Cell Dysfunction Research Articles

Decoding the Significance of Alpha Cell Function in the Pathophysiology of Type 1 Diabetes.

Alpha cells in the pancreas, traditionally known for their role in secreting glucagon to regulate blood glucose levels, are gaining recognition for their involvement in the pathophysiology of type 1 diabetes (T1D). In T1D, autoimmune destruction of beta cells results in insulin deficiency, which in turn may dysregulate alpha cell function, leading to elevated glucagon levels and impaired glucose homeostasis. This dysfunction is characterized by inappropriate glucagon secretion, augmenting the risk of life-threatening hypoglycemia. Moreover, insulin deficiency and autoimmunity alter alpha cell physiological responses, further exacerbating T1D pathophysiology. Recent studies suggest that alpha cells undergo transdifferentiation and interact with beta cells through mechanisms involving gamma-aminobutyric acid (GABA) signaling. Despite these advances, the exact pathways and interactions remain poorly understood and are often debated. Understanding the precise role of alpha cells in T1D is crucial, as it opens up avenues for developing new therapeutic strategies for T1D. Potential strategies include targeting alpha cells to normalize glucagon secretion, utilizing glucagon receptor antagonists, enhancing GABA signaling, and employing glucagon-like peptide-1 (GLP-1) receptor agonists. These approaches aim to improve glycemic control and reduce the risk of hypoglycemic events in individuals with T1D. This review provides an overview of alpha cell function in T1D, highlighting the emerging focus on alpha cell dysfunction in the context of historically well-developed beta cell research.

Arachidonic acid impairs natural killer cell functions by disrupting signaling pathways driven by activating receptors and reactive oxygen species

BackgroundHigh levels of the polyunsaturated fatty acid arachidonic acid (AA) within the ovarian carcinoma (OC) microenvironment correlate with reduced relapse-free survival. Furthermore, OC progression is tied to compromised immunosurveillance, partially attributed to the impairment of natural killer (NK) cells. However, potential connections between AA and NK cell dysfunction in OC have not been studied.MethodsWe employed a combination of phosphoproteomics, transcriptional profiling and biological assays to investigate AA’s impact on NK cell functions.ResultsAA (i) disrupts interleukin-2/15-mediated expression of pro-inflammatory genes by inhibiting STAT1-dependent signaling, (ii) hampers signaling by cytotoxicity receptors through disruption of their surface expression, (iii) diminishes phosphorylation of NKG2D-induced protein kinases, including ERK1/2, LYN, MSK1/2 and STAT1, and (iv) alters reactive oxygen species production by transcriptionally upregulating detoxification. These modifications lead to a cessation of NK cell proliferation and a reduction in cytotoxicity.ConclusionOur findings highlight significant AA-induced alterations in the signaling network that regulates NK cell activity. As low expression of several NK cell receptors correlates with shorter OC patient survival, these findings suggest a functional linkage between AA, NK cell dysfunction and OC progression.

Reconstructing the Stem Leydig Cell Niche via the Testicular Extracellular Matrix for the Treatment of Testicular Leydig Cell Dysfunction

AbstractTherapies involving the use of stem Leydig cells (SLCs), as testicular mesenchymal stromal cells, have shown great promise in the treatment of Leydig cell (LC) dysfunction in aging males. However, the outcomes of these therapies are not satisfactory. In this study, it is demonstrated that the aging microenvironment of the testicular interstitium impairs the function of SLCs, leading to poor regeneration of LCs and, consequently, inefficient functional restoration. The study develops a decellularized testicular extracellular matrix (dTECM) hydrogel from young pigs and evaluates its safety and feasibility as a supportive niche for the expansion and differentiation of SLCs. dTECM hydrogel facilitates the steroidogenic differentiation of SLCs into LCs, the primary producers of testosterone. The combination of SLCs with a dTECM hydrogel leads to a significant and sustained increase in testosterone levels, which promotes the restoration of spermatogenesis and fertility in an LC‐deficient and aged mouse model. Mechanistically, collagen 1 within the dTECM is identified as a key factor contributing to these effects. Notably, symptoms associated with testosterone deficiency syndrome are significantly alleviated in aged mice. These findings may aid the design of therapeutic interventions for patients with testosterone deficiency in the clinic.

