Endogenous targeting lipid nanoparticles for systemic mRNA delivery to lung cancer tumors.

Diabetes mellitus (DM) is a frequent and debilitating complication in patients with β-thalassemia major (β-TM), driven by chronic iron overload-induced pancreatic and hepatic dysfunction. Validated biomarkers for early diabetes detection in this population remain limited. Follistatin (FST), a secreted glycoprotein modulating TGF-β/activin signaling and glucose metabolism, has been associated with Type 2 diabetes (T2D), though its relevance to thalassemia-associated diabetes is unexplored. This cross-sectional study examined plasma FST levels and gene expression relative to diabetic status and glycemic control in 134 participants: 80 β-TM/non-diabetic, 34 β-TM/diabetic patients, and 20 healthy controls. Iron modulation effects were explored in HepG2 hepatocytes using ferric ammonium citrate (FAC) or deferoxamine (DFO), and metabolic stress effects were assessed in healthy PBMCs exposed to high glucose or insulin. Plasma FST was significantly elevated in β-TM/diabetic patients versus β-TM/non-diabetic patients or controls (p < 0.001). FST correlated positively with glucose and fructosamine (r = 0.23; p ≤ 0.02), but not with serum ferritin. A stepwise increase in plasma FST was observed across glycemic control subgroups, from 2421 pg/mL in well-controlled to 3831 pg/mL in poorly controlled diabetics (p < 0.0001). FAC upregulated FST mRNA in HepG2 cells, while DFO suppressed it (p < 0.05). Peripheral Blood Mononuclear Cells (PBMCs) FST mRNA was paradoxically lower in β-TM/diabetic patients (p = 0.02), a pattern reproduced in PBMCs under high glucose or insulin, indicating tissue-specific metabolic regulation. Collectively, these findings suggest that elevated plasma FST may reflect a convergence of iron-induced hepatic stress and hyperglycemia-related dysregulation and warrant further cross-sectional investigations as a candidate biomarker in this population.

Pulmonary arterial hypertension remains a life-threatening disease despite advances in vasodilator therapy. Vascular remodeling, partly driven by pulmonary artery endothelial cell dysfunction, is accompanied by vasoactive mediators imbalance such as ET-1 (endothelin-1). Although endothelin receptor antagonists alleviate vasoconstriction, they incompletely address the remodeling process. We previously reported how endothelial-derived activin A promotes vascular remodeling, leading to the clinical development of the activin signaling inhibitor sotatercept, which improves outcomes when added to endothelin receptor antagonists. As both activin A and ET-1 originate from endothelial cells and promote remodeling, we investigated whether activin A regulates ET-1 production and activity in pulmonary arterial hypertension. In vitro, we used pulmonary artery endothelial cell models of activin A overabundance alone or cocultured with pulmonary artery smooth muscle cells. Cells were treated with either the activin A inhibitor FST (follistatin), the endothelin receptor antagonist bosentan, the FST/bosentan combination, or vehicle for analysis. In vivo, we exposed wild-type or endothelial-specific INHBA (inhibin β-A)-overexpressing mice (VEcadherin-INHBA-Transgenic/VEcad-INHBA-Tg) to chronic hypoxia pulmonary hypertension model, with the addition of FST, bosentan, FST and bosentan, or vehicle treatments after the first week of hypoxia exposure. Activin A upregulated ET-1 expression via canonical SMAD2/3 (small mother against decapentaplegic family member 2/3) signaling in pulmonary artery endothelial cells. This induction, as well as ET-1-driven downstream effects-including reduced eNOS (endothelial NO synthase), pulmonary artery smooth muscle cell phenotypic switching, oxidative stress, and endothelial-to-mesenchymal transition-was reversed by FST alone or in combination with bosentan. In vivo, FST-based therapy achieved greater hemodynamic, right ventricular remodeling, and vascular structural normalization in wild-type and VEcad-INHBA-Tg mice than bosentan alone, accompanied by stronger ET-1 suppression. We identified ET-1 as a downstream effector of activin A in pulmonary arterial hypertension development, supporting activin A blockade as a strategy to inhibit ET-1-mediated vasoconstriction and remodeling. This mechanistic link provides a rationale for the rapid clinical benefits observed with sotatercept and suggests its potential role earlier in the pulmonary arterial hypertension treatment paradigm.

