Enteroendocrine cells (EECs) are specialized sensory cells widely distributed throughout the villi and crypts of the intestinal epithelium. EECs secrete the majority of all gut hormones in response to nutrient and microbial cues which regulate various systemic processes, including glucose regulation and satiety. With new model systems, like organoids, novel roles for EECs in regulating their local environment are beginning to emerge, particularly in the intestinal epithelium. We recently identified EECs as important regulators of intestinal epithelial barrier integrity and permeability via unknown mechanisms. Here, we used human intestinal enteroids to test the hypothesis that EECs regulate the stability of junctional proteins embedded in sphingolipid-rich lipid membrane microdomains. We compared control human intestinal enteroids with those harboring a genetic loss of NEUROG3 (EEC-deficient). Loss of EECs altered gene expression of many enzymes involved in sphingolipid metabolism alongside significant increases in several ceramide species, but only in crypt-like enteroids. Manipulation of ceramide levels by knockdown of key enzymes or exogenous addition of long-chain and very long-chain ceramides directly impacted barrier permeability. Treatment of EEC-deficient enteroids with exogenous PYY or octreotide (OCT, a synthetic analog of somatostatin) restored gene expression and ceramide levels to wild-type, visibly tightened the apical-lateral junctions between cells by TEM, restored mRNA levels of junctional proteins, and improved their subcellular localization. PYY and OCT activated the PI3K/AKT and mTOR signaling pathways, which are upstream regulators of sphingolipid metabolism and abundance. These findings in human enteroids correlate with increased barrier permeability in EEC-deficient mice. Our data support a novel mechanism by which EECs regulate intestinal barrier permeability. To our knowledge, this is the first report connecting nutrient-sensitive EECs with structural and bioactive sphingolipids. Funding sources: This project was supported by the NIH, 1K01 DK125341 (HAM) and R01 DK132079 (AJS). The Microscopy Services Laboratory, Department of Pathology and Laboratory Medicine, is supported in part by P30 CA016086 Cancer Center Core Support Grant to the UNC Lineberger Comprehensive Cancer Center. The Advanced Analytics Core is supported in part by P30 DK034987 to the Center for Gastrointestinal Biology and Disease at UNC. The Analytical Chemistry Shared Resource Core in the University of Arizona Cancer Center is supported in part by P30 CA023074 and S10 OD032134 (JMS). This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Dibutyl phthalate, benzyl butyl phthalate, and di-2-ethylhexyl phthalates leach from consumer and medical products, leading to chronic daily exposure in women. Phthalates are associated with impaired ovarian function and metabolic syndrome in women. In mice, oral exposure to human relevant levels of a mixture of these phthalates disrupted the ovarian follicle proteome causing dysregulation of lipid metabolism proteins. This study aimed to establish the consequences of those alterations on the follicular lipid profile and identify relevant systemic impacts of phthalates. Adult CD-1 female mice were pipet fed vehicle (corn oil) or the phthalate mixture (32 µg/kg/day) for 10 days. Antral follicles were isolated and subjected to targeted lipid profiling, neutral lipid and triglyceride quantification, and expression analyses of key lipid homeostasis enzymes. Liver and serum samples were also tested for systemic effects. Lipid profiling revealed that phthalate-treated mice had significantly increased follicular free fatty acid (FFA), acylcarnitine, and lysophospholipid content with some changes also observed in liver and serum. Neutral lipid content was unaffected, but decreased follicle and increased hepatic triglyceride content were observed in phthalate-treated mice. Phthalate exposure increased follicular fatty acid synthase expression, decreased carnitine o-palmitoyltransferase 2 and altered some key triglyceride hydrolysis enzymes. These results strongly suggest that human relevant phthalate mixture exposure leads to lipid, gene and protein changes consistent with increased FFA synthesis, impaired beta oxidation, and decreased triglyceride abundance in antral follicles. These findings add key mechanistic information to the poorly understood associations between phthalate burden, antral follicle function, and metabolic dysfunction in women.

