Seahorse Analyzer Research Articles

Abstract 2029: Augmenting Mitochondrial Respiration In Immature Smooth Muscle Cells Is A Therapeutic Target For Moyamoya-like Cerebrovascular Disease

Introduction: Moyamoya disease (MMD), characterized by occlusive lesions in the distal internal carotid arteries, is a malignant cause of pediatric stroke. Surgical revascularization is the mainstay treatment, but peri- and postoperative ischemic complications are common. Multiple identified genes account for only a small percentage of cases. Identifying a common pathway of disease pathogenesis would enhance care for MMD patients. Goal and Approach: We used cellular and animal models for a rare cause of MMD-like cerebrovascular disease, heterozygous ACTA2 p.R179 pathogenic variants, to define molecular mechanisms and assess therapeutic strategies. Our prior work showed that smooth muscle cells (SMCs) with ACTA2 p.R179 variants are incompletely differentiated. We hypothesized that these immature phenotypes could be rescued by manipulation of cellular metabolism. We validated our results with cellular models of other pediatric MMD-associated variants: loss-of-function variants in Pcnt (explanted from a mouse model) and a pathogenic variant in RNF213 (generated via Crispr/Cas9 in human iPSCs). Results: Acta2 SMC-R179C/+ SMCs have increased glycolysis and reduced oxidative respiration on Seahorse analyses. Molecular characterization showed that Acta2 SMC-R179C/+ SMCs have reduced mitochondrial mass and decreased complex I activity, which is rescued by nicotinamide riboside (NR) treatment. NR surprisingly also increases SMC differentiation and decreases migration. Acta2 SMC-R179C/+ mice and littermate controls were subjected to carotid artery ligation, and 33% of Acta2 SMC-R179C/+ mice died due to strokes while all control mice survived (Kaplan-Meier p<0.01). Surviving Acta2 SMC-R179C/+ mice have significant cerebrovascular remodeling and persistent occlusive carotid artery lesions. Treatment with NR reduces death (Kaplan-Meier p=0.08) and resolves persistent lesions (p<0.05) in Acta2 SMC-R179C/+ mice after carotid ligation. Pcnt and RNF213 mutant SMCs have a similar phenotype of incomplete differentiation and decreased mitochondrial respiration as ACTA2 p.R179 and are responsive to NR treatment. Conclusions: These data provide evidence for a common and therapeutically targetable pathway in pediatric MMD patients.

Effects of heat perturbation to rhinoceros’ cellular metabolism

Climate change is associated with increasing global mean temperatures and extreme heat events. These drastic changes in temperature can have significant deleterious physiological effects, particularly on large strict homeothermic mammals such as the threatened Southern White Rhino (SWR - Ceratotherium simum simum). Developing alternative strategies to understand how climatic events impact species that are logistically challenging to study are crucial. Dermal fibroblasts have proven to be effective in vitro models for studying species resilience to climate since they can reflect an organism's thermosensitivity. These cells can provide an integrative and mechanistic explanation on how cellular function can inform organismal responses to environmental changes. We investigated the effects of heat on mitochondrial function and cellular biology by exposing SWR primary dermal fibroblasts (n = 10 biological replicates) to 24 hours of heat (41°C) or 37°C as a control in culture. Then, we evaluated mitochondrial function using mitochondrial stress test, glycolytic rate test and immunofluorescence (IF) to track nucleus size and mitochondrial distribution in the cytoplasm. Metabolic function was assessed using Agilent Seahorse analyzer and IF images were analyzed on CellProfilerTM software. Our results indicate that compared to the control (37°C), heat exposure decreases mitochondrial respiration (basal: t = 3.7, df = 9, p < 0.01; maximum: t = 3.0, df = 9, p < 0.02), ATP production (t = 5.8, df = 9, p < 0.001), and coupling effciency (t = 7.6, df = 9, p < 0.001). No changes were observed in spare respiratory capacity (t = 0.7, df = 9, p = 0.5), proton leak (t = 0.7, df = 9, p = 0.5) and glycolysis (basal: t = 0.3, df = 4, p = 0.4; compensatory: t = 0.6, df = 4, p = 0.6). Furthermore, heat exposure increases nucleus size and mitochondrial distribution area (Nucleus area: t = 6.8, d.f. = 9, P < 0.001, Mitochondrial coverage area: t = 5.3, d.f. = 9, p < 0.001) and decreases fluorescence intensity of the mitochondrial dye and solidity area covered by mitochondria (Fluorescence: t = 2.3, d.f. = 9, p = 0.04, Mitochondrial area solidity: t = 5.6, d.f. = 9, p < 0.001). These findings show that acute heatwaves trigger a series of complex and interconnected responses in SWR fibroblasts resulting in mitochondrial disfunction, possibly mitophagy and/or mitochondria disintegration, and larger dispersion through the cytoplasm. The thermosensitive phenotype exhibited by SWR fibroblasts provides insights into the likely consequences of global warming on the performance of this species, as cellular and mitochondrial functioning plays a fundamental role in an organism's ability to adjust and thrive under environmental change. Funded by NSF and San Diego Zoo Wildlife Alliance. 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.