The pathogenesis of endometriosis (EM) remains intricate and multifactorial. This study aimed to investigate the biological functions of microRNA-769-5p (miR-769-5p) in an in vitro model of EM. Serum was obtained from 120 EM patients and 100 healthy controls. miRNA and mRNA expression were quantified by RT-qPCR. Functional analyses of miR-769-5p were performed in vitrousing cell counting kit-8 (CCK-8) cell proliferation assays, Transwell assays, and dual-luciferase assays in an endometriosis-derived stromal cell line (hEM15A). miR-769-5p was markedly down-regulated in EM sera and correlated inversely with disease severity (p < 0.001). Moreover, miR-769-5p exhibited high diagnostic accuracy for EM (AUC = 0.9166, p < 0.001). In the hEM15A cell model, overexpression of miR-769-5p effectively suppressed the proliferation, migration, and invasion capabilities of EM-derived stromal cells (p < 0.001). miR-769-5p targeted follistatin (FST) and negatively regulated its expression in vitro (p < 0.001). In contrast, FST overexpression could partially reverse the inhibitory effects of miR-769-5p-mimic on these EM-derived cells. Our findings indicate that miR-769-5p is downregulated in EM serum. Furthermore, in vitro experiments suggest that it can target FST to inhibit proliferation, migration, and invasion of ectopic endometrial stromal cells.

Hepatocellular carcinoma (HCC) is a malignant tumor originating from hepatocytes, characterized by high mortality rates. Follistatin (FST), an inhibitor of follicle-stimulating hormone, correlates with poor prognosis in some cancers, but its role in HCC is unclear. This study intends to clarify FST's impact on HCC prognosis and clinical characteristics. FST expression was compared between HCC and adjacent tissues using Clinical Proteomic Tumor Analysis Consortium (CPTAC) and The Cancer Genome Atlas (TCGA) databases. 78 HCC patients were dichotomized into FST-high and FST-low groups based on the median expression of FST assessed by immunohistochemistry. Kaplan-Meier analysis evaluated recurrence-free (RFS) and overall survival (OS). Univariate/multivariate Cox regression identified OS risk factors; logistic regression analyzed FST's association with clinical features. FST was upregulated in HCC tissues. High FST correlated with Larger tumor size (OR = 2.030, P = 0.025), Microvascular invasion (OR = 1.933, P = 0.037), Elevated AFP (OR = 2.046, P = 0.024). Patients in the FST-high group had shorter mean OS (53.189 ± 6.37 vs. 94.832 ± 7.739 months, P = 0.0084). Multivariate Cox confirmed FST as an independent OS predictor (HR = 3.88, P = 0.0003), alongside tumor size (HR = 4.33, P = 0.0025) and gender (HR = 0.309, P = 0.0421). FST is upregulated in HCC tissues and serves as an independent prognostic factor for poor overall survival. High FST expression was strongly correlated with aggressive clinicopathological features, including larger tumor burden, microvascular invasion (MVI), and elevated AFP.

High hyaluronan binding and RHAMM expression identify an invasive and metastatic subpopulation in androgen-resistant prostate cancer cells.