Background:Global dietary guidelines for polyunsaturated fatty acids (PUFAs), especially linoleic acid (LA) and its metabolite arachidonic acid (ARA), remain debated. Almost all research to date has used fatty acid (FA) data expressed as percent of total FA (% total). Objective:The objective of this study was to determine whether expressing fatty acid (FA) data as % of total or as absolute concentrations alters associations with clinical biomarkers. Methods:Serum FA data obtained via electron capture negative-ion mass spectrometry was obtained from NHANES. Each FA was expressed both as % total and absolute concentration (µmol/L). Associations were examined between individual and total FAs and a panel of lipid and non-lipid biomarkers, including total cholesterol, LDL-C, HDL-C, triglycerides, blood pressure, body mass index, waist circumference, glucose, and insulin. Results:Associations between LA and clinical biomarkers including triglycerides, cholesterol, HDL-C, BMI, glucose, and insulin, reversed direction depending on whether LA was expressed as % total or as a concentration. Similar reversals were observed for ARA, DHA, DPA, and stearic acid. Increases in total FA levels were accompanied by decreases in % total of several PUFAs and HUFAs, despite rising absolute concentrations. Total FA was positively associated, often strongly, with nine clinical markers and negatively associated with HDL-C. Conclusions:Expression format significantly impacts observed FA associations. Reliance on % total FA values alone may misrepresent true associations between individual FAs and clinical endpoints, especially when the total fatty acid pool also changes size. To develop effective dietary guidance or clinical recommendations, it is essential to consider the underlying FA biology and total FA pool size when determining whether % total or absolute FA concentrations are more appropriate.

Firefighters face regular exposure to known and probable human carcinogens, such as polycyclic aromatic hydrocarbons (PAHs), benzene, and formaldehyde, leading to an increased risk of various cancers compared to the general population. Hispanic and black firefighters are at increased risk of additional cancers not elevated in non-Hispanic white firefighters, yet biological pathways underlying these differences are unknown. The study objectives were to evaluate differences in the urinary metabolome between Hispanic and non-Hispanic firefighters, pre-and post-fireground exposure. To investigate the metabolic patterns, we employed a comprehensive metabolomics pipeline that leveraged liquid chromatography coupled with high-resolution mass spectrometry. We applied linear mixed effects regression to identify the differential metabolites at an FDR < 0.05 among 19 Hispanic and 81 non-Hispanic firefighters. We also performed overrepresentation analysis using Mummichog to identify enriched pathways at FDR < 0.05. Out of 175 features in HILIC(−) mode and 1847 features in RP(+) mode, we found 26 and 276 differential urinary features, respectively, when comparing Hispanic and non-Hispanic firefighters. We noted pathway enrichment in tryptophan and galactose metabolism. However, post-exposure, we did not observe differences in the metabolomic response by ethnicity despite differing fireground exposures. Dysregulation in the tryptophan and galactose pathway is an important contributor to cancer risks and may explain the increased cancer risk among Hispanic firefighters.

Lipid droplets (LDs) are dynamic organelles with a neutral lipid core surrounded by a phospholipid monolayer. Solid tumors exhibit LD accumulation, and it is believed that LDs promote cell survival by providing an energy source during energy deprivation. However, the precise mechanisms controlling LD accumulation and utilization in prostate cancer are not well known. Here, we show peroxisome proliferator-activated receptor α (PPARα) acts downstream of PIM1 kinase to accelerate LD accumulation and promote cell proliferation in prostate cancer. Mechanistically, PIM1 inactivates glycogen synthase kinase 3 beta (GSK3β) via serine 9 phosphorylation. GSK3β inhibition stabilizes PPARα and enhances the transcription of genes linked to peroxisomal biogenesis (PEX3 and PEX5) and LD growth (Tip47). The effects of PIM1 on LD accumulation are abrogated with GW6471, a specific inhibitor for PPARα. Notably, LD accumulation downstream of PIM1 provides a significant survival advantage for prostate cancer cells during nutrient stress, such as glucose depletion. Inhibiting PIM reduces LD accumulation in vivo alongside slow tumor growth and proliferation. Furthermore, TKO mice, lacking PIM isoforms, exhibit suppression in circulating triglycerides. Overall, our findings establish PIM1 as an important regulator of LD accumulation through GSK3β-PPARα signaling axis to promote cell proliferation and survival during nutrient stress.