Immune Metabolic Reprogramming by Excess Dietary Salt in Salt Sensitive Hypertension via CXCL8

Salt sensitivity of blood pressure (SSBP) is when blood pressure changes parallel to salt loading/ depletion and is an independent risk factor for cardiovascular disease. Despite its importance, there is no feasible diagnosis, which limits investigational studies to identify therapeutic targets. We have found that immune cells mediate SSBP via Epithelial Sodium Channel (ENaC). CXCL8/(IL-8), a known chemoattractant plays a well-known role in glycolysis. We hypothesize that CXCL8 mediates immune metabolic reprogramming of immune cells to glycolysis via ENaC. To test this hypothesis, we performed bulk and single cell RNA sequencing in human monocytes from individuals from a modified Weinberger inpatient salt loading/ depletion protocol. The participants were instructed to refrain from taking their blood pressure medication for 2 weeks prior to the salt loading/depletion protocol. Blood and urine samples were collected, as well as ambulatory blood pressure recording. We found that changes in immune cell activation via isolevuglandin (IsoLG) protein-adducts mirror changes in salt balance and blood pressure in salt sensitive but not salt resistant individuals. We confirmed that these in vivo changes can be recapitulated in vitro. In addition, we found that salt-loading both in vivo and in vitro increases antigen presenting cell production of IL-8, which was reversed after salt depletion in salt sensitive, but not salt resistant people. ENaC inhibition by amiloride prevented the high salt-induced production of IL-8. Moreover, high salt induced blood pressure changes of expression of CXCL8 (p = 0.01553, r = 0.8067) were significant along with varying expression of glycolytic genes, including hexokinase 1(HK1)(p = 0.04190, r = -0.7249), phosphofructokinase (PFKP)(p = 0.05932, r = 0.6879) and galactose mutarotase (GALM) (p = 0.04176, r = 0.5138). Seahorse analysis revealed increased extracellular acidification rate (ECAR) in high salt conditioned monocytes, which was associated with mitochondrial dysfunction. Our results suggest that high salt intake reprograms antigen presenting cell metabolism to glycolysis via IL-8 signaling. Furthermore, targeting IL-8 and mitochondrial switching to glycolysis may not only provide novel therapeutic approaches, but also important biomarkers for diagnosis of salt sensitivity of blood pressure. 1R03HL155041-01 (Kirabo, PI), 1R01HL144941-01A1 (Kirabo, PI), 5R01HL147818-22 (Kleyman & Kirabo), 1R01HL157584-01 (Shibao, Kirabo — MPI), R01DK135764 (Kon, Shelton, Kirabo — MPI), R21TW012635 (Kirabo, Masenga — MPI), Meharry Medical College: School of Graduate Studies T32AI007281, T32GM14492, andT32HL00773. 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.

Metabolic pathways in T cells are modulated by CCR7 and IL-7R to facilitate T cell motility.

Abstract T cells play an essential role in eliminating infections by migrating through tissues and interacting with antigen-presenting dendritic cells within lymph nodes, leading to activation. A continuous supply of energy is required for T cell movement to enable scanning of dendritic cells. Many studies have established that signaling through chemokine receptor CCR7 facilitates rapid movement, however, how CCR7 signaling induces T cell metabolism remains poorly understood. IL-7 signaling regulates glycolysis to promote T cell survival, but how signaling through the IL-7R affects T cell movement is unexplored. This study investigates the roles of CCR7 and IL-7R in metabolism to drive naïve T cell movement. Utilizing Seahorse analysis, we observe that CCL21 signaling to CCR7 has no effect on glycolysis, yet decreases cellular respiration. Conversely, IL-7 has no effect on respiration but promotes glycolysis. We then determined whether oxidative phosphorylation and glycolysis play a role in CCL21 and IL-7 driven T cell movement. We employ inhibitors of oxidative phosphorylation such as rotenone to examine the influence of different metabolic pathways on T cell mobility. Our findings provide new insights into the intersection of metabolism and motility, suggesting potential differential mechanisms of ATP production that drive interactions.