In Vivo modeling of leukemic evolution in ELANE-associated severe congenital neutropenia

BackgroundMyostatin (MSTN) is a well-known negative regulator of skeletal muscle growth, and its pharmacological blockade, such as with follistatin (FST), an endogenous MSTN inhibitor, is under active investigation as a treatment for muscle-wasting conditions. However, the dynamics of MSTN signaling during in vitro myogenesis and its modulation by culture conditions remain incompletely understood.MethodsPrimary myoblasts were isolated from wild-type (WT), Mstn−/−, and FST transgenic (F66) mice to evaluate the effects of MSTN inhibition on myotube formation. Myoblasts were differentiated on Matrigel-coated surfaces in the presence of horse serum. Myotube maturation was evaluated by confocal microscopy and reverse transcription-polymerase chain reaction; protein synthesis was assessed using a puromycin incorporation assay; and MSTN concentrations in serum were quantified by enzyme-linked immunosorbent assay.ResultsMstn expression peaked at 24 hr of differentiation, coinciding with early myotube formation, and progressively declined as myotubes matured. Both Mstn−/− and F66 myotubes showed increased thickness and protein synthesis compared to WT controls, with more pronounced effects observed in F66 myotubes. Consistent with this, F66 myotubes also displayed higher expression of myogenic maturation markers. Notably, horse serum used in the culture medium contained detectable levels of MSTN, which may have partially restored MSTN signaling in Mstn−/− cultures and masked the full knockout phenotype.ConclusionsFST overexpression induces greater myotube hypertrophy and protein synthesis than Mstn deletion, likely due to its broader inhibition of both endogenous and serum-derived MSTN. These findings highlight the importance of serum composition in interpreting phenotypes from in vitro knockout models targeting secreted factors such as MSTN.

To investigate the causal relationship between follistatin (FST) levels and endocrine diseases such as polycystic ovary syndrome (PCOS), type 2 diabetes (T2DM), obesity, and osteoporosis (OP) using a 2-sample Mendelian randomization (MR) analysis. Instrumental variables closely associated with FST levels were obtained from large-scale genome-wide association study data in the IEU database. Summary-level data for 4 endocrine diseases were sourced from the latest version of the FinnGen database. Our primary method for MR analysis was the inverse-variance weighted (IVW) method, supplemented by the MR-Egger and Weighted Median methods. We conducted a series of sensitivity tests to assess the reliability of our MR results. The IVW analysis revealed a significant causal relationship between elevated levels of FST and both PCOS (odds ratio [OR] = 1.129, 95% confidence interval [CI]: 1.042–1.224, P = .003) and T2DM (OR = 1.103, 95% CI: 1.02–1.187, P = .01). However, the IVW model did not indicate a causal connection between FST levels and either OP (OR = 1.061, 95% CI: 0.909–1.238, P = .452) or obesity (OR = 1.082, 95% CI: 0.983–1.192, P = .108). The reverse MR analysis results indicated a causative association between T2DM (OR = 1.047, 95% CI: 1.006–1.089, P = .023) and an elevation in FST levels, as well as a causal link between OP (OR = 0.889, 95% CI: 0.804–0.982, P = .021) and a reduction in FST levels. There is no direct causality between PCOS (OR = 0.925, 95% CI: 0.778–1.098, P = .372), obesity (OR = 1.035, 95% CI: 0.968–1.107, P = .312), and FST levels. In addition, our sensitivity tests, which included a pleiotropy test, heterogeneity test, and “leave-one-out” analysis, consistently confirmed the reliability of our results. Genetically predicted high FST levels are causally associated with increased risks of PCOS and T2DM, indicating a potential role in endocrine disease pathogenesis. Moreover, reverse MR analysis revealed a significant causal link between OP and decreased FST levels, suggesting that FST may serve as a promising biomarker or therapeutic target in bone metabolism.

