Pancreatic cancer cells overexpressing interleukin 6 induce T-cell-mediated tumor clearance and durable anti-tumor immune response.

Tumor immune resistance is recognized as a contributor to low survivorship in pancreatic ductal adenocarcinoma (PDAC). We developed a novel murine model of spontaneous PDAC clearance, generated by overexpressing interleukin-6 (IL-6) in orthotopically implanted PDAC cancer cells (OT-PDACIL6). Circulating IL-6 was 100-fold higher in OT-PDACIL6 than in OT-PDACparental mice. OT-PDACIL6 tumors were present at 5 days post-implantation, and undetectable by 10 days post implantation. Flow cytometry revealed increased T cells and NK cells, and decreased T regulatory cells in OT-PDACIL6 as compared to OT-PDACparental tumors. Increased lymphoid aggregates were apparent by histological assessment and may account for elevated T cell content. Antibody-based depletion of CD4+ and CD8+ T cells prevented tumor clearance and significantly reduced survival of OT-PDACIL6 mice. The anti-tumor immune response to OT-PDACIL6 rendered mice immune to re-challenge with OT-PDACparental tumors. In high concentrations, IL-6 acts in opposition to previously described pro-tumorigenic effects by enhancing the T cell-mediated anti-tumor response to PDAC.

GDF15 and LCN2 for early detection and prognosis of pancreatic cancer.

BACKGROUND: The prognosis of pancreatic ductal adenocarcinomas (PDAC) remains very poor, emphasizing the critical importance of early detection, where biomarkers offer unique potential. Although growth differentiation factor 15 (GDF15) and Lipocalin 2 (LCN2) have been linked to PDAC, their precise roles as biomarkers are uncertain. METHODS: Circulating levels of GDF15 and LCN2 were examined in human PDAC patients, heathy controls, and individuals with benign pancreatic diseases. Circulating levels of IL-6, CA19-9, and neutrophil-to-lymphocyte ratio (NLR) were measured for comparisons. Correlations between PDAC progression and overall survival were assessed. A mouse PDAC model was employed for comprehensive analyses, complementing the human studies by exploring associations with various metabolic and inflammatory parameters. Sensitivity and specificity of the biomarkers were evaluated. FINDINGS: Our results demonstrated elevated levels of circulating GDF15 and LCN2 in PDAC patients compared to both healthy controls and individuals with benign pancreatic diseases, with higher GDF15 levels associated with disease progression and increased mortality. In PDAC mice, circulating GDF15 and LCN2 progressively increased, correlating with tumor growth, behavioral manifestations, tissue and molecular pathology, and cachexia development. GDF15 exhibited highly sensitive and specific for PDAC patients compared to CA19-9, IL-6, or NLR, while LCN2 showed even greater sensitivity and specificity in PDAC mice. Combining GDF15 and LCN2, or GDF15 and CA19-9, enhanced sensitivity and specificity. INTERPRETATION: Our findings indicate that GDF15 holds promise as a biomarker for early detection and prognosis of PDAC, while LCN2 could strengthen diagnostic panels.

Lipid Nanoparticles Elicit Reactogenicity and Sickness Behavior in Mice Via Toll-Like Receptor 4 and Myeloid Differentiation Protein 88 Axis.

