone helped to maintain a significantly higher viability (p< 0.05) in ITT organoids at day 28. These organoids could be cryopreserved for storage and thawed as needed. The successful generation of ITT organoids provides a valuable tool for establishing in vitro spermatogenesis, propagating human germ cells, investigating testicular physiology and the origin of germ cell tumors, and testing the toxicity of new drugs in future clinical applications.

Bioinformatic analysis of endometrial miRNA expression profile at day 26–28 of pregnancy in the mare

The establishment of the fetomaternal interface depends on precisely regulated communication between the conceptus and the uterine environment. Recent evidence suggests that microRNAs (miRNAs) may play an important role in embryo-maternal dialogue. This study aimed to determine the expression profile of endometrial miRNAs during days 26–28 of equine pregnancy. Additionally, the study aimed to predict target genes for differentially expressed miRNAs (DEmiRs) and their potential role in embryo attachment, adhesion, and implantation. Using next-generation sequencing, we identified 81 DEmiRs between equine endometrium during the pre-attachment period of pregnancy (day 26–28) and endometrium during the mid-luteal phase of the estrous cycle (day 10–12). The identified DEmiRs appear to have a significant role in regulating the expression of genes that influence cell fate and properties, as well as endometrial receptivity formation. These miRNAs include eca-miR-21, eca-miR-126-3p, eca-miR-145, eca-miR-451, eca-miR-491-5p, members of the miR-200 family, and the miRNA-17-92 cluster. The target genes predicted for the identified DEmiRs are associated with ion channel activity and sphingolipid metabolism. Furthermore, it was noted that the expression of mucin 1 and leukemia inhibitory factor, genes potentially regulated by the identified DEmiRs, was up-regulated at day 26–28 of pregnancy. This suggests that miRNAs may play a role in regulating specific genes to create a favorable uterine environment that is necessary for proper attachment, adhesion, and implantation of the embryo in mares.

In vivo PSC differentiation as a platform to identify factors for improving the engraftability of cultured muscle stem cells.

Producing an adequate number of muscle stem cells (MuSCs) with robust regenerative potential is essential for the successful cell therapy of muscle-wasting disorders. We have recently developed a method to produce skeletal myogenic cells with exceptional engraftability and expandability through an in vivo pluripotent stem cell (PSC) differentiation approach. We have subsequently mapped engraftment and gene expression and found that leukemia inhibitory factor receptor (Lifr) expression is positively correlated with engraftability. We therefore investigated the effect of LIF, the endogenous ligand of LIFR, on cultured MuSCs and examined their engraftment potential. We found that LIF-treated MuSCs exhibited elevated expression of PAX7, formed larger colonies from single cells, and favored the retention of PAX7+ "reserve cells" upon myogenic differentiation. This suggested that LIF promoted the maintenance of cultured MuSCs at a stem cell stage. Moreover, LIF enhanced the engraftment capability of MuSCs that had been expanded in vitro for 12 days by 5-fold and increased the number of MuSCs that repopulated the stem cell pool post-transplantation. These results thereby demonstrated the effectiveness of our in vivo PSC differentiation platform to identify positive regulators of the engraftability of cultured MuSCs.

IDH2/R140Q mutation confers cytokine-independent proliferation of TF-1 cells by activating constitutive STAT3/5 phosphorylation