Abstract 118: Dennd5b Affects Dietary Lipid Absorption And Metabolism In Male And Female Mice

Background: Dietary lipid absorption by the intestine is essential for systemic lipid homeostasis. We previously demonstrated a role for Dennd5b in intestinal lipid absorption. In humans, DENND5B gene variants are correlated with BMI in females, but not males. Female Dennd5b -/- mice have reduced appearance of dietary triacylglyceride (TAG) in plasma and are resistant to diet-induced obesity, however, the metabolic impact of Dennd5b -/- in male mice has not been reported. In this study, we test the hypothesis that the Dennd5b -/- mouse model recapitulates the sex disparity observed in humans with DENND5B polymorphisms. We also investigate the fate of unsecreted intestinal TAG in this model of impaired dietary lipid absorption. Methods and Results: A non-absorbable fatty acid (FA) tracer was used to quantify the impact of Dennd5b -deficiency on lipid absorption in male and female mice (n=7-9 mice/genotype/sex). We observed significant, but relatively modest, reductions in lipid absorption efficiency in Dennd5b -/- mice in both sexes, despite a complete absence of plasma TAG after oil gavage. We hypothesized that this was due to metabolic utilization of TAG by enterocytes. Metabolic cage studies showed that both wildtype and Dennd5b -/- mice shift toward utilization of FAs when fed high-fat diet and Seahorse analysis revealed altered metabolic activity in Dennd5b -/- intestinal tissue. Electron microscopy (EM) revealed large electron dense structures that resemble autophagosomes in Dennd5b -/- enterocytes. Western blots detected altered Lc3 levels in Dennd5b -/- intestine, implicating autophagy. RNA sequencing of intestinal tissue detected changes in expression of genes involved in beta oxidation and EM imaging revealed altered mitochondrial morphology in Dennd5b -/- enterocytes. Conclusions: We conclude that autophagy contributes to degradation of unsecreted TAG in the Dennd5b -/- enterocytes, liberating free FAs which are utilized in mitochondrial oxidation. The sexually dimorphic effect of Dennd5b does not appear to be as prominent in mice as it is in humans. Overall, our findings demonstrate that Dennd5b plays a critical role in TAG secretion by intestinal tissue and that disruption of this process can impact systemic metabolism in both sexes.

The e-liquid flavoring cinnamaldehyde induces cellular stress responses in human proximal tubule (HK-2) kidney cells

Flavored e-liquid use has become popular among e-cigarette users recently, but the effects of such products outside the lung are not well characterized. In this work, acute exposure to the popular flavoring cinnamaldehyde (CIN) was performed on human proximal tubule (HK-2) kidney cells. Cells were exposed to 0–100 µM CIN for 24–48 h and cellular stress responses were assessed. Mitochondrial viability via MTT assay was significantly decreased at 20 µM for 24 and 48 h exposure. Seahorse XFp analysis showed significantly decreased mitochondrial energy output at 20 µM by 24 h exposure, in addition to significantly reduced ATP Synthase expression. Seahorse analysis also revealed significantly decreased glycolytic function at 20 µM by 24 h exposure, suggesting inability of glycolytic processes to compensate for reduced mitochondrial energy output. Cleaved caspase-3 expression, a mediator of apoptosis, was significantly increased at the 24 h mark. C/EBP homologous protein (CHOP) expression, a mediator of ER-induced apoptosis, was induced by 48 h and subsequently lost at the highest concentration of 100 µM. This decrease was accompanied by a simultaneous decrease in its downstream target cleaved caspase-3 at the 48 h mark. The autophagy marker microtubule-associated protein 1 A/1B light chain 3 (LC3B-I and LC3B-II) expression was significantly increased at 100 µM by 24 h. Autophagy-related 7 (ATG7) protein and mitophagy-related proteins PTEN-induced putative kinase 1 (PINK1) and PARKIN expression were significantly reduced at 24 and 48 h exposure. These results indicate acute exposure to CIN in the kidney HK-2 model induces mitochondrial dysfunction and cellular stress responses.