Sphingolipids are an important class of lipids present in all eukaryotic cells that regulate critical cellular processes. Disturbances in sphingolipid homeostasis have been linked to several diseases in humans. Ceramides are central in sphingolipid metabolism and are largely synthesized by six ceramide synthase (CerS) isoforms (CerS1-6), each with a preference for different fatty acyl chain lengths. Although the tissue distribution of CerS mRNA expression in humans and the roles of CerS isoforms in synthesizing ceramides with different acyl chain lengths are known, it is unknown how CerS expression dictates ceramides and downstream metabolites within tissues. In this study, we analyzed sphingolipid levels and CerS mRNA expression in 3month -old C57BL/6J mouse brain, heart, kidney, liver, lung, and skeletal muscle. The results showed that CerS expression and sphingolipid species abundance varied by tissue and that CerS expression was a predictor of ceramide species within tissues. Interestingly, although CerS expression was not predictive of complex sphingolipid species within all tissues, composite scores for CerSs contributions to total sphingolipids measured in each tissue correlated to CerS expression. Lastly, we determined that the most abundant ceramide species in mouse tissues aligned with CerS mRNA expression in corresponding human tissues (based on chain length preference), suggesting that mice are relevant preclinical models for ceramide and sphingolipid research. SIGNIFICANCE STATEMENT The current study demonstrates that ceramide synthase (CerS) expression in specific tissues correlates not only with ceramide species but contributes to the generation of complex sphingolipids as well. As many of the CerSs and/or specific ceramide species have been implicated in disease, these studies suggest the potential for CerSs as therapeutic targets and the use of sphingolipid species as diagnostics in specific tissues.

Background: Stroke remains a leading cause of mortality and disability. The narrow temporal window and limited availability of, and eligibility for thrombolytic therapy or endovascular thrombectomy are major therapeutic limitations in treating stroke. Neuroprotective therapies that could be given early to replace or augment these existing therapies are needed to improve stroke outcomes. We showed that monosialoganglioside (GM1) containing nanoliposomes composed of phosphatidylcholine, cholesterol and GM1 (70/25/5% molar ratios, NLGM1) protect against hypoxic injury likely through Nrf2-dependent upregulation of antoxidant enzymes. Aims: To test if post-occlusion NLGM1 treatment could reduce 1) acute stroke injury following middle cerebral artery occlusion (MCAO) and 2) chronic injury following photothrombotic (PT) stroke injury. Methods: 20 week old C57BL/6 mice underwent MCAO for 60 minutes and then injected with saline or NLGM1 (1 or 2 mg IV) prior to reperfusion. Neurologic deficit score and brain infarct % area were measured the next day. Separately, mice underwent PT injury followed by injection of saline or NLGM1 (1 or 2 mg immediately and 2 hours post-injury) and cognitive/behavior tests done 1-90 days post injury. Results: Following MCAO, there was reduced neurologic impairment, infarct volume and brain edema with NLGM1 versus saline control (Fig. 1). Following PT injury, there was reduced neurologic, cognitive and motor impairment from Day 2-90 post-injury with NLGM1 versus saline control (Fig. 2). In both stroke models, there was no difference in efficacy between 1 and 2 mg NLGM1 doses. Conclusions: Treatment of mice with NLGM1 following MCAO or PT stroke injury resulted in improved structural (infarct size, edema) and functional (cognitive, behavior, motor) outcomes in the acute (MCAO) and chronic (PT) timeframes. NLGM1 is a potential novel therapeutic agent for stroke.