BackgroundR140Q mutation in isocitrate dehydrogenase 2 (IDH2) promotes leukemogenesis. Targeting IDH2/R140Q yields encouraging therapeutic effects in the clinical setting. However, therapeutic resistance occurs in 12% of IDH2/R140Q inhibitor treated patients. The IDH2/R140Q mutant converted TF-1 cells to proliferate in a cytokine-independent manner. This study investigated the signaling pathways involved in TF-1(R140Q) cell proliferation conversion as alternative therapeutic strategies to improve outcomes in patients with acute myeloid leukemia (AML) harboring IDH2/R140Q.MethodsThe effects of IDH2/R140Q mutation on TF-1 cell survival induced by GM-CSF withdrawal were evaluated using flow cytometry assay. The expression levels of apoptosis-related proteins, total or phosphorylated STAT3/5, ERK, and AKT in wild-type TF-1(WT) or TF-1(R140Q) cells under different conditions were evaluated using western blot analysis. Cell viability was tested using MTT assay. The mRNA expression levels of GM-CSF, IL-3, IL-6, G-CSF, leukemia inhibitory factor (LIF), oncostatin M (OSM), and IL-11 in TF-1(WT) and TF-1(R140Q) cells were quantified via RT-PCR. The secretion levels of GM-CSF, OSM, and LIF were determined using ELISA.ResultsOur results showed that STAT3 and STAT5 exhibited aberrant constitutive phosphorylation in TF-1(R140Q) cells compared with TF-1(WT) cells. Inhibition of STAT3/5 phosphorylation suppressed the cytokine-independent proliferation of TF-1(R140Q) cells. Moreover, the autocrine GM-CSF, LIF and OSM levels increased, which is consistent with constitutive STAT5/3 activation in TF-1(R140Q) cells, as compared with TF-1(WT) cells.ConclusionsThe autocrine cytokines, including GM-CSF, LIF, and OSM, contribute to constitutive STAT3/5 activation in TF-1(R140Q) cells, thereby modulating IDH2/R140Q-mediated malignant proliferation in TF-1 cells. Targeting STAT3/5 phosphorylation may be a novel strategy for the treatment of AML in patients harboring the IDH2/R140Q mutation.BWcAJaJC8rGH42opvAqQCZVideo

Engineered exosome as a biological nanoplatform for drug delivery of Rosmarinic acid to improve implantation in mice with induced endometritis

Endometritis is an inflammatory and histopathologic disease in uterine tissues that interferes with the proper decidualization and implantation of the embryo. In this study, rosmarinic acid (RA) is used as an anti-inflammatory agent that encapsulates in exosomes and is used to attenuate lipopolysaccharide (LPS)-induced endometritis and improve implantation. For this purpose, exosomes were loaded with RA and then administrated into the animal groups, including RA, exosome, RA plus exosome (RA + Exo), and RA-loaded exosomes (RALExo) groups. The concentrations of RA or exosomes used in this study were 10 mg/kg, and the compounds were injected into the uterine horn 24 h following the induction of endometritis. Upon the presence of inflammation detected by the histopathological method, the most proper groups were mated with male mice. The effect of the treatment group on the implantation rate, progesterone levels, and gene expressions were assessed by Chicago Blue staining, enzyme-linked immunosorbent assay (ELISA), and Quantitative PCR (qPCR), respectively. Results showed RALExo10 and RA10 + Exo10 groups improved pathological alterations, enhanced progesterone levels, increased implantation rate, as well as heightened expression levels of Leukemia inhibitory factor (LIF) and Mucin-16 (MUC-16) genes. Besides, the expression levels of inflammatory cytokines, including Transforming growth factor-β (TGF-β), Interlukine-10 (IL-10), Interlukine-15 (IL-15), and Interlukine-18 (IL-18), were regulated. Our findings indicated that the expression of LIF, Muc-16 genes as well as IL-18, were significantly correlated with serum progesterone concentrations and the implantation rate in the treatment groups. The RALExo10 and RA10 + Exo10 groups showed ameliorated implantation rates in experimental groups.