Energy Metabolism in Human Pluripotent Stem and Differentiated Cells Compared Using a Seahorse XF96 Extracellular Flux Analyzer.

Evaluating cell metabolism is crucial during pluripotent stem cell (PSC) differentiation and somatic cell reprogramming as it affects cell fate. As cultured stem cells are heterogeneous, a comparative analysis of relative metabolism using existing metabolic analysis methods is difficult, resulting in inaccuracies. In this study, we measured human PSC basal metabolic levels using a Seahorse analyzer. We used fibroblasts, human induced PSCs, and human embryonic stem cells to monitor changes in basal metabolic levels according to cell number and determine the number of cells suitable for analysis. We evaluated normalization methods using glucose and selected the most suitable for the metabolic analysis of heterogeneous PSCs during the reprogramming stage. The response of fibroblasts to glucose increased with starvation time, with oxygen consumption rate and extracellular acidification rate responding most effectively to glucose 4 hours after starvation and declining after 5 hours of starvation. Fibroblasts and PSCs achieved appropriate responses to glucose without damaging their metabolism 2∼4 and 2∼3 hours after starvation, respectively. We developed a novel method for comparing basal metabolic rates of fibroblasts and PSCs, focusing on quantitative analysis of glycolysis and oxidative phosphorylation using glucose without enzyme inhibitors. This protocol enables efficient comparison of energy metabolism among cell types, including undifferentiated PSCs, differentiated cells, and cells undergoing cellular reprogramming, and addresses critical issues, such as differences in basal metabolic levels and sensitivity to normalization, providing valuable insights into cellular energetics.

The troglitazone derivative EP13 disrupts energy metabolism through respiratory chain complex I inhibition in breast cancer cells and potentiates the antiproliferative effect of glycolysis inhibitors

BackgroundThe metabolism of cancer cells generally differs from that of normal cells. Indeed, most cancer cells have a high rate of glycolysis, even at normal oxygen concentrations. These metabolic properties can potentially be exploited for therapeutic intervention. In this context, we have developed troglitazone derivatives to treat hormone-sensitive and triple-negative breast cancers, which currently lack therapeutic targets, have an aggressive phenotype, and often have a worse prognosis than other subtypes. Here, we studied the metabolic impact of the EP13 compound, a desulfured derivative of Δ2-troglitazone that we synthetized and is more potent than its parent compounds.MethodsEP13 was tested on two triple-negative breast cancer cell lines, MDA-MB-231 and Hs578T, and on the luminal cell line MCF-7. The oxygen consumption rate (OCR) of the treated cell lines, Hs578T mammospheres and isolated mitochondria was measured using the XFe24 Seahorse analyser. ROS production was quantified using the MitoSOX fluorescent probe. Glycolytic activity was evaluated through measurement of the extracellular acidification rate (ECAR), glucose consumption and lactate production in extracellular medium. The synergistic effect of EP13 with glycolysis inhibitors (oxamate and 2-deoxyglucose) on cell cytotoxicity was established using the Chou-Talalay method.ResultsAfter exposure to EP13, we observed a decrease in the mitochondrial oxygen consumption rate in MCF7, MDA-MB-231 and Hs578T cells. EP13 also modified the maximal OCR of Hs578T spheroids. EP13 reduced the OCR through inhibition of respiratory chain complex I. After 24 h, ATP levels in EP13-treated cells were not altered compared with those in untreated cells, suggesting compensation by glycolysis activity, as shown by the increase in ECAR, the glucose consumption and lactate production. Finally, we performed co-treatments with EP13 and glycolysis inhibitors (oxamate and 2-DG) and observed that EP13 potentiated their cytotoxic effects.ConclusionThis study demonstrates that EP13 inhibits OXPHOS in breast cancer cells and potentiates the effect of glycolysis inhibitors.