Muscle growth is a critical determinant of meat yield and quality in livestock. Although follistatin (FST) is recognized as a key regulator of skeletal muscle development and fat metabolism, its specific function in geese remains largely unexplored. In this study, we identified two transcript variants of goose FST (gFST) in Zhedong White geese: gFST-X1 (1125 bp), encoding a 343-amino acid protein with a 28-amino acid signal peptide and four conserved domains, and gFST-X2, which contains a 243 bp insertion within the gFST-X1 transcript. RT-qPCR analysis revealed that gFST mRNA expression varied across tissues from female embryos (25 days), adults (70 days), and laying geese (270 days), as well as in skeletal muscle satellite cells (SMSCs) at embryonic day 16 (E16d). Overexpression of gFST in SMSCs resulted in 3596 differentially expressed genes (DEGs), including 2247 upregulated and 1349 downregulated genes (padj < 0.01). Key stemness markers (PAX7, PAX3) and myogenic regulators (MYOG, MYOD, MYF5) were significantly downregulated, whereas genes associated with lipid metabolism (PPARG, FABP5, ACSL5) and myosin-related processes (MYO1D, MYO1F, MYO1E) were markedly upregulated (padj < 0.01). Functional enrichment analysis linked these DEGs to the TGF-β, PPAR signaling, fatty acid metabolism, and Notch signaling pathways. These transcriptomic findings were further validated by qRT-PCR. Collectively, our results demonstrate the dual regulatory role of gFST in skeletal muscle development and provide new mechanistic insights into muscle development in geese.

ABSTRACTThe Gouldian finch exhibits a head color polymorphism, which is tightly coupled to distinct differences in aggression, stress responses, and parental care. In competitive environments, red‐headed birds are more aggressive, are less parental, and exhibit a heightened stress response relative to black‐headed birds. The head color polymorphism has been associated with genetic variation in a small noncoding region near the follistatin (FST) gene. Given the regulatory nature of this gene, we hypothesized that FST mRNA would be differentially expressed in association with morph‐ and sex‐specific differences in the brains of red‐ and black‐headed morphs. To test this hypothesis, we analyzed FST gene expression in the Gouldian finch brain using RNAscope in situ hybridization assay. Our results revealed significant differences in FST gene expression between morphs and sex. Specifically, black‐headed morphs, regardless of sex, displayed higher FST mRNA levels across multiple brain regions associated with aggression, stress responses, and parental care compared to red‐headed morphs. Furthermore, males consistently showed greater FST mRNA levels within the same morph type than females. These findings suggest that head color morph‐ and sex‐specific differences in FST gene expression may underlie the observed morph‐ and sex‐specific differences in aggression, stress responses, and parental care in Gouldian finches.

High-grade serous ovarian carcinoma (HGSOC) is associated with high mortality rates due to late-stage diagnosis and limited treatment options. We investigated the role of FSTL3 in ovarian cancer progression both as a prognostic biomarker and as a potential therapeutic target.We measured levels of follistatin (FST) and follistatin-like 3 (FSTL3) in 96 ovarian cancer patient ascites samples and found that FSTL3 overexpression was more predominant than FST and associated with poorer survival outcomes. Mice implanted with an HGSOC syngeneic cell line bearing common alterations in ovarian cancer (KRASG12 V, P53R172H, CCNE1oe, AKT2oe) had increasing levels of FST and FSTL3 in serum during tumor growth. Further alteration of this model to generate a knockout of FST (KPCA.FSTKO) and an overexpression of human FSTL3 (KPCA.FSTKO_hFSTL3) revealed that FSTL3 expression was associated with a more fibrotic tumor microenvironment, correlating with an increased abundance of cancer-associated myofibroblasts (myCAFs), and cancer cells with a more mesenchymal phenotype. Tumors overexpressing FSTL3 also had significantly less immunocyte infiltration, reduced intratumoral T-cell abundance, and increased CD8+ T cell exhaustion. FSTL3 overexpression completely abrogated tumor response to PPC treatment (Prexasertib combined with PD-1 and CTLA-4 blockade) compared to controls, suggesting that FSTL3 may be involved in immunotherapy resistance. In conclusion, this study suggests a role for FSTL3 as a prognostic marker and as therapeutic target in HGSOC, where it may play a role in promoting a mesenchymal tumor phenotype, maintaining an immunosuppressive tumor microenvironment, and driving immunotherapy resistance.