IFNT-induced IRF1 enhances bovine endometrial receptivity by transactivating LIFR

Interferon-τ (IFN-τ) participates in the establishment of endometrial receptivity in ruminants. However, the precise mechanisms by which IFN-τ establishes bovine endometrial receptivity remain largely unknown. Interferon regulatory factor 1 (IRF1) is a classical interferon-stimulated gene (ISG) induced by type I interferon, including IFN-τ. Leukemia inhibitory factor receptor (LIFR) is a transmembrane receptor for leukemia inhibitory factor (LIF), which is a key factor in regulating embryo implantation in mammals. This study aimed to investigate the roles of IRF1 and LIFR in the regulation of bovine endometrial receptivity by IFN-τ. In vivo, we found IRF1 and LIFR were upregulated in the bovine endometrial luminal epithelium on Day 18 of pregnancy compared to Day 18 of the estrous cycle. In vitro, IFN-τ could upregulate IRF1, LIFR, and endometrial receptivity markers (LIF, HOXA10, ITGAV, and ITGB3) expression, downregulate E-cadherin expression and reduce the quantity of microvilli of bovine endometrial epithelial cells (bEECs). Overexpression of IRF1 had similar effects to IFN-τ on endometrial receptivity, and interference of LIFR could block these effects, suggesting the positive effects of IRF1 on endometrial receptivity were mediated by LIFR. Dual luciferase reporter assay verified that IRF1 could transactivate LIFR transcription by binding to its promoter. In conclusion, IFN-τ can induce IRF1 expression in bovine endometrial epithelial cells, and IRF1 upregulates LIFR expression by binding to LIFR promoter, contributing to the enhancement of bovine endometrial receptivity.

Utility of Visual Evoked Potentials (VEPs) study in the evaluation of visual pathway dysfunction in diabetics without retinopathy: correlations with diabetic peripheral neuropathy and other clinical findings.

Aim and objectives: Visual dysfunction in diabetes mellitus (DM) is multifactorial and can be due to vascular disease, and metabolic abnormalities that can affect the retina, optic nerve, and visual pathways. Visual evoked potential (VEP) is an electrophysiological test that can quantify the functional integrity of the visual pathways from the retina via the optic nerves, and optic tracts to the visual cortices. In this study, we aimed to investigate the visual pathway dysfunction among diabetics without retinopathy compared with healthy controls and to look for any correlation with diabetic neuropathy, duration of diabetes, or HbA1c level. Methods: The study included 75 diabetic patients and 75 age and sex-matched controls. VEPs were recorded using the pattern reversal stimulation method on the Medtronic EMG EP machine, and P100 latency and N75-P100 amplitude were recorded in both diabetic patients and healthy controls. Results: Mean P100 latency was significantly prolonged and N75-P100 amplitude significantly reduced among diabetic cases compared to healthy controls (p < 0.001). Among diabetics with peripheral neuropathy, P100 latency was significantly prolonged and N75-P100 amplitude was significantly reduced compared to diabetics without peripheral neuropathy. A significant positive correlation of VEP P100 latency (p < 0.001) and a negative correlation with N75-P100 amplitude (p < 0.001) with duration of disease were also found. Conclusion: VEP changes are observed in diabetics before the development of retinopathy or peripheral neuropathy indicating optic pathway dysfunction, which precedes the development of these complications. Early preclinical visual pathway dysfunction can warrant taking the necessary measures to reduce diabetic complications. Abbreviations: DM = Diabetes Mellitus, VEP = Visual Evoked Potential, HbA1c = Hemoglobin A1 c, MRI = Magnetic Resonance Imaging, EEG = Electroencephalography, P100 = Positive wave peak at latency 100 ms (millisecond), N75 = Negative wave peak at latency 75 ms (millisecond), N145 = Negative wave peak at latency 145 ms (millisecond), OCT = Optical coherence tomography, PRVEP = Pattern Reversal Visual Evoked Potential, NCS = Nerve Conduction Study, SSR = Sympathetic Skin Response, IL1 = Interleukin-1, LIF = Leukemia inhibitory factor, CNTF = Ciliary neurotrophic factor, TNF alpha = Tumor necrosis factor-alpha, TGF-beta = Transforming growth factor-beta.