Ze-Qi-Tang formula inhibits MDSCs glycolysis through the down-regulation of p21/Hif1α/Glut1 signal in psoriatic-like mice

BackgroundPsoriasis is a chronic immune-mediated inflammatory skin disease that affects the quality of life and mental health of approximately 150 million people worldwide. Ze-Qi-Tang (ZQT) is a classic compound used in China for lung disease; however, its mechanism of action in psoriasis remains unclear. This study aimed to investigate the therapeutic effect of the ZQT formula on psoriasis and explore the underlying molecular mechanisms. MethodsPeripheral blood samples were collected from patients with psoriasis and healthy individuals. Flow cytometry was used to detect changes in the proportions of myeloid-derived suppressor cells (MDSCs) and other immune cells. Psoriasis was induced in mice by the daily application of imiquimod. ZQT was administered separately or in combination with anti-Gr1 antibody to deplete MDSC. The glycolysis levels of the MDSCs were detected using a Seahorse analyzer. The p21/Hif1α/Glut1 pathway was identified and validated by mRNA sequence, RT-qPCR, WB, IF, and the application of p21 inhibitor UC2288. ResultsThe number of MDSCs was significantly increased in patients with psoriasis, with the increased expression of p21, Hif1α, and Glut1 in MDSCs. ZQT significantly alleviated psoriasis-like skin lesions in mice. ZQT formula significantly reduced the number of MDSCs in psoriatic-like mice and enhanced their suppressive capacity for T cells. The efficacy of ZQT in alleviating psoriatic dermatitis is compromised by MDSC depletion. ZQT decreased the expressions of p21, Hif1α, and Glut1-induced glycolysis in MDSCs, thereby inhibiting Th17 cell differentiation. ConclusionThese suggest that ZQT alleviates IMQ-induced psoriatic dermatitis, by inhibiting p21/Hif1α/Glut1-induced glycolysis in MDSCs.

Abstract 3158: TUSC2 suppresses energy metabolism in lung cancer cells with opposite effects in normal bronchial epithelial cells

Abstract Introduction: TUSC2 (TUmor Suppressor Candidate 2) is a tumor suppressor gene from the human 3p21.3 chromosomal region frequently deleted in lung cancers. Independently of deletion, reduced expression of TUSC2 is observed in ~ 80% of lung cancers, mesothelioma, breast, head-and neck, osteosarcoma, glioblastoma, and other cancers, suggesting a critical anti-tumor role of TUSC2. TUSC2 resides in mitochondria and is involved mitochondrial respiration/energy metabolism, ROS production, Ca2+ flux to/from mitochondria, etc. Studies on tumor-bearing mice treated with TUSC2-containing plasmid encapsulated in lipid nanoparticles (quaratusugene ozeplasmid) and Phase I and II clinical trials of quar oze in lung cancer patients provides a strong rationale to further study the tumor-protective mechanisms of TUSC2 protein. Main scientific question. We asked if and how human lung cancer cells, A549 and H358, that have ~80-90% decrease in TUSC2 expression, change their energy metabolism in response to re-introduction of TUSC2 as compared to Beas2B, a normal bronchial epithelial cell line. Cancer cells rely heavily on anaerobic glycolysis, while normal epithelial cells use mitochondrial respiration as a primary way of energy production. Methods: Transient transfection of TUSC2-expressing plasmid to 3 cell lines; qPCR to confirm TUSC2 delivery; Seahorse analysis of energy production that measures in real time Oxygen Consumption Rate (OCR) and Extracellular Acidification Rate (ECAR) in different conditions. Results: All three cell lines demonstrated high transfection efficiency. Seahorse analysis revealed that TUSC2 re-introduction to TUSC2-deficient cancer cells consistently suppressed both glycolytic and mitochondrial ATP production, thus leaving cells without sufficient energy to support their vital functions. We found a significant decrease in basal and maximal respiration, spare respiratory capacity, ATP production, glycolysis and glycolytic capacity in cancer cell lines transfected with TUSC2-expressing plasmid. Unlike cancer cells, both glycolytic and mitochondrial metabolism of normal epithelial cells Beas2B were significantly strengthened after the introduction of TUSC2, suggesting a beneficial role of TUSC2 for the metabolic health of normal cells. The experiments were repeated four times; Student T-test was used to calculate statistical significance. Therapeutic Significance: These experiments suggest that TUSC2 plasmid delivery to cancer patients, and thus quar oze therapeutic use, may target and disrupt the metabolism of cancer cells, triggering either senescence or apoptotic pathways, while on the other hand, supporting the metabolism of normal epithelial cells. These data have high therapeutic significance, suggesting that one of the anti-tumor mechanisms of quar oze action in patients is the suppression of cancer cell metabolism resulting in cancer cell death. Citation Format: Jane Tonello, Mark Berger, Anil Shanker, Alla Ivanova. TUSC2 suppresses energy metabolism in lung cancer cells with opposite effects in normal bronchial epithelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3158.