mRNA therapeutics encapsulated in lipid nanoparticles (LNPs) offer promising avenues for treating various diseases. While mRNA vaccines anticipate immunogenicity, the associated reactogenicity of mRNA-loaded LNPs poses significant challenges, especially in protein replacement therapies requiring multiple administrations, leading to adverse effects and suboptimal therapeutic outcomes. Historically, research has primarily focused on the reactogenicity of mRNA cargo, leaving the role of LNPs understudied in this context. Adjuvanticity and pro-inflammatory characteristics of LNPs, originating at least in part from ionizable lipids, may induce inflammation, activate toll-like receptors (TLRs), and impact mRNA translation. Knowledge gaps remain in understanding LNP-induced TLR activation and its impact on induction of animal sickness behavior. We hypothesized that ionizable lipids in LNPs, structurally resembling lipid A from lipopolysaccharide, could activate TLR4 signaling via MyD88 and TRIF adaptors, thereby propagating LNP-associated reactogenicity. Our comprehensive investigation utilizing gene ablation studies and pharmacological receptor manipulation proves that TLR4 activation by LNPs triggers distinct physiologically meaningful responses in mice. We show that TLR4 and MyD88 are essential for reactogenic signal initiation, pro-inflammatory gene expression, and physiological outcomes like food intake and body weight─robust metrics of sickness behavior in mice. The application of the TLR4 inhibitor TAK-242 effectively reduces the reactogenicity associated with LNPs by mitigating TLR4-driven inflammatory responses. Our findings elucidate the critical role of the TLR4-MyD88 axis in LNP-induced reactogenicity, providing a mechanistic framework for developing safer mRNA therapeutics and offering a strategy to mitigate adverse effects through targeted inhibition of this pathway.

TSG-6-Mediated Extracellular Matrix Modifications Regulate Hypoxic-Ischemic Brain Injury.

Proteoglycans containing link domains modify the extracellular matrix (ECM) to regulate cellular homeostasis and can also sensitize tissues/organs to injury and stress. Hypoxic-ischemic (H-I) injury disrupts cellular homeostasis by activating inflammation and attenuating regeneration and repair pathways. In the brain, the main component of the ECM is the glycosaminoglycan hyaluronic acid (HA), but whether HA modifications of the ECM regulate cellular homeostasis and response to H-I injury is not known. In this report, employing both male and female mice, we demonstrate that link-domain-containing proteoglycan, TNFα-stimulated gene-6 (TSG-6), is active in the brain from birth onward and differentially modifies ECM HA during discrete neurodevelopmental windows. ECM HA modification by TSG-6 enables it to serve as a developmental switch to regulate the activity of the Hippo pathway effector protein, yes-associated protein 1 (YAP1), in the maturing brain and in response to H-I injury. Mice that lack TSG-6 expression display dysregulated expression of YAP1 targets, excitatory amino acid transporter 1 (EAAT1; glutamate-aspartate transporter) and 2 (EAAT2; glutamate transporter-1). Dysregulation of YAP1 activation in TSG-6-/- mice coincides with age- and sex-dependent sensitization of the brain to H-I injury such that 1-week-old neonates display an anti-inflammatory response in contrast to an enhanced proinflammatory injury reaction in 3-month-old adult males but not females. Our findings thus support that a key regulator of age- and sex-dependent H-I injury response in the mouse brain is modulation of the Hippo-YAP1 pathway by TSG-6-dependent ECM modifications.

Grb7 Ablation in Mice Improved Glycemic Control, Enhanced Insulin Signaling, and Increased Abdominal fat Mass in Females.

OBJECTIVES: Growth factor receptor bound protein 7 (GRB7) is a multidomain signaling adaptor. Members of the Grb7/10/14 family, specifically Gbrb10/14, have important roles in metabolism. We ablated the Grb7 gene in mice to examine its metabolic function. METHODS: Global ablation of Grb7 in FVB/NJ mice was generated. Growth, organ weight, food intake, and glucose homeostasis were measured. Insulin signaling was examined by Western blotting. Fat and lean body mass was measured by nuclear magnetic resonance, and body composition after fasting or high-fat diet was assessed. Energy expenditure was measured by indirect calorimetry. Expression of adiposity and lipid metabolism genes was measured by quantitative PCR. RESULTS: Grb7-null mice were viable, fertile, and without obvious phenotype. Grb7 ablation improved glycemic control and displayed sensitization to insulin signaling in the liver. Grb7-null females but not males had increased gonadal white adipose tissue mass. Following a 12-week high-fat diet, Grb7-null female mice gained fat body mass and developed relative insulin resistance. With fasting, there was less decrease in fat body mass in Grb7-null female mice. Female mice with Grb7 ablation had increased baseline food intake, less energy expenditure, and displayed a decrease in the expression of lipolysis and adipose browning genes in gonadal white adipose tissue by transcript and protein analysis. CONCLUSION: Our study suggests that Grb7 is a negative regulator of glycemic control. Our results reveal a role for Grb7 in female mice in the regulation of the visceral adipose tissue mass, a powerful predictor of metabolic dysfunction in obesity.