Previous research suggests that group IIA-secreted phospholipase A(2) (sPLA(2)-IIA) plays a role in and predicts lethal COVID-19 disease. The current study reanalyzed a longitudinal proteomic data set to determine the temporal relationship between levels of several members of a family of sPLA(2) isoforms and the severity of COVID-19 in 214 ICU patients. The levels of six secreted PLA(2) isoforms, sPLA(2)-IIA, sPLA(2)-V, sPLA(2)-X, sPLA(2)-IB, sPLA(2)-IIC, and sPLA(2)-XVI, increased over the first 7 ICU days in those who succumbed to the disease but attenuated over the same time period in survivors. In contrast, a reversed pattern in sPLA(2)-IID and sPLA(2)-XIIB levels over 7 days suggests a protective role of these two isoforms. Furthermore, decision tree models demonstrated that sPLA(2)-IIA outperformed top-ranked cytokines and chemokines as a predictor of patient outcome. Taken together, proteomic analysis revealed temporal sPLA(2) patterns that reflect the critical roles of sPLA(2) isoforms in severe COVID-19 disease.

Abstract Background Previous studies have linked sports-related concussions and repeated subconcussive head impacts in contact sport athletes to elevated brain injury biomarkers. Docosahexaenoic acid (DHA), the primary omega-3 (n-3) highly unsaturated fatty acid (HUFA) in the brain, has shown neuroprotective effects in animal models after brain injury, but clinical research has shown mixed results. Methods We conducted a randomized, double-blind, placebo-controlled study on 29 Division 1 collegiate American football players, exploring the impact of DHA (2.5 g) and eicosapentaenoic acid (EPA) (1.0 g) supplied as ethyl esters, on levels of plasma lipids shown to cross the blood-brain barrier. Dietary intake data was collected using food frequency questionnaires (FFQ). Complex lipids and unesterified fatty acids were isolated from plasma, separated via reversed-phase liquid chromatography and analyzed by targeted lipidomics analysis. Results FFQ results indicated that participants had low dietary n-3 HUFA intake and high omega-6 (n-6):n-3 polyunsaturated fatty acids (PUFA) and HUFA ratios at baseline. After DHA + EPA supplementation, plasma lysophosphatidylcholine (LPC) containing DHA and EPA significantly increased at all timepoints (weeks 17, 21, and 26; p < 0.0001), surpassing placebo at Weeks 17 (p < 0.05) and 21 (p < 0.05). Phosphatidylcholine (PC) molecular species containing DHA or EPA, PC38:6 PC36:6, PC38:7, PC40:6, and PC40:8, increased significantly in the DHA + EPA treatment group at Weeks 17 (and 21. Plasma concentrations of non-esterified DHA and EPA rose post-supplementation in Weeks 17 and 21. Conclusions This study demonstrates that n-3 HUFA supplementation, in the form of ethyl esters, increased the DHA and EPA containing plasma lipid pools the have the capacity to enrich brain lipids and the potential to mitigate the effects of sports-related concussions and repeated subconcussive head impacts. Trial Registration All deidentified data are available at ClinicalTrials.gov #NCT0479207.