This study provides the first evidence related to the identification of microbial strains closely associated with muscle strength enhancement, independent of the host’s genetic background. Fecal transplants from humans into mice revealed a significant impact of gut bacteria on muscle strength, with some mice experiencing increases, while others showed no change or decreases. Interestingly, analysis of the fecal and gastrointestinal tract bacteria from each mouse classified by the degree of muscle strength revealed significant differences based on muscle strength. Furthermore, a more diverse microbial community was observed in the gastrointestinal tract compared to the feces. Further investigation identified two bacterial species, Lactobacillus johnsonii (L. johnsonii) and Limosilactobacillus reuteri (L. reuteri), that are related to improved muscle strength. Indeed, we confirmed that the supplementation with these bacteria in aged mice significantly enhanced their muscle strength by increasing the mRNA expression levels of follistatin (FST) and insulin-like growth factor-1 (IGF1) in muscle tissue. Overall, this study provides the first evidence that specific gut bacteria can directly improve muscle strength and introduces a novel approach to studying the gut microbiome’s influence on complex traits.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-15222-2.

ABSTRACTAimTo perform a genome‐wide association study (GWAS) for periodontitis in the FinnGen cohort, as genetic factors contribute to periodontitis.Materials and MethodsWe included nearly 250,000 Finnish individuals who had visited a dentist in the public healthcare sector for a clinical oral examination. We designed three periodontitis phenotypes based on diagnosis and procedure codes and CPI indexes in national health registers.ResultsWe identified 11 independent genetic loci associated with periodontitis, among which 6 were common and novel. A locus near the FST gene was associated with two phenotypes, whereas other lead SNPs were located near ARL15, MFHAS1, DEFB130A and APOE. Additionally, all phenotypes in the discovery and replication cohorts were associated with genetic variations in the HLA region. Furthermore, imputed HLA allele frequencies identified independent associations between HLA‐DRB1, HLA‐DPB1 and HLA‐DQA1 and periodontitis. Based on single‐cell RNA sequencing, the expression of genes near our lead SNPs across all three phenotypes was particularly enriched in gingival cell lineages important in the pathogenesis of periodontitis. Phenotypical and genetic correlations revealed associations between periodontitis and bacterial diseases, as well as autoimmune and cardiometabolic phenotypes.ConclusionsOur GWAS suggests that genetic variation contributing to immune dysregulation is involved in the pathogenesis of periodontitis, which has considerable genetic similarity with other complex traits.

Background/Objectives: To investigate associations between Follistatin (FST) gene polymorphisms (SNPs) and baseline musculoskeletal traits, and their interactions with 16-week exercise interventions. Methods: A cohort of 470 untrained Northern Han Chinese adults (208 males, 262 females), sourced from the "Research on Key Technologies for an Exercise and Fitness Expert Guidance System" project, was analyzed. These participants were previously randomly assigned to one of four exercise groups (Hill, Running, Cycling, Combined) or a non-exercising Control group, and completed their respective 16-week protocols. Body composition, bone mineral content (BMC), bone mineral density (BMD), and serum follistatin levels were all assessed pre- and post-intervention. Dual-energy X-ray absorptiometry (DXA) was utilized for the body composition, BMC, and BMD measurements. FST SNPs (rs3797296, rs3797297) were genotyped using matrix assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF MS) or microarrays. To elucidate the biological mechanisms, we performed in silico functional analyses for rs3797296 and rs3797297. Results: Baseline: In females only, the rs3797297 T allele was associated with higher muscle mass (β = 1.159, 95% confidence interval (CI): 0.202-2.116, P_adj = 0.034) and BMC (β = 0.127, 95% CI: 0.039-0.215, P_adj = 0.009), with the BMC effect significantly mediated by muscle mass. Exercise Response: Interventions improved body composition, particularly in females. Gene-Exercise Interaction: A significant interaction occurred exclusively in women undertaking hill climbing: the rs3797296 G allele was associated with attenuated muscle mass gains (β = -1.126 kg, 95% CI: -1.767 to -0.485, P_adj = 0.034). Baseline follistatin correlated with body composition (stronger in males) and increased post-exercise (primarily in males, Hill/Running groups) but did not mediate SNP effects on exercise adaptation. Functional annotation revealed that rs3797297 is a likely causal variant, acting as a skeletal muscle eQTL for the mitochondrial gene NDUFS4, suggesting a mechanism involving muscle bioenergetics. Conclusions: Findings indicate that FST polymorphisms associate with musculoskeletal traits in Northern Han Chinese. Mechanistic insights from functional annotation reveal potential pathways for these associations, highlighting the potential utility of these genetic markers for optimizing training program design.