Diabetes Mellitus Associated with Maternally Inherited Diabetes and Deafness (MIDD): From Pathogenic Variant to Phenotype.

Maternally inherited diabetes and deafness (MIDD) is a monogenic mitochondrial disorder caused by a pathogenic variant in the MT-TL1 gene encoding for a leucine transfer RNA. We propose a new hypothesis that explains how the MT-TL1 variant causes impaired glucose tolerance and diabetes in MIDD. We suggest that diabetes in MIDD primarily depends on a variable combination of insulin resistance and impaired beta cell function that seems more likely to occur in the presence of high skeletal muscle heteroplasmy and moderate beta cell heteroplasmy for m.3243A>G. The underlying genetic defect generates oxidative stress and disrupts the tricarboxylic acid cycle, leading to mTORC1 hyperactivity and modifying mitochondrial retrograde signalling. mTORC1 hyperactivity contributes to insulin resistance and beta cell dysfunction and to an increased load of the m.3243A>G phenotypic variant. Abnormal mitochondrial signalling affects the nuclear epigenome and influences MIDD phenotype. Despite being an apparent pathogenic factor, we highlight evidence showing that heteroplasmy in the blood and in tissues, does not fully explain the phenotypic variability of this condition, and that other factors, including mtDNA copy number, additional nuclear or mitochondrial variants, environmental factors and metabolic characteristics of the patient may be contributing factors. A better understanding of the mechanisms leading to MIDD diabetes will help inform novel management strategies for this form of diabetes.

An Insulin Upstream Open Reading Frame (INSU) Is Present in Skeletal Muscle Satellite Cells: Changes with Age.

Insulin resistance, stem cell dysfunction, and muscle fiber dystrophy are all age-related events in skeletal muscle (SKM). However, age-related changes in insulin isoforms and insulin receptors in myogenic progenitor satellite cells have not been studied. Since SKM is an extra-pancreatic tissue that does not express mature insulin, we investigated the levels of insulin receptors (INSRs) and a novel human insulin upstream open reading frame (INSU) at the mRNA, protein, and anatomical levels in Baltimore Longitudinal Study of Aging (BLSA) biopsied SKM samples of 27-89-year-old (yrs) participants. Using RT-qPCR and the MS-based selected reaction monitoring (SRM) assay, we found that the levels of INSR and INSU mRNAs and the proteins were positively correlated with the age of human SKM biopsies. We applied RNAscope fluorescence in situ hybridization (FISH) and immunofluorescence (IF) to SKM cryosections and found that INSR and INSU were co-localized with PAX7-labeled satellite cells, with enhanced expression in SKM sections from an 89 yrs old compared to a 27 yrs old. We hypothesized that the SKM aging process might induce compensatory upregulation of INSR and re-expression of INSU, which might be beneficial in early embryogenesis and have deleterious effects on proliferative and myogenic satellite cells with advanced age.

CD36 inhibition corrects lipid-FetuinA mediated insulin secretory defects by preventing intracellular lipid accumulation and inflammation in the pancreatic beta cells