Following spinal cord injury (SCI), there is a short-lived recovery phase that ultimately plateaus. Understanding changes within the spinal cord over time may facilitate targeted approaches to prevent and/or reverse this plateau and allow for continued recovery. Untargeted metabolomics revealed distinct metabolic profiles within the injured cord during recovery (7 days postinjury [DPI]) and plateau (21 DPI) periods in a mouse model of severe contusion SCI. Alterations in lipid metabolites, particularly those involved in phospholipid (PL) metabolism, largely contributed to overall differences. PLs are hydrolyzed by phospholipases A2 (PLA2s), yielding lysophospholipids (LPLs) and fatty acids (FAs). PL metabolites decreased between 7 and 21 DPI, whereas LPLs increased at 21 DPI, suggesting amplified PL metabolism during the plateau phase. Expression of various PLA2s also differed between the two time points, further supporting dysregulation of PL metabolism during the two phases of injury. FAs, which can promote inflammation, mitochondrial dysfunction, and neuronal damage, were increased regardless of time point. Carnitine can bind with FAs to form acylcarnitines, lessening FA-induced toxicity. In contrast to FAs, carnitine and acylcarnitines were increased at 7 DPI, but decreased at 21 DPI, suggesting a loss of carnitine-mediated mitigation of FA toxicity at the later time point, which may contribute to the cessation of recovery post-SCI. Alterations in oxidative phosphorylation and tricarboxylic acid cycle metabolites were also observed, indicating persistent although dissimilar disruptions in mitochondrial function. These data aid in increasing our understanding of lipid metabolism following SCI and have the potential to lead to new biomarkers and/or therapeutic strategies.

Measurements of sphingolipid metabolism are most accurately performed by LC-MS. However, this technique is expensive, not widely accessible, and without the use of specific probes, it does not provide insight into metabolic flux through the pathway. Employing the fluorescent ceramide analogue NBD-C6-ceramide as a tracer in intact cells, we developed a comprehensive HPLC-based method that simultaneously measures the main nodes of ceramide metabolism in the Golgi. Hence, by quantifying the conversion of NBD-C6-ceramide to NBD-C6-sphingomyelin, NBD-C6-hexosylceramides, and NBD-C6-ceramide-1-phosphate (NBD-C1P), the activities of Golgi resident enzymes sphingomyelin synthase 1, glucosylceramide synthase, and ceramide kinase (CERK) could be measured simultaneously. Importantly, the detection of NBD-C1P allowed us to quantify CERK activity in cells, a usually difficult task. By applying this method, we evaluated the specificity of commonly used sphingolipid inhibitors and discovered that 1-phenyl-2-decanoylamino-3-morpholino-1-propanol, which targets glucosylceramide synthase, and fenretinide (4HPR), an inhibitor for dihydroceramide desaturase, also suppress CERK activity. This study demonstrates the benefit of an expanded analysis of ceramide metabolism in the Golgi, and it provides a qualitative and easy-to-implement method.