Unraveling the lost balance: Adrenergic dysfunction in cancer cachexia.

Cancer cachexia, characterized by muscle wasting and widespread inflammation, poses a significant challenge for patients with cancer, profoundly impacting both their quality of life and treatment management. However, existing treatment modalities remain very limited, accentuating the necessity for innovative therapeutic interventions. Many recent studies demonstrated that changes in autonomic balance is a key driver of cancer cachexia. This review consolidates research findings from investigations into autonomic dysfunction across cancer cachexia, spanning animal models and patient cohorts. Moreover, we explore therapeutic strategies involving adrenergic receptor modulation through receptor blockers and agonists. Mechanisms underlying adrenergic hyperactivity in cardiac and adipose tissues, influencing tissue remodeling, are also examined. Looking ahead, we present a perspective for future research that delves into autonomic dysregulation in cancer cachexia. This comprehensive review highlights the urgency of advancing research to unveil innovative avenues for combatting cancer cachexia and improving patient well-being.

From Bench to Bedside: Implications of Lipid Nanoparticle Carrier Reactogenicity for Advancing Nucleic Acid Therapeutics.

In biomedical applications, nanomaterial-based delivery vehicles, such as lipid nanoparticles, have emerged as promising instruments for improving the solubility, stability, and encapsulation of various payloads. This article provides a formal review focusing on the reactogenicity of empty lipid nanoparticles used as delivery vehicles, specifically emphasizing their application in mRNA-based therapies. Reactogenicity refers to the adverse immune responses triggered by xenobiotics, including administered lipid nanoparticles, which can lead to undesirable therapeutic outcomes. The key components of lipid nanoparticles, which include ionizable lipids and PEG-lipids, have been identified as significant contributors to their reactogenicity. Therefore, understanding the relationship between lipid nanoparticles, their structural constituents, cytokine production, and resultant reactogenic outcomes is essential to ensure the safe and effective application of lipid nanoparticles in mRNA-based therapies. Although efforts have been made to minimize these adverse reactions, further research and standardization are imperative. By closely monitoring cytokine profiles and assessing reactogenic manifestations through preclinical and clinical studies, researchers can gain valuable insights into the reactogenic effects of lipid nanoparticles and develop strategies to mitigate undesirable reactions. This comprehensive review underscores the importance of investigating lipid nanoparticle reactogenicity and its implications for the development of mRNA-lipid nanoparticle therapeutics in various applications beyond vaccine development.

Development and Perspectives: Multifunctional Nucleic Acid Nanomedicines for Treatment of Gynecological Cancers.

Gynecological malignancies are a significant cause of morbidity and mortality across the globe. Due to delayed presentation, gynecological cancer patients are often referred late in the disease's course, resulting in poor outcomes. A considerable number of patients ultimately succumb to chemotherapy-resistant disease, which reoccurs at advanced stages despite treatment interventions. Although efforts have been devoted to developing therapies that demonstrate reduced resistance to chemotherapy and enhanced toxicity profiles, current clinical outcomes remain unsatisfactory due to treatment resistance and unfavorable off-target effects. Consequently, innovative biological and nanotherapeutic approaches are imperative to strengthen and optimize the therapeutic arsenal for gynecological cancers. Advancements in nanotechnology-based therapies for gynecological malignancies offer significant advantages, including reduced toxicity, expanded drug circulation, and optimized therapeutic dosing, ultimately leading to enhanced treatment effectiveness. Recent advances in nucleic acid therapeutics using microRNA, small interfering RNA, and messenger RNA provide novel approaches for cancer therapeutics. Effective single-agent and combinatorial nucleic acid therapeutics for gynecological malignancies have the potential to transform cancer treatment by giving safer, more tailored approaches than conventional therapies. This review highlights current preclinical studies that effectively exploit these approaches for the treatment of gynecological malignant tumors and malignant ascites.