Leukemia inhibitory factor suppresses hepatic de novo lipogenesis and induces cachexia in mice

Cancer cachexia is a systemic metabolic syndrome characterized by involuntary weight loss, and muscle and adipose tissue wasting. Mechanisms underlying cachexia remain poorly understood. Leukemia inhibitory factor (LIF), a multi-functional cytokine, has been suggested as a cachexia-inducing factor. In a transgenic mouse model with conditional LIF expression, systemic elevation of LIF induces cachexia. LIF overexpression decreases de novo lipogenesis and disrupts lipid homeostasis in the liver. Liver-specific LIF receptor knockout attenuates LIF-induced cachexia, suggesting that LIF-induced functional changes in the liver contribute to cachexia. Mechanistically, LIF overexpression activates STAT3 to downregulate PPARα, a master regulator of lipid metabolism, leading to the downregulation of a group of PPARα target genes involved in lipogenesis and decreased lipogenesis in the liver. Activating PPARα by fenofibrate, a PPARα agonist, restores lipid homeostasis in the liver and inhibits LIF-induced cachexia. These results provide valuable insights into cachexia, which may help develop strategies to treat cancer cachexia.

Impairment of adenosine signaling disrupts early embryo development: unveiling the underlying mechanisms.

Purinergic signaling has been implicated in many biological functions, including development. In this study, we investigate the functions of extracellular adenosine and adenosine receptors using a mouse embryonic stem cell (ESC) line and morula stages isolated from mouse embryos. Feeder-free mouse ESC was investigated in the absence and presence of the leukemia inhibitory factor (LIF), configuring undifferentiated cells and cells undergoing spontaneous differentiation. High alkaline phosphatase (ALPL) and low CD73 levels resulting in low adenosine (eADO) levels were characteristic for pluripotent cells in the presence of the LIF, while LIF deprivation resulted in augmented adenosine levels and reduced pluripotency marker expression, which indicated differentiation. Tracing ESC proliferation by BrdU labeling revealed that the inhibition of ALPL by levamisole resulted in a decrease in proliferation due to less eADO accumulation. Furthermore, caffeine and levamisole treatment, inhibiting adenosine receptor and eADO accumulation, respectively, reduced ESC migration, similar to that observed in the absence of the LIF. Pharmacological approaches of selective adenosine receptor subtype inhibition triggered specific adenosine receptor activities, thus triggering calcium or MAP kinase pathways leading to differentiation. In line with the in vitro data, mouse embryos at the morula stage were sensitive to treatments with A1 and A3 receptor antagonists, leading to the conclusion that A1 receptor and A3 receptor inhibition impairs proliferation and self-renewal and triggers inappropriate differentiation, respectively. The findings herein define the functions of eADO signaling in early development with implications for developmental disorders, in which adenosine receptors or ectonucleotidase dysfunctions are involved, and which could lead to malformations and miscarriages, due to exposure to caffeine.

Interleukin-6 Family of Cytokines in Cancers.

Nine soluble ligands [interleukin-6 (IL-6), interleukin-11 (IL-11), leukemia inhibitory factor (LIF), oncostatin M (OSM), ciliary neurotrophic factor (CNTF), cardiotrophin-1 (CT-1), cardiotrophin-like cytokine, interleukin-27 (IL-27), and interleukin-31] share the ubiquitously expressed transmembrane protein-glycoprotein-130 beta-subunit (gp130) and thus form IL-6 family cytokines. Proteins that may be important for cancerogenesis, CT-1, IL-11, IL-27, LIF, OSM, and CNTF, belong to the superfamily of IL-6. Cytokines such as IL-6, IL-11, and IL-27 are better investigated in comparison with other members of the same family of cytokines, eg, CT-1. Gp130 is one of the main receptors through which these cytokines exert their effects. The clinical implication of understanding the pathways of these cytokines in oncology is that targeted therapy to inhibit or potentiate cytokine activity may lead to remission in some cases.