Inflammatory bowel disease (IBD) is characterized by chronic inflammation in the colon and drastically increases the risk in the development of colorectal cancer (CRC). Over expression of acid ceramidase (AC) resulting in accumulation of sphingosine-1-phosphate (S1P), which amplify inflammatory pathways, has been implicated in patients with IBD and CRC. The conditional loss of AC in myeloid cells (ACMYE) has demonstrated promise as a therapeutic target in an acute colitis model. We sought to expand the investigation of AC loss in a physiologically relevant model of IBD using IL-10 deficient mice. IL10−/− mice were crossed with AC conditional knockout mice where AC is deleted in myeloid cells to generate ACMYE/IL10−/−, with ACfl/fl/IL10−/− mice used as control. Male and female animals were collected at 8, 12, and 24 weeks of age and assessed for intestinal inflammation. Upon examination of basic parameters of colitis, ACMYE mice exhibited reduced body weight and enlarged spleens at 12 weeks of age. Histologic assessment revealed no difference between genotypes in colitis severity at 8 or 12 weeks of age. Flow cytometry analysis was utilized to define the role of AC in immune cell recruitment. ACMYE mice exhibit reduced MHC II surface expression on antigen presenting cells (APCs) as well as reductions in neutrophil activation markers in peripheral tissues and the colon at 8 weeks of age. Interestingly, at 12 weeks of age ACfl/fl mice demonstrate significant immunosuppression across various cell types in the colon compared to ACMYE mice, whose colonic immune cells demonstrate little change over time. Furthermore, CD8+ cytotoxic T cell populations were significantly elevated in peripheral tissues despite continued reductions of MHC II surface expression on APCs in ACMYE mice at 12 weeks of age. These data indicate that loss of AC may provide protection in early age while resulting in altered inflammation in later age; however, loss of AC may also disrupt MHC II expression as seen consistently in APCs, and may indicate interference with cell-to-cell cross talk. Realtime qPCR revealed that ACMYE mice exhibited significant ablation in the expression of several pro-inflammatory cytokines at 24 weeks of age. Together these data implicate AC as a potential therapeutic target in reducing inflammation in chronic IBD. This work is supported by the National Institute of Diabetes and Digestive and Kidney Diseases R01 DK132079 (AJS). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Charcot-Marie-Tooth Disease (CMT) is a commonly inherited peripheral polyneuropathy. Clinical manifestations for this disease include symmetrical distal polyneuropathy, altered deep tendon reflexes, distal sensory loss, foot deformities, and gait abnormalities. Genetic mutations in heat shock proteins have been linked to CMT2. Specifically, mutations in the heat shock protein B1 (HSPB1) gene encoding for heat shock protein 27 (Hsp27) have been linked to CMT2F and distal hereditary motor and sensory neuropathy type 2B (dHMSN2B) subtype. The goal of the study was to examine the role of an endogenous mutation in HSPB1 in vivo and to define the effects of this mutation on motor function and pathology in a novel animal model. As sphingolipids have been implicated in hereditary and sensory neuropathies, we examined sphingolipid metabolism in central and peripheral nervous tissues in 3-month-old HspS139F mice. Though sphingolipid levels were not altered in sciatic nerves from HspS139F mice, ceramides and deoxyceramides, as well as sphingomyelins (SMs) were elevated in brain tissues from HspS139F mice. Histology was utilized to further characterize HspS139F mice. HspS139F mice exhibited no alterations to the expression and phosphorylation of neurofilaments, or in the expression of acetylated α-tubulin in the brain or sciatic nerve. Interestingly, HspS139F mice demonstrated cerebellar demyelination. Locomotor function, grip strength and gait were examined to define the role of HspS139F in the clinical phenotypes associated with CMT2F. Gait analysis revealed no differences between HspWT and HspS139F mice. However, both coordination and grip strength were decreased in 3-month-old HspS139F mice. Together these data suggest that the endogenous S139F mutation in HSPB1 may serve as a mouse model for hereditary and sensory neuropathies such as CMT2F.

Salivary gland hypofunction is an adverse side effect associated with radiotherapy for head and neck cancer patients. This study delineated metabolic changes at acute, intermediate, and chronic radiation damage response stages in mouse salivary glands following a single 5 Gy dose. Ultra-high performance liquid chromatography-mass spectrometry was performed on parotid salivary gland tissue collected at 3, 14, and 30 days following radiation (IR). Pathway enrichment analysis, network analysis based on metabolite structural similarity, and network analysis based on metabolite abundance correlations were used to incorporate both metabolite levels and structural annotation. The greatest number of enriched pathways are observed at 3 days and the lowest at 30 days following radiation. Amino acid metabolism pathways, glutathione metabolism, and central carbon metabolism in cancer are enriched at all radiation time points across different analytical methods. This study suggests that glutathione and central carbon metabolism in cancer may be important pathways in the unresolved effect of radiation treatment.