Abstract Background: Proteins secreted by cancer-associated fibroblasts (CAFs) can influence inflammation and recruit external immune cells to the tumor microenvironment. However, the cross[1]talk between CAFs and immune cells in the tumor microenvironment is not well understood. Therefore, in this study, we determined the association of proteins secreted by the CAF secretome with immune cell infiltration in patients diagnosed with breast cancer (BC) and ovarian cancer (OC). Methods: The Human Protein Atlas was explored to identify potential gene targets associated with the CAFs secretome, including inhibin subunit beta A (INHBA), Dickkopf-related protein 3 (DKK3), and follistatin (FST). BC and OC patient data sets (n=14 samples) in the Gene Expression Omnibus were analyzed as discovery cohorts to compare CAFs and normal fibroblasts. Kaplan-Meier Plotter, Gene Expression Profiling Interactive Analysis, and Tumor Immune Estimation Resource bioinformatics databases were explored to computationally understand relationships between gene expression, tumor prognosis, and immune infiltration. Single cell analysis datasets and opal multiple staining were used to validate the contribution of CAFs to overexpression of the gene targets in BC and OC cancer patients. Proteogenomic BC and OC data sets (n=46) from Clinical Proteomic Tumor Analysis Consortium were used for validation of the correlation between marker expression and immune cell infiltration. Results: Increased expression of INHBA, DKK3, and FST correlated with shortened survival of patients with BC and OC. All three genes were negatively associated with multiple tumor-infiltrating immune cell types, both in RNA and protein levels. INHBA expression was positively correlated with the expression of T cell exhaustion markers. Pathway analysis demonstrates that INHBA’s role in immune infiltration may be a result of its association with the Smad2 signaling pathway, which promotes the transition of fibroblasts to activated CAFs via an inflammatory response. In BC and OC, the three genes were generally positively correlated with immune cells characterized as “protumor”, such as macrophages, CAFs, and neutrophils and negatively correlated with those considered as “antitumor,” such as B cells. Conclusion: INHBA, DKK3, and FST are candidate therapeutic targets for the treatment of breast, ovarian, and other cancers. Their therapeutic effect should be evaluated in the context of regulating infiltration and inflammation of the tumor microenvironment. Citation Format: Anjali Agrawal, Elaine Stur, Emine Bayraktar, Sara Corvigno, Kirill Pevzner, Gali Arad, Yibo Dai, Sisy Chen, Robiya Joseph, Nitzan Simchi, Eran Seger, Cristina Ivan, Anil Sood. Potential Impact of the Cancer-Associated Fibroblast Secretome in the Tumor Microenvironment [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P2-06-04.