CD36 is a multifunctional protein involved in long chain fatty acid uptake and immune modulation in different cells. Recently it was reported that increased expression of CD36 is evident in the islets of diabetic obese individuals. In this present study we investigated the role of CD36 in regulating intracellular lipid accumulation and inflammation in beta cells and its implication on secretory dysfunction. Additionally, we have elucidated the potential role of fetuinA, a circulatory glycoprotein and an endogenous ligand of TLR4, for aggravating lipid accumulation and insulin secretory defects in beta cells. MIN6 mouse insulinoma cells when incubated with palmitate and fetuinA together showed activation of TLR4-NFkB inflammatory cascade and increased uptake of palmitate, which was rescued by CD36 functional inhibition or knockdown. Moreover, glucose stimulated insulin secretion was restored with consequent downregulation of IL-1β secretion. TLR4 inhibition also decreased intracellular lipid content with a reduction of CD36, suggesting functional crosstalk between them. At physiological level, excess fetuinA in the islet milieu of HFD fed C57BL/6J mice or exogenous fetuinA administration (i.p.) promoted lipid accumulation in the islets resulting in decreased insulin secretion with increased CD36 expression. Interestingly, CD36 inhibition in HFD mice with a pharmacological inhibitor Salvianolic acid B attenuated inflammation, reduced intracellular lipid accumulation in beta cells and restored insulin secretory function. Therefore, our results suggest that inhibition of CD36 protects beta cells from the derogatory effects of lipid and fetuinA and restores secretory function and can be considered as a therapeutic target for obesity mediated beta cell dysfunction.

High Doses of Vitamin D and Specific Metabolic Parameters in Type 2 Diabetes Patients: Systematic Review.

Background/Objectives: Type II diabetes mellitus (T2DM) is recognized as a condition of mild chronic inflammation, marked by increased levels of acute-phase proteins and various inflammatory indicators. These inflammatory substances, along with inflammation of adipose tissue and the secretion of adipocytokines, can contribute to insulin resistance and β cell dysfunction. By influencing both innate and adaptive immunity, vitamin D can inhibit the production of inflammatory cytokines and help mitigate the low-grade chronic inflammation associated with T2DM. Several strategies have been proposed to increase vitamin D levels effectively and safely, but the recent and strong ones have common tactics. Short-term high doses increase the level acutely, and long-term lower doses maintain sufficient levels. Methods: The aim of our work was to determine and verify the effectiveness of high doses of vitamin D to safely increase its level in patients with type 2 diabetes mellitus, as well as the effect of these doses on selected metabolic parameters. Data from 20 studies (vitamin D group n = 612, and control group n = 592) regarding the influence of vitamin D supplementation with doses above 4000 IU on serum 25(OH)D, fasting blood glucose (FBG), hemoglobin A1c (HbA1c), blood pressure, serum calcium, and parathormone were pooled. Results: Vitamin D supplementation significantly improved serum 25(OH)D levels, with an average increase after intervention versus baseline at 177.09%. Our studies suggest that vitamin D supplementation may benefit various parameters in T2DM patients, including glycemic control, blood pressure, and PTH levels. Conclusions: Vitamin D supplementation may have beneficial effects on various parameters in type 2 diabetes patients, including glycemic control, blood pressure, and parathormone levels. However, the results are only sometimes consistent across all studies. Further examination is needed.

1-Deoxynojirimycin affects high glucose-induced pancreatic beta-cell dysfunction through regulating CEBPA expression and AMPK pathway.

This study aims to explore the role of 1-Deoxynojirimycin (DNJ) in high glucose-induced β-cells and to further explore the molecular mechanism of DNJ effect on β-cells through network pharmacology. In the study, high glucose treatment of mouse INS-1 cells inhibited cell proliferation and insulin secretion, decreased the expression of Bcl-2 protein and Ins1 and Ins2 genes, promoted apoptosis, and increased cleaved caspase-3 and cleaved caspase-9 expression levels as well as intracellular reactive oxygen species (ROS) production. DNJ treatment significantly restored the dysfunction of INS-1 cells induced by high glucose, and DNJ showed no toxicity to normal INS-1 cells. Silencing CEBPA promoted, while overexpression of CEBPA relieved the dysfunction of pancreatic β-cells induced by high glucose. DNJ treatment partially restored the pancreatic β-cell dysfunction caused by silencing CEBPA. In conclusion, DNJ can inhibit high glucose-induced pancreatic β-cell dysfunction by promoting the expression of CEBPA.