Firefighters have elevated rates of urinary tract cancers and other adverse health outcomes, which may be attributable to environmental occupational exposures. Untargeted metabolomics was applied to characterize this suite of environmental exposures and biological changes in response to occupational firefighting. 200 urine samples from 100 firefighters collected at baseline and two to four hours post-fire were analyzed using untargeted liquid-chromatography and high-resolution mass spectrometry. Changes in metabolite abundance after a fire were estimated with fixed effects linear regression, with false discovery rate (FDR) adjustment. Partial least squares discriminant analysis (PLS-DA) was also used, and variable important projection (VIP) scores were extracted. Systemic changes were evaluated using pathway enrichment for highly discriminating metabolites. Metabolome-wide-association-study (MWAS) identified 268 metabolites associated with firefighting activity at FDR q < 0.05. Of these, 20 were annotated with high confidence, including the amino acids taurine, proline, and betaine; the indoles kynurenic acid and indole-3-acetic acid; the known uremic toxins trimethylamine n-oxide and hippuric acid; and the hormone 7a-hydroxytestosterone. Partial least squares discriminant analysis (PLS-DA) additionally implicated choline, cortisol, and other hormones. Significant pathways included metabolism of urea cycle/amino group, alanine and aspartate, aspartate and asparagine, vitamin b3 (nicotinate and nicotinamide), and arginine and proline. Firefighters show a broad metabolic response to fires, including altered excretion of indole compounds and uremic toxins. Implicated pathways and features, particularly uremic toxins, may be important regulators of firefighter’s increased risk for urinary tract cancers.

Background Ovarian cancer (OC) is the most lethal gynecologic malignancy. Despite the initial high response rate to chemotherapy, most patients relapse. Immunotherapy offers potential for long-term remission but single checkpoint inhibition benefits less than 15% of patients. Growing evidence suggests that immune-checkpoint blockade (ICB) is enhanced when combined with therapies that target tumor tolerance. Transforming growth factor beta (TGF-β) is associated with resistance to immunotherapy and tumor tolerance. Integrin αvβ8 controls cell-type-specific activation of TGF-β and αvβ8 antagonism promotes anti-tumor immunity leading to tumor regression in ICB refractory tumors.1,2 We explored the impact of αvβ8 inhibition to restore ICB response in a murine ovarian carcinoma model and performed blood cytokine profiling to search for pharmacodynamic markers of response to treatment. Methods Bioinformatic analysis on bulk and single-cell levels of public OC datasets was performed to evaluate ITGB8 and TGF-β-related gene signatures. qPCR was used for the detection of TGF-β and ITGB8 expression in ID8, a murine ovarian carcinoma model (ID8-Luc-mCh-Puro.TD1). ID8 tumors unresponsive to PD-1/L1 inhibition were established to mimic advanced-stage disease. The efficacy of αvβ8 mAbs in combination with PD-L1 blockade was evaluated. Mice were evaluated for survival for 65 days. Blood samples were harvested before treatment and on days 1,8 and 14. Plasma was analyzed via Luminex for a total of 30 ­­­cytokines. Transcriptomic analyses of MC38 and EMT6 mouse tumors were performed by bulk RNA seq. Results In high-grade serous OC, ITGB8 expression is associated with shorter overall survival and increased gene signatures of TGF-β1/3 pathways. The ID8 murine ovarian carcinoma model is unresponsive to PD-1/L1 inhibition and expresses Itgb8 together with TGF-β 1/3. The combination of αvβ8 and PD-L1 mAbs led to complete tumor regression in 9/10 mice relative to 0/10 in the PD-L1 group, resulting in superior survival (P=0.0001) (Fig 1 A, B). The combination therapy led to upregulation of blood granzyme B (P=0.05), IL-27(P=0.03), and IL-1a (P= 0.008) at 14-day post-treatment and transient upregulation of CCL3 (P<0.0001) and CCL7 (P= 0.0005) at 8-day post-treatment. Cross-analysis of tumor tissues from the colon and breast syngeneic models after treatment showed similar transcript upregulation for cytokines. Conclusions Inhibition of αvβ8 renders advanced ID8 tumors sensitive to immune checkpoint therapy, leading to tumor eradication and superior survival. These data provide evidence that a treatment modality targeting αvβ8-mediated TGF-β 1/3 pathways may enhance patients' sensitivity to checkpoint therapies. Plasma blood-based biomarkers serve as a non-invasive method for response assessment to αvβ8-based therapy. References Reszka-Blanco NJ, Yadav V, Krumpoch M, Cappellucci L, Cui D, Dowling JE, et al. Inhibition of integrin avß8 enhances immune checkpoint induced anti-tumor immunity by acting across immunologic synapse in syngeneic models of breast cancer. AACR; Cancer Res 2021;81(13_Suppl): Abstract nr 1559 Dodagatta-Marri E, Ma H-Y, Liang B, Li J, Meyer DS, Chen S-Y, et al. Integrin avß8 on T cells suppresses anti-tumor immunity in multiple models and is a promising target for tumor immunotherapy. Cell Report. 2021; 36(1): 109309