Abstract B095: Leukemia inhibitory factor fosters tumor growth in KRAS-driven pancreatic ductal adenocarcinoma

Abstract While inflammation is a known and well-validated risk factor for developing pancreatic ductal adenocarcinoma (PDA), it remains unclear how Leukemia inhibitory factor (LIF), a cytokine of the IL-6 class family, influences tumor initiation and progression in PDA. LIF is highly upregulated in PDA when compared to the normal healthy pancreas. Using a human single-cell RNAseq PDA data set, we identified pancreatic cancer cells as the main source of LIF, followed by fibroblasts and stellate cells. After confirming that Pdx1-expressing cells represent the majority of Lif-expressing cells in the pancreas, we crossed our newly developed Lif flox/flox allele into the well-established KrasLSL-G12D/+, Trp53flox/flox, Pdx1-Cre (KPC) mouse model. Lif flox/flox, Pdx1-Cre (LC) mice present with normal pancreas histology, mouse weight, and mouse size at 12 months, indicating that Lif is dispensable for mouse pancreas development and function. Lif flox/flox, Trp53flox/flox, Pdx1-Cre (LPC) mice also showed no differences in pancreas histology when compared to normal Lif-expressing PC mice. Lif flox/floxKPC (LKPC) mice develop PDA tumors. Cell lines from LKPC tumors (n=4) presented with undetectable Lif expression and no LIF protein secretion. Control KPC tumor cell lines (n=4) showed Lif expression and LIF secretion, confirming the successful deletion of Lif in our newly developed mouse model. LKPC mice (n=21) showed a significant increase in disease-specific overall survival at 72 days when compared to control KPC mice (n=26) at 62 days (p&amp;lt;0.05). When mice reached endpoint, LKPC mice presented with significantly smaller tumors with an average tumor weight of 0.7g compared to KPC control animals with an average tumor weight of 1.3g (p&amp;lt;0.05). In summary, we find that cancer-cell expressed LIF augments tumor growth and shortens disease-specific overall survival, nominating leukemia inhibitory factor depletion as a promising therapeutic target in KRAS-driven pancreatic cancer. Citation Format: Lisa Miller-Phillips, Imene Boukhalfa Hanafi, Sarah Umetsu, Adam Olshen, Ritu Roy, Ashley Kiemen, Man-Tzu Wang, Eric Collisson. Leukemia inhibitory factor fosters tumor growth in KRAS-driven pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B095.

Abstract B031: Targeting epigenetic machinery to revoke stromal pro-tumorigenicity and enhance efficacy in pancreatic cancer therapy

Abstract Oncogenic lesions in pancreatic ductal adenocarcinoma (PDAC) induce prominent desmoplasia in part by activating fibroblasts or their progenitors in the stroma. The resultant cancer-associated fibroblasts (CAFs) often switch on the pro-desmoplastic, pro-inflammatory and/or pro-tumorigenic transcriptional programs, contributing to tumor progression and therapeutic resistance. Identifying and targeting epigenetic machinery essential for stromal fibroblast activation provides potential strategies to revoke stromal pro-tumorigenicity and overcome therapeutic resistance in PDAC. Here we identify Class I histone deacetylases (HDACs) as key epigenetic regulators facilitating the induction of pro-desmoplastic and pro-tumorigenic transcriptional programs in stromal fibroblasts. Mechanistically, HDACs coordinate the establishment of chromatin architecture that enables the activation of pro-desmoplastic programs directed by the transcriptional factor axis of serum response factor (SRF) and forkhead box M1 (FOXM1). HDACs also facilitate the induction of pro-inflammatory program in stromal fibroblasts, supporting leukemia inhibitory factor (LIF)-mediated pro-tumorigenic crosstalk. Both HDAC depletion in CAFs and treatment of the HDAC inhibitor entinostat (Ent) in PDAC mouse models reduce stromal activation and curb tumor progression. Notably, HDAC inhibition (HDACi) enriches a novel lipogenic fibroblast subpopulation in CAFs, a potential precursor population for activated myofibroblasts in vivo. Our work pinpoints a critical role of HDACs in driving stromal pro-tumorigenicity and highlights the stromal targeting capacity of HDACi therapy. Furthermore, the clinical utility of HDACi therapy is often hindered by limited potency and nonnegligible systemic toxicity of the HDAC inhibitors. To facilitate the translation of HDACi-based therapy, we develop a polymer-based bottlebrush pro-drug (BPD) nanoparticle incorporating Ent. The Ent-BPD nanoparticle overcomes the limitations of conventional HDACi therapy by sustaining long-lasting release of HDACi activity specifically at the tumor sites. Administration of Ent-BPD in PDAC mouse models achieves superior intratumoral HDACi potency and enhanced therapeutic efficacy compared to the free drug Ent treatment; meanwhile, it avoids the systemic toxicity associated with the free drug treatment. Our work further demonstrates a promising strategy for the clinical adaptation of HDACi-based therapy. Citation Format: Gaoyang Liang, Jeremiah A. Johnson, Ronald M. Evans. Targeting epigenetic machinery to revoke stromal pro-tumorigenicity and enhance efficacy in pancreatic cancer therapy [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B031.