Impact of psychological stressors on natural killer cell function: A comprehensive analysis based on stressor type, duration, intensity, and species

Patients with natural killer (NK) cell deficiency or dysfunction are more susceptible to infections by Herpesviridae viruses, herpesvirus-related cancers, and macrophage activation syndromes. This review summarizes research on NK cell dysfunction following psychological stress, focusing on stressor type, duration, age of exposure, and species studied. Psychological stressors negatively affect NK cell activity (NKCA) across species. Prolonged stress leads to more significant decreases in NK cell number and function, with rehabilitation efforts proving ineffective in reversing these effects. Early life and prolonged stress exposure particularly increases the risk of infections and cancer due to impaired NKCA. The review also highlights that stress impacts males and females differently, with females exhibiting a more immunosuppressed NK cell phenotype. Notably, mice respond differently compared to humans and other animals, making them unsuitable for NK cell stress-related studies. Most studies measured NKCA using cytolytic assays against K-562 or YAC-1 cells. Although the exact mechanisms of NK cell dysfunction under stress remain unclear, potential causes include reduced release of secretory lysosomes with perforin or granzyme, impaired NK cell synapse formation, decreased expression of synapse-related molecules like CD2 or LFA-1 (CD11a), altered activating receptor expression, and dysregulated signaling pathways, such as decreased Erk1/2 phosphorylation and NFkB signaling.These mechanisms are not mutually exclusive, and future research is needed to clarify these pathways and develop therapeutic interventions for stress-induced immune dysregulation.

Germ Cell Dysfunction is Universal in Male Patients with β-Thalassemia Following Hematopoietic Stem Cell Transplantation During Childhood and Adolescence.

To assess gonadal function in adolescent male patients with β-thalassemia who underwent successful hematopoietic stem cell transplantation (HSCT) during childhood or adolescence. Fifty-two male patients with β-thalassemia, aged ≥10 years, who had undergone HSCT ≥2 years were included. Clinical data, such as age, genital Tanner (GT) stage at HSCT and enrollment, serum ferritin levels, and cumulative doses of alkylating agents, were collected. Gonadal function was evaluated through measurements of serum luteinizing hormone (LH), follicle-stimulating hormone (FSH), testosterone, inhibin B levels, and semen analysis. Age at enrollment and HSCT were 17 (10-31) and 9 (1-19) years, respectively. The duration from HSCT to enrollment was 7.5 (2-20) years. Of 52 patients, 46 (88%) exhibited Sertoli cell dysfunction. Thirty-one patients had relatively small testes for their GT stage, 34 of 44 with GT V had elevated FSH of ≥5 IU/L, and 20 of 49 with GT stages II-V had low serum inhibin B levels. None of the patients with GT stage V showed Leydig cell dysfunction or gonadotropin deficiency. Serum FSH ≥8 IU/L showed the best diagnostic accuracy for detecting oligo- and azoospermia. All 39 patients who underwent semen analysis had >1 abnormal parameters. Having relatively small testes for GT stage and serum FSH ≥8 IU/L were associated with oligo- and azoospermia (p <0.01). Male patients with β-thalassemia after HSCT experienced universal spermatogenesis impairment and frequent Sertoli cell dysfunction but their Leydig cell function appears to be preserved. The high likelihood of future subfertility should be informed before HSCT.

Palmitoyl-L-carnitine induces tau phosphorylation and mitochondrial dysfunction in neuronal cells.

Alzheimer's disease (AD) is characterized by cognitive decline and memory loss, involving mechanisms such as tau hyperphosphorylation and mitochondrial dysfunction. Increasing evidence suggests that age-related alterations in metabolite levels are crucial for the pathogenesis of AD. Here, we analyzed serum metabolites from mice of various ages (2, 4, 14, and 21 months old) using mass spectrometry. We identified palmitoyl-L-carnitine as a key metabolite with significantly increased levels in aged mice. In vitro experiments with SH-SY5Y neuronal cells demonstrated that palmitoyl-L-carnitine treatment enhanced tau phosphorylation, increased mitochondrial fission, and elevated intracellular calcium levels. Furthermore, the increased levels of tau phosphorylation were significantly reduced by the inhibition of GSK-3β, CDK5, and calpain, indicating that tau kinases activated by calcium overload are directly involved in the increase of tau phosphorylation. Considering that mitochondrial fission is related to mitochondrial dysfunction, we propose that the elevated level of serum palmitoyl-L-carnitine during aging contributes to AD pathology through these pathways. These findings highlight the significant role of lipid metabolism in neurodegeneration and offer potential therapeutic targets for age-related diseases, including AD.