Nanoparticle-Based Follistatin Messenger RNA Therapy for Reprogramming Metastatic Ovarian Cancer and Ameliorating Cancer-Associated Cachexia.

This study presents the first messenger RNA (mRNA) therapy for metastatic ovarian cancer and cachexia-induced muscle wasting based on lipid nanoparticles that deliver follistatin (FST) mRNA predominantly to cancer clusters following intraperitoneal administration. The secreted FST protein, endogenously synthesized from delivered mRNA, efficiently reduces elevated activin A levels associated with aggressive ovarian cancer and associated cachexia. By altering the cancer cell phenotype, mRNA treatment prevents malignant ascites, delays cancer progression, induces the formation of solid tumors, and preserves muscle mass in cancer-bearing mice by inhibiting negative regulators of muscle mass. Finally, mRNA therapy provides synergistic effects in combination with cisplatin, increasing the survival of mice and counteracting muscle atrophy induced by chemotherapy and cancer-associated cachexia. The treated mice develop few nonadherent tumors that are easily resected from the peritoneum. Clinically, this nanomedicine-based mRNA therapy can facilitate complete cytoreduction, target resistance, improve resilience during aggressive chemotherapy, and improve survival in advanced ovarian cancer.

Neural Mechanisms of Cancer Cachexia.

Nearly half of cancer patients suffer from cachexia, a metabolic syndrome characterized by progressive atrophy of fat and lean body mass. This state of excess catabolism decreases quality of life, ability to tolerate treatment and eventual survival, yet no effective therapies exist. Although the central nervous system (CNS) orchestrates several manifestations of cachexia, the precise mechanisms of neural dysfunction during cachexia are still being unveiled. Herein, we summarize the cellular and molecular mechanisms of CNS dysfunction during cancer cachexia with a focus on inflammatory, autonomic and neuroendocrine processes and end with a discussion of recently identified CNS mediators of cachexia, including GDF15, LCN2 and INSL3.

Chronic cerebral lipocalin 2 exposure elicits hippocampal neuronal dysfunction and cognitive impairment.

Lipocalin 2 (LCN2) is a pleiotropic molecule that is induced in the central nervous system (CNS) in several acute and chronic pathologies. The acute induction of LCN2 evolved as a beneficial process, aimed at combating bacterial infection through the sequestration of iron from pathogens, while the role of LCN2 during chronic, non-infectious disease remains unclear, and recent studies suggest that LCN2 is neurotoxic. However, whether LCN2 is sufficient to induce behavioral and cognitive alterations remains unclear. In this paper, we sought to address the role of cerebral LCN2 on cognition in both acute and chronic settings. We demonstrate that LCN2 is robustly induced in the CNS during both acute and chronic inflammatory conditions, including LPS-based sepsis and cancer cachexia. In vivo, LPS challenge results in a global induction of LCN2 in the central nervous system, while cancer cachexia results in a distribution specific to the vasculature. Similar to these in vivo observations, in vitro modeling demonstrated that both glia and cerebral endothelium produce and secrete LCN2 when challenged with LPS, while only cerebral endothelium secrete LCN2 when challenged with cancer-conditioned medium. Chronic, but not short-term, cerebral LCN2 exposure resulted in reduced hippocampal neuron staining intensity, an increase in newborn neurons, microglial activation, and increased CNS immune cell infiltration, while gene set analyses suggested these effects were mediated through melanocortin-4 receptor independent mechanisms. RNA sequencing analyses of primary hippocampal neurons revealed a distinct transcriptome associated with prolonged LCN2 exposure, and ontology analysis was suggestive of altered neurite growth and abnormal spatial learning. Indeed, LCN2-treated hippocampal neurons display blunted neurite processes, and mice exposed to prolonged cerebral LCN2 levels experienced a reduction in spatial reference memory as indicated by Y-maze assessment. These findings implicate LCN2 as a pathologic mediator of cognitive decline in the setting of chronic disease.

Epigenetic factors Dnmt1 and Uhrf1 coordinate intestinal development.