Synbiotics can be used to reduce intestinal inflammation and mitigate dysbiosis in dogs with chronic inflammatory enteropathy (CIE). Prior research has not assessed the colonic mucosal ultrastructure of dogs with active CIE treated with synbiotics, nor has it determined a possible association between morphologic injury and signaling pathways. Twenty client-owned dogs diagnosed with CIE were randomized to receive either a hydrolyzed diet (placebo; PL) or a hydrolyzed diet supplemented with synbiotic-IgY (SYN) for 6 weeks. Endoscopic biopsies of the colon were obtained for histopathologic, ultrastructural, and molecular analyses and were compared before and after treatment. Using transmission electron microscopy (TEM), an analysis of the ultrastructural alterations in microvilli length (MVL), mitochondria (MITO), and rough endoplasmic reticulum (ER) was compared between treatment groups. To explore potential signaling pathways that might modulate MITO and ER stress, a transcriptomic analysis was also performed. The degree of mucosal ultrastructural pathology differed among individual dogs before and after treatment. Morphologic alterations in enterocytes, MVL, MITO, and ER were detected without significant differences between PL and SYN dogs prior to treatment. Notable changes in ultrastructural alterations were identified post-treatment, with SYN-treated dogs exhibiting significant improvement in MVL, MITO, and ER injury scores compared to PL-treated dogs. Transcriptomic profiling showed many pathways and key genes to be associated with MITO and ER injury. Multiple signaling pathways and their associated genes with protective effects, including fibroblast growth factor 2 (FGF2), fibroblast growth factor 7 (FGF7), fibroblast growth factor 10 (FGF10), synaptic Ras GTPase activating protein 1 (SynGAP1), RAS guanyl releasing protein 2 (RASGRP2), RAS guanyl releasing protein 3 (RASGRP3), thrombospondin 1 (THBS1), colony stimulating factor 1 (CSF1), colony stimulating factor 3 (CSF3), interleukin 21 receptor (IL21R), collagen type VI alpha 6 chain (COL6A6), ectodysplasin A receptor (EDAR), forkhead box P3 (FoxP3), follistatin (FST), gremlin 1 (GREM1), myocyte enhancer factor 2B (MEF2B), neuregulin 1 (NRG1), collagen type I alpha 1 chain (COL1A1), hepatocyte growth factor (HGF), 5-hydroxytryptamine receptor 7 (HTR7), and platelet derived growth factor receptor beta (PDGFR-β), were upregulated with SYN treatment. Differential gene expression was associated with improved MITO and ER ultrastructural integrity and a reduction in oxidative stress. Conversely, other genes, such as protein kinase cAMP-activated catalytic subunit beta (PRKACB), phospholipase A2 group XIIB (PLA2G12B), calmodulin 1 (CALM1), calmodulin 2 (CALM2), and interleukin-18 (IL18), which have harmful effects, were downregulated following SYN treatment. In dogs treated with PL, genes including PRKACB and CALM2 were upregulated, while other genes, such as FGF2, FGF10, SynGAP1, RASGRP2, RASGRP3, and IL21R, were downregulated. Dogs with CIE have colonic ultrastructural pathology at diagnosis, which improves following synbiotic treatment. Ultrastructural improvement is associated with an upregulation of protective genes and a downregulation of harmful genes that mediate their effects through multiple signaling pathways.

INTRODUCTION: Metastatic ovarian cancer, often accompanied by cachexia, is a deadly gynecological malignancy. Activin A (ActA), overexpressed in ovarian, endometrial, and cervical cancers, promotes tumor growth, metastasis, and muscle atrophy. Follistatin (FST), an ActA inhibitor, is a promising therapeutic target. Lipid nanoparticles (LNPs), proven effective in COVID-19 vaccines, offer efficient mRNA delivery for transient protein expression. This study investigates LNP-based Fst mRNA therapy to inhibit ActA activity in ovarian cancer and cachexia. Given the scarcity of murine gynecological cancer models overexpressing ActA, we also evaluated efficacy in ActA-overexpressing head and neck and lung cancer models. METHODS: Fst mRNA LNPs were administered intraperitoneally in murine models of metastatic ovarian cancer, alone and combined with cisplatin. Outcomes included cancer progression, serum ActA levels, muscle mass, cachexia markers, and survival. RESULTS: Fst mRNA LNP therapy reduced serum ActA levels twofold. Combined with cisplatin, it extended survival to 33.8 ± 2.4 days versus 21.2 ± 1.66 days (cisplatin-only). It reversed 37% muscle loss in controls, increased muscle fiber cross-sectional area by 90%, and decreased malignant ascites fivefold, forming easily resectable nonadherent tumors. Similar reductions in cachexia and metastasis were observed in head and neck and lung cancer models overexpressing ActA. CONCLUSIONS/IMPLICATIONS: Fst mRNA LNP therapy offers a novel approach to treating metastatic ovarian cancer and associated cachexia by inhibiting ActA. It delays tumor progression, reduces muscle wasting, and significantly decreases malignant ascites. Leveraging LNPs, proven effective in COVID-19 vaccines, this therapy shows promise for improving outcomes in ovarian cancer patients, warranting further clinical investigation.