Engineered Biomimetic Cancer Cell Membrane Nanosystems Trigger Gas-Immunometabolic Therapy for Spinal-Metastasized Tumors.

Despite great progress in enhancing tumor immunogenicity, conventional gas therapy cannot effectively reverse the tumor immunosuppressive microenvironment (TIME), limiting immunotherapy. The development of therapeutic gases that are tumor microenvironment responsive and efficiently reverse the TIME for precisely targeted tumor gas-immunometabolic therapy remains a great challenge. In this study, a novel cancer cell membrane-encapsulated pH-responsive nitric oxide (NO)-releasing biomimetic nanosystem (MP@AL) is prepared. Lactate oxidase (Lox) in MP@AL consumed oxygen to promote the decomposition of lactate, a metabolic by-product of tumor glycolysis, and the generation of H2O2, while L-arginine (L-Arg) in MP@AL is oxidized by H2O2 to generate nitric oxide (NO). For one thing, NO led to mitochondrial dysfunction in tumor cells to reduce oxygen consumption and promote the efficiency of Lox in lactate decomposition, thus reversing lactate-induced TIME; for another, NO effectively triggered immunogenic cell death, activated anti-tumor immune response and long-term immune memory, and ensured a favorable effect in the synergistic interaction with PD-L1 antibody for inhibiting tumor growth and recurrence. Therefore, a novel gas-immunometabolic therapy dual closed-loop nanosystem for enhancing tumor immunogenicity and remodeling lactate-induced TIME is established. Overall, this work will provide new ideas for gas therapy to effectively remodel the TIME to enhance cancer immunotherapy.

Abstract 4137609: Lentiviral shRNA Library Screen Using Human Pluripotent Stem Cell-derived Cardiomyocytes Identified Mediators of Protection via MAP4K4 Inhibition against Doxorubicin-induced Cardiotoxicity

Background: Doxorubicin (Dox) is a common component in chemotherapy for a wide range of tumors, but the cardiotoxicity has restricted its use. There is an unmet need for novel therapeutics of Dox-induced cardiotoxicity (DIC). We previously identified the protein kinase MAP4K4 as a mediator of DIC. Notably, we showed that blocking MAP4K4 activity via DMX-5804 protected against Dox-induced cell death and dysfunction in human iPSCs derived cardiomyocytes (hiPSC-CMs). As a powerful tool to study the protective mechanism, pooled library screen performs high-throughput phenotypic examination, but its potential on hiPSC-CMs is poorly explored. Hypothesis: A pooled lentiviral shRNA library screen in hiPSC-CMs treated with Dox ± DMX-5804 could identify mediators of protection against DIC. Methods: To maximize the chances to uncover key factors mediating DMX-5804 protection, we performed a high-throughput loss-of-function screen. A pooled library of 1731 lentiviral shRNAs was generated. hiPSC-CMs were transduced, then treated with Dox ± DMX-5804. Cardiac troponin I ELISA was used to examine myocyte injury. Genomic DNA was isolated, followed by amplification and sequencing of the integrated shRNAs. Log2 fold change was calculated and transformed into Z score to identify significantly shifted shRNAs. Results: To build the shRNA library we included 1) genes identified via transcriptomic analysis of hiPSC-CMs treated with Dox ± DMX-5804; 2) genes in the MAP4K4 signaling cascade; 3) previously defined pro-death or pro-survival genes in DIC. To ensure single gene silenced per cell, we used a low MOI of 0.5. After transduction, cells were treated with Dox ± DMX-5804. Confirming the cardiotoxicity and protection, Dox induced a 2-fold increase of cardiac troponin I release and DMX-5804 significantly reversed it. Next, we recovered and sequenced the integrated shRNAs, and quantified the frequency of each. We identified 146 significantly enriched or depleted shRNAs (|Z score| &gt; 2). When comparing Dox treated group to vehicle or DMX-5804 protected group, shRNA enrichment indicates pro-death function of the targeted gene. Conversely, pro-survival genes can be inferred from shRNA depletion. Amongst the 5 most consistently targeted genes, we identified SIRT6 and GATA4 which are known pro-survival in DIC, suggesting the screen is potent to define key genes. Conclusion: We performed a proof-of-concept pooled library screen in human cardiomyocytes and identified mediators of protection against DIC.