Abstract C041: Oncostatin-M and transforming growth factor-beta promote loss of PTEN in PDAC cancer associated fibroblasts

Abstract Pancreatic Ductal Adenocarcinoma (PDAC) is a deadly malignancy with poor prognosis and treatment options. Cancer associated fibroblasts (CAFs) play a complex role in the PDAC tumor microenvironment (TME), contributing significantly to the progression of the tumor and the dense desmoplastic stroma. Phosphatase and tensin homolog (PTEN) is a tumor suppressor and its loss in both the tumor and in CAFs is associated with increased tumor aggressiveness and resistance to therapy. Our group previously demonstrated PTEN loss occurs in CAFs in ~25% of PDAC patient samples, which correlated with worsened outcomes. In addition, PTEN expression was lost in 80% of SMA+ CAFs in mouse PDAC models. Genetic or pharmacologic inhibition of the hedegehog pathway leads to ubiquitin-dependent destruction of PTEN by the proteosome. However, little is known of the signals produced by tumor cells that trigger PTEN degradation in CAFs. In order to address this problem, we developed an in vitro assay system based on wild type murine PDAC-CAFs that expresses a PTEN-GFP fusion protein. Initially, we focused on IL6 and IL6 family members Leukemia Inhibitory Factor (LIF) and Oncostatin-M (OSM) and Transforming Growth Factor beta 1 (TGFb1). The SMO inhibitor GDC-0449 was used as a positive control. PTEN stability was evaluated using the LSM 880 confocal microscope and GFP+ cells were quantified with QuPath-0.4.3 and analyzed with GraphPad Prism. Among the repertoire of secreted factors, we found that only OSM and TGFb1 could destabilize PTEN after 48H of treatment. When queried against the single cell RNA sequence dataset produced by our lab, we observed that CD45+ leukocytes were the likely source of OSM whereas TGFb1 was produced by tumor cells, fibroblasts, and immune cells. In conclusion, OSM and TGFb1 can downregulate PTEN in CAFs in vitro. Current efforts are aimed at testing whether inhibition of these pathways can restore PTEN expression in mouse PDAC models, and in identifying signaling pathways and E3-ligases that mediate PTEN destruction. These pathways and interactions could be further exploited for selective inhibition aimed at therapeutic benefits in curbing PDAC progression. Citation Format: Ivo N. Woogeng, Lu Han, Samaneh Saberi, Cameron Bumbleburg, Joseph Beaudet, Sudarshana Sharma, Michael Ostrowski. Oncostatin-M and transforming growth factor-beta promote loss of PTEN in PDAC cancer associated fibroblasts [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C041.