Chronic pain, encompasses neuropathic and inflammatory pain, is a major public health burden. The insulin-like growth factor 1 receptor (IGF1R) has emerged as a critical player in pain modulation, exhibiting dual roles in both pain promotion and resolution. Recent studies have identified Follistatin (FST) as a novel ligand for IGF1R in neuropathic pain, where it activates the ERK/AKT signaling pathway, enhances Nav1.7-mediated sodium channel function, and induces neuronal hyperexcitability. Targeting the FST-IGF1R interaction with antagonist peptides has shown promise in alleviating neuropathic pain, highlighting its therapeutic potential. Conversely, IGF1/IGF1R signaling has also been implicated in pain resolution, particularly through CD11c+ microglia in the spinal dorsal horn, which promote recovery from neuropathic pain by phagocytosing myelin debris and modulating inflammatory responses. These findings underscore the context-dependent nature of IGF1R signaling, which can drive both nociceptive hypersensitivity and pain relief. This work synthesizes recent advances in understanding the multifaceted roles of IGF1R in chronic pain, offering novel insights into its mechanisms and therapeutic applications, and paving the way for the development of targeted therapies to address the complex challenges of chronic pain management.

Abstract Head and Neck Squamous Cell Carcinoma (HNSCC) is a common malignancy with limited therapeutic options. The initiation and progression of HNSCC is closely tied to epithelial cell fate decisions influenced by the tumor microenvironment (TME). The Transforming Growth Factor-β (TGF-β) Superfamily of ligands are pleiotropic growth factors and key mediators of cell-cell communication within the TME. Although TGF-β has been extensively studied for its critical role in cancer progression, therapeutic strategies targeting TGF-β have been largely unsuccessful. Such efforts have often overlooked the potential contribution of myriad factors that influence TGF-β signaling such as follistatin (FST), a secreted glycoprotein and potent inhibitor of TGF-β. FST is frequently upregulated in solid tumors where it is associated with dismal survival outcomes and reduced therapeutic responses. However, the molecular mechanisms by which FST counterbalances TGF-β and potentially other signaling pathways to modulate the TME is not well understood. This underscores the critical need for a more nuanced and molecular understanding of the FST driven networks to develop more effective therapeutic approaches. Here we examined the tumor cell intrinsic and extrinsic roles of FST within the TME of HNSCC by leveraging single cell RNA-seq and spatial transcriptomics datasets from HNSCC patients and analyzing transcriptional changes following depletion of FST in human HNSCC cell lines. We find that while fibroblasts and epithelial cells both express FST, tumor epithelial cells are the primary source of heightened FST levels in HNSCC. Notably, we discover that FST expression is intrinsically linked to a transcriptional program promoting a basal stem-like state in cancer epithelial cells, suggesting a role for FST in tumor plasticity. Consistently, tumor cells that express FST exhibit increased signaling and molecular attributes of the epithelial-to-mesenchymal transition (EMT) program and reduced differentiation. Since cancer stem cells are enriched at the tumor leading edge, where they leverage EMT processes for dissemination, we investigated the spatial distribution of FST-enriched tumor cells and their interactions with the surrounding extracellular milieu. Our analyses show that FST is enriched within the tumor leading edge invasive niche, and further, that tumor-stroma crosstalk via COL1A1-CD44 interactions is spatially localized within FST-rich domains. Finally, we find CD44 positively correlates with FST expression in patient tumors and cancer cells at the transcriptional and protein level. Collectively, our findings define the functional role of FST within the HNSCC TME and supports a model in which FST counterbalances TGF-β signaling and facilitates distinct epithelial-stroma interactions at the dynamic leading edge to enhance tumor aggression. Citation Format: Jennifer Sosa, Satrajit Sinha. Follistatin shapes the tumor microenvironment in head and neck squamous cell carcinoma through COL1A1-CD44-mediated tumor-stroma crosstalk [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5231.