Type 2 diabetes pathway-specific polygenic risk scores elucidate heterogeneity in clinical presentation, disease progression and diabetic complications in 18,217 Chinese individuals with type 2 diabetes.

Type 2 diabetes is a complex and heterogeneous disease and the aetiological components underlying the heterogeneity remain unclear in the Chinese and East Asian population. Therefore, we aimed to investigate whether specific pathophysiological pathways drive the clinical heterogeneity in type 2 diabetes. We employed newly developed type 2 diabetes hard-clustering and soft-clustering pathway-specific polygenic risk scores (psPRSs) to characterise individual genetic susceptibility to pathophysiological pathways implicated in type 2 diabetes in 18,217 Chinese patients from Hong Kong. The 'total' type 2 diabetes polygenic risk score (PRS) was summed by genome-wide significant type 2 diabetes signals (n=1289). We examined the associations between psPRSs and cardiometabolic profile, age of onset, two glycaemic deterioration outcomes (clinical requirement of insulin treatment, defined by two consecutive HbA1c values ≥69 mmol/mol [8.5%] more than 3 months apart during treatment with two or more oral glucose-lowering drugs, and insulin initiation), three renal (albuminuria, end-stage renal disease and chronic kidney disease) outcomes and five cardiovascular outcomes. Although most psPRSs and total type 2 diabetes PRS were associated with an earlier and younger onset of type 2 diabetes, the psPRSs showed distinct associations with clinical outcomes. In particular, individuals with normal weight showed higher psPRSs for beta cell dysfunction and lipodystrophy than those who were overweight. The psPRSs for obesity were associated with faster progression to clinical requirement of insulin treatment (adjusted HR [95% CI] 1.09 [1.05, 1.13], p<0.0001), end-stage renal disease (1.10 [1.04, 1.16], p=0.0007) and CVD (1.10 [1.05, 1.16], p<0.0001) while the psPRSs for beta cell dysfunction were associated with reduced incident end-stage renal disease (0.90 [0.85, 0.95], p=0.0001) and heart failure (0.83 [0.73, 0.93], p=0.0011). Major findings remained significant after adjusting for a set of clinical variables. Beta cell dysfunction and lipodystrophy could be the driving pathological pathways in type 2 diabetes in individuals with normal weight. Genetic risks of beta cell dysfunction and obesity represent two major genetic drivers of type 2 diabetes heterogeneity in disease progression and diabetic complications, which are shared across ancestry groups. Type 2 diabetes psPRSs may help inform patient stratification according to aetiology and guide precision diabetes care.

Abstract 4124070: Dual-action Nanomatrix Coated Stent for Improving Endothelialization while Suppressing Restenosis and Inflammation