Intestinal tract development is a coordinated process involving signaling among the progenitors and developing cells from all three germ layers. Development of endoderm-derived intestinal epithelium has been shown to depend on epigenetic modifications, but whether that is also the case for intestinal tract cell types from other germ layers remains unclear. We found that functional loss of a DNA methylation machinery component, ubiquitin-like protein containing PHD and RING finger domains 1 (uhrf1), leads to reduced numbers of ectoderm-derived enteric neurons and severe disruption of mesoderm-derived intestinal smooth muscle. Genetic chimeras revealed that Uhrf1 functions both cell-autonomously in enteric neuron precursors and cell-non-autonomously in surrounding intestinal cells, consistent with what is known about signaling interactions between these cell types that promote one another's development. Uhrf1 recruits the DNA methyltransferase Dnmt1 to unmethylated DNA during replication. Dnmt1 is also expressed in enteric neurons and smooth muscle progenitors. dnmt1 mutants have fewer enteric neurons and disrupted intestinal smooth muscle compared to wildtypes. Because dnmt1;uhrf1 double mutants have a similar phenotype to dnmt1 and uhrf1 single mutants, Dnmt1 and Uhrf1 must function together during enteric neuron and intestinal muscle development. This work shows that genes controlling epigenetic modifications are important to coordinate intestinal tract development, provides the first demonstration that these genes influence development of the ENS, and advances uhrf1 and dnmt1 as potential new Hirschsprung disease candidates.

Evolution of Endothelin signaling and diversification of adult pigment pattern in Danio fishes.

Fishes of the genus Danio exhibit diverse pigment patterns that serve as useful models for understanding the genes and cell behaviors underlying the evolution of adult form. Among these species, zebrafish D. rerio exhibit several dark stripes of melanophores with sparse iridophores that alternate with light interstripes of dense iridophores and xanthophores. By contrast, the closely related species D. nigrofasciatus has an attenuated pattern with fewer melanophores, stripes and interstripes. Here we demonstrate species differences in iridophore development that presage the fully formed patterns. Using genetic and transgenic approaches we identify the secreted peptide Endothelin-3 (Edn3)-a known melanogenic factor of tetrapods-as contributing to reduced iridophore proliferation and fewer stripes and interstripes in D. nigrofasciatus. We further show the locus encoding this factor is expressed at lower levels in D. nigrofasciatus owing to cis-regulatory differences between species. Finally, we show that functions of two paralogous loci encoding Edn3 have been partitioned between skin and non-skin iridophores. Our findings reveal genetic and cellular mechanisms contributing to pattern differences between these species and suggest a model for evolutionary changes in Edn3 requirements for pigment patterning and its diversification across vertebrates.

A TLR/AKT/FoxO3 immune tolerance-like pathway disrupts the repair capacity of oligodendrocyte progenitors.

Cerebral white matter injury (WMI) persistently disrupts myelin regeneration by oligodendrocyte progenitor cells (OPCs). We identified a specific bioactive hyaluronan fragment (bHAf) that downregulates myelin gene expression and chronically blocks OPC maturation and myelination via a tolerance-like mechanism that dysregulates pro-myelination signaling via AKT. Desensitization of AKT occurs via TLR4 but not TLR2 or CD44. OPC differentiation was selectively blocked by bHAf in a maturation-dependent fashion at the late OPC (preOL) stage by a noncanonical TLR4/TRIF pathway that induced persistent activation of the FoxO3 transcription factor downstream of AKT. Activated FoxO3 selectively localized to oligodendrocyte lineage cells in white matter lesions from human preterm neonates and adults with multiple sclerosis. FoxO3 constraint of OPC maturation was bHAf dependent, and involved interactions at the FoxO3 and MBP promoters with the chromatin remodeling factor Brg1 and the transcription factor Olig2, which regulate OPC differentiation. WMI has adapted an immune tolerance-like mechanism whereby persistent engagement of TLR4 by bHAf promotes an OPC niche at the expense of myelination by engaging a FoxO3 signaling pathway that chronically constrains OPC differentiation.