Oncogenic reprogramming of cellular metabolism is a hallmark of many cancers, but our mechanistic understanding of how such dysregulation is linked to tumor behavior remains poor. In this study, we have identified dihydroceramide desaturase (DES1)-which catalyzes the last step in de novo sphingolipid synthesis-as necessary for the acquisition of anchorage-independent survival (AIS), a key cancer enabling biology, and establish DES1 as a downstream effector of HER2-driven glucose uptake and metabolism. We further show that DES1 is sufficient to drive AIS and in vitro tumorigenicity and that increased DES1 levels-found in a third of HER2+ breast cancers-are associated with worse survival outcomes. Taken together, our findings reveal a novel pro-tumor role for DES1 as a transducer of HER2-driven glucose metabolic signals and provide evidence that targeting DES1 is an effective approach for overcoming AIS. Results further suggest that DES1 may have utility as a biomarker of aggressive and metastasis-prone HER2+ breast cancer.

Objective: Obesity is one the main risk factors promoting the progression of aortic stenosis. Today, there is a critical need to identify new therapeutic targets to reverse or stop the progression of aortic stenosis. This study aimed to evaluate the modulation of extracellular matrix proteins (ECM) and its impact on metabolic enzymes during aortic valve mineralization.Methods: We analyzed ECM markers expression by mass spectrometry and RNA sequencing on explanted human aortic valves and isolated human valve cells, in 2 independent human cohorts (n= 15 vs n=17).Results: We identified a complex linkage between calcified and fibrotic phases, with an elaborate interaction between ECM, metabolic, complement and lipid transporter proteins. Aldehyde Dehydrogenase 1 Family Member A1 (ALDH1), a major enzyme in alcohol metabolism, was down regulated in both fibrotic and calcified stages. We identified Fibronectin Type III Domain Containing 1 (FNDC1) and Matrix-remodeling Associated 5 (MXRA5) as the common modulated ECM protein in calcified valve tissue in both proteomic and RNAseq data sets.Conclusion: This work highlights the interactome between metabolic and ECM markers, and identified FNDC1 and MXRA5 as novel ECM markers in calcified valves, electing them as potential targets in the development and the progression of AS.

Sphingolipids (SLs) have been implicated in numerous important cellular biologies; however, their study has been hindered by the complexities of SL metabolism. Furthermore, enzymes of SL metabolism represent a dynamic and interconnected network in which one metabolite can be transformed into other bioactive SLs through further metabolism, resulting in diverse cellular responses. Here we explore the effects of both lethal and sublethal doses of doxorubicin (Dox) in MCF-7 cells. The two concentrations of Dox resulted in the regulation of SLs, including accumulations in sphingosine, sphingosine-1-phosphate, dihydroceramide, and ceramide, as well as reduced levels of hexosylceramide. To further define the effects of Dox on SLs, metabolic flux experiments utilizing a d17 dihydrosphingosine probe were conducted. Results indicated the regulation of ceramidases and sphingomyelin synthase components specifically in response to the cytostatic dose. The results also unexpectedly demonstrated dose-dependent inhibition of dihydroceramide desaturase and glucosylceramide synthase in response to Dox. Taken together, this study uncovers novel targets in the SL network for the action of Dox, and the results reveal the significant complexity of SL response to even a single agent. This approach helps to define the role of specific SL enzymes, their metabolic products, and the resulting biologies in response to chemotherapeutics and other stimuli.