Introduction: The most common cause of cardiovascular disease (CVD) is atherosclerosis. The progression of atherosclerosis is characterized by endothelial cell dysfunction, proliferation of smooth muscle cell proliferation, and plaques which lead to progressive arterial stenosis. Bare metal stents (BMS) were developed to hinder stenosis, but re-stenosis has been observed following deployment. Drug-eluting stents (DES) have been developed to mitigate this, by releasing anti-proliferative drugs to suppress smooth muscle cell proliferation. However, these drugs also suppress endothelial growth. To address this, we are developing and evaluating a dual action nanomatrix coated stent comprised of a nitric oxide (NO) releasing peptide amphiphile (PA) and everolimus-encapsulated liposomes to improve endothelial proliferation and suppressing smooth muscle cell proliferation and inflammation. Materials and Methods: The nanomatrix coating contains a PA generated via solid phase peptide synthesis consisting of a nitric oxide donor and a cell adhesive ligand. The everolimus-encapsulated liposomes were prepared with DPPC, DOTAP, DSPE-PEG, and cholesterol via thin film rehydration, followed by encapsulation with everolimus. TEM was performed on liposomes to assess their stability. The release kinetics were evaluated via UV-vis of samples containing released everolimus. To evaluate the dual effect of the nanomatrix coating on metabolism, NO and everolimus were used to treat endothelial and smooth muscle cells, and an MTT assay was done. Results and Discussion: TEM showed that liposomes maintained stability over time. It was found that 60% of everolimus was released by day 30. The MTT assay showed that NO with everolimus increased endothelial cell metabolic activity and maintained or lowered smooth muscle cell activity compared to everolimus alone. Conclusions: Everolimus-encapsulated liposomes have potential as an effective delivery method of everolimus. NO and everolimus have a synergistic effect of improving endothelial function and suppressing smooth muscle cells.

BIOS-07. GRADUAL METABOLIC ALTERATIONS RESTRUCTURE THE SPATIAL ARCHITECTURE AND IMMUNITY IN GLIOBLASTOMA

Abstract The organized layered structure of the human neocortex contrasts sharply with the chaotic architecture of malignant CNS tumors. The heterogenous nature of glioblastoma (GBM) poses significant challenges to research and treatment, given that effective strategies require consistent biological patterns. Here, we leverage recent advances in spatial biology, integrating spatially resolved transcriptomics and metabolomics to identify drivers of GBM-specific architectural evolution. We performed spatial metabolomics (MALDI) and transcriptomics (Visium n=43 and MERFISH n=25) on glioblastoma patients, with computational analysis conducted using the SPATA2 R package. Our findings reveal that most spatial recurrent patterns in glioblastoma emerge in a gradient-like organization, particularly influenced by proximity to areas of necrosis and hypoxia. Samples without hypoxic regions demonstrated less spatial organization. Metabolomic and transcriptomic profiling indicated enhanced responses to hypoxia, reduced cell proliferation, and elevated expression indicative of the S phase in cell division near necrotic regions. The vicinity to hypoxic areas was associated with increased extracellular matrix remodeling and epithelial-to-mesenchymal transition, suggesting that the invasive capabilities of GBM cells originate from these hypoxic niches. By further integrating spatially resolved TCR-sequencing, we identified that hypoxic gradients drive defined T cell responses with a higher likelihood of T cell dysfunction within a distance of 235 µm (sd: 132 µm) to hypoxia. This suggests that the presence of necrosis, hypoxia, and hypoxia-associated metabolites could serve as potential predictive markers for T-cell behavior and exhaustion in glioblastoma. Our study offers an in-depth analysis of the influence of two prevalent niches in glioblastoma: necrosis and hypoxia. We reveal that the complex architecture of glioblastoma is not solely dictated by genetic randomness and stochastic occurrences but is significantly influenced by spatial proximity to key factors like necrosis and hypoxia. This insight contributes to an evolving model that seeks to unravel the intricate biology underpinning this formidable disease.

IMMU-09. REVERSING SYSTEMIC T CELL DYSFUNCTION TO LICENSE IMMUNOTHERAPY IN INTRACRANIAL TUMORS

Abstract Intracranial tumors present unique challenges for immunotherapy. These can include both local and systemic modes of immune suppression, the mechanistic underpinnings of which are incompletely understood. Our work reveals that intracranial tumors elicit systemic increases in circulating catecholamine levels, and that this chronic sympathetic hyperactivity results in T cell dysfunction that impedes immunotherapeutic efficacy. We show that treatment with b-adrenergic blockade can partially overcome the negative impacts of chronic sympathetic hyperactivity by increasing NF-kB activity in immune cells, restoring T cell polyfunctionality, favorably modifying the tumor microenvironment, and extending survival in murine models of glioblastoma treated with immune-based therapies. Furthermore, we demonstrate that extended survival is also observed in glioblastoma patients receiving b-adrenergic blockade, as well as in patients with melanoma and lung cancer brain metastases who received b-blockade alongside concomitant immune checkpoint inhibition. Importantly, while local sympathetic hyperactivity in the tumor microenvironment and b-blockade also impact the anti-tumor immune response in the context of extracranial disease, these effects are markedly more pronounced in intracranial disease. Taken together, these data reveal that sympathetic hyperactivity facilitates systemic immune dysfunction in the setting of intracranial tumors and highlight a novel role for the application of b-adrenergic blockade to license immunotherapy in intracranial malignancies.

Cell type–specific gene therapy confers protection against motor neuron disease caused by a TFG variant

Inherited forms of motor neuron disease (MND), including hereditary spastic paraplegias (HSP), are associated with the death or dysfunction of nerve cells that control skeletal muscle activity. However, in some cases, the impacts of genetic variants underlying MND act in a non-cell autonomous manner, instead affecting the function of other cell types necessary for neuronal maintenance. Pathological mutations in TFG, which have been implicated in HSP, lead to axonopathy within the corticospinal tract, but it remains unclear whether this problem arises due to perturbations within neurons or supporting neuroglia. To address this question, we leveraged a rat model harboring the recessive TFG p.R106C mutation (mRATBN7.2, g.11:43897639C>T, c.316C>T), which recapitulates multiple phenotypes associated with HSP in humans, including progressive motor deficits, leg spasticity, and indications of an inflammatory response within the motor cortex. In particular, we took advantage of cell type-specific gene therapies to demonstrate that the reintroduction of wild-type TFG into synapsin 1-positive neurons provides robust protection against MND, whereas its expression in GFAP-positive glial cells provides no significant improvement in quantitative measures of gait, despite a dramatic reduction in the presence of reactive astrocytes throughout the brain. These data strongly suggest that therapeutic approaches targeting neurons should be pursued in cases of TFG-HSP, with our animal model offering a unique platform for preclinical assessment.

Generation of a novel constitutive smooth muscle cell-specific Myh11 -driven Cre mouse model.

Dysfunction in either embryonic or postnatal vascular smooth muscle cells (SMCs) significantly contributes to the progression of various cardiovascular diseases. Therefore, elucidating the molecular mechanisms governing VSMC development and homeostasis is crucial. MYH11 is the most reliable lineage gene for SMCs and has been utilized to develop tamoxifen-inducible Cre driver lines for achieving SMC-specific gene manipulation by crossing with mice carrying the lox P -flanked gene, particularly in adult mice. For studies involving SMCs during embryogenesis, the commonly used constitutive Cre driver is controlled by the Tagln ( Sm22α ) promoter. However, this Cre driver exhibits activity in multiple non-SMC populations, including cardiomyocytes and skeletal muscle precursors, introducing confounding effects. Additionally, most existing SMC-specific Cre drivers are generated using a transgenic approach, raising concerns about random site integration and variable gene copy numbers. To address these limitations, we report a novel Cre mouse model generated by knock-in (KI) of a nuclear-localized Cre recombinase into the Myh11 gene locus using homologous recombination. We confirmed that the Cre activity precisely recapitulates endogenous Myh11 expression by crossing with Rosa26 mTmG or tdTomato reporter mice. Moreover, Myh11 -driven Cre can efficiently delete the floxed allele of the transcription factor Tead1 specifically in SMCs. The Tead1 SMC-specific knockout mice did not exhibit an overt phenotype, thereby circumventing the embryonic lethal phenotype mediated by Tagln -driven Cre, as we previously reported. These findings establish this novel Cre driver line as a robust tool for tracing the Myh11 -positive SMC lineage and manipulating gene function specifically in SMCs during embryonic development in mice.

Upregulated let-7 expression in the follicular fluid of patients with endometriomas leads to dysfunction of granulosa cells through targeting of IGF1R.

Upregulated let-7 expression in the follicular fluid of patients with endometriomas leads to dysfunction of granulosa cells through targeting of IGF1R.