Abstract 2887: Impact of ionizing radiation on the energy metabolism of normal and tumor cells in the brain

Abstract This study aims to analyze irradiation-induced metabolic alterations in glioblastoma cells and astrocytes to enhance understanding of cellular responses and facilitate the development of more effective radiotherapeutic strategies. Metabolic reprogramming of tumor cells is considered one of the hallmarks of cancer. Studies have shown that abnormal activation of oncogenes and cancer-related signaling pathways, as well as the inactivation of tumor suppressor genes can induce metabolic reprogramming. These metabolic aberrations facilitate rapid proliferation, continuous growth, invasion, metastasis, and immune evasion. Following the application of radiotherapy, the activity of several metabolic pathways significantly changes, potentially leading to the development of radioresistance. However, a differential effect of ionizing radiation on tumor and normal cells is not well understood. Hence, quantifying irradiation-induced metabolic changes in glioblastoma cells and astrocytes is of high importance. We have developed a novel approach to measure dynamic metabolite changes upon irradiation, in vivo, in situ, and in real-time. This tool combines imaging of recently developed fluorescent biosensors via 2-photon microscopy and millimeter-scaled irradiation. Murine glioblastoma cells and astrocytes were generated with stable expression of biosensors for the quantification of intracellular concentrations of various metabolites, including lactate. Cytosolic concentrations of lactate depend on the balance between glycolytic production, mitochondrial consumption of pyruvate and lactate, and the exchange with the extracellular lactate pool via monocarboxylate transporters. Many cancer cells are characterized by an excessive conversion of glucose to lactate even under normoxic conditions. We quantified this so-called Warburg Effect in glioblastoma cells and astrocytes as the ratio between basal lactate production and the lactate increase after OXPHOS inhibition and determined irradiation-induced metabolic changes on the single-cell level. Complementarily, we used metabolomics and Seahorse analyses before and after irradiation. Our data indicate that irradiation increases oxygen consumption in glioblastoma cells but not in astrocytes. Also, we established an orthotopic glioblastoma mouse model via injection of sensor-expressing tumor cells for in vivo measurements of metabolite changes during and after irradiation. These data are expected to significantly advance the understanding of the cellular response to irradiation on a molecular level in situ and in real-time. This knowledge will help to develop combination treatments and increase the efficiency of radiotherapy. Citation Format: Marvin Kreuzer, Pascal Imseng, Bruno Weber, Martin Pruschy. Impact of ionizing radiation on the energy metabolism of normal and tumor cells in the brain [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2887.

Abstract 4064: hetIL-15 and Fenofibrate combination alters the metabolic fitness of tumor-infiltrating CD8+T cells leading to TNBC tumor eradication in mice

Abstract Introduction: Tumor-infiltrating cytotoxic CD8+T cells frequently undergo an altered state of differentiation referred to as "exhaustion" and, as a result, they fail to control tumor growth. IL-15 is a cytokine which stimulates the generation, proliferation and cytotoxic function of tumor CD8+T and NK cells. We have produced the native heterodimeric form of IL-15 (hetIL-15) which has advanced in clinical trials. The aim of this study was to overcome the exhaustion of the tumor-infiltrating CD8+T cells and to increase their cytotoxicity, using Fenofibrate (FF), a PPAR-α agonist, in combination with hetIL-15. Experimental Procedures: We have evaluated the therapeutic efficacy of hetIL-15 immunotherapy as a monotherapy and in combination with FF in the murine EO771 orthotopic breast cancer model. The effects of hetIL-15 and/or FF on immune cells and on tumor microenvironment were analyzed by flow cytometry, transcriptomics, metabolomics and proteomics. The metabolic profile of tumor-infiltrating T cells was performed with Seahorse analysis. Results and Conclusions: hetIL-15 monotherapy resulted in tumor eradication in 40% of treated mice and increased survival. Seahorse analysis of tumor-infiltrating CD8+T cells confirmed a rise in oxygen consumption rate (OCR) with substantial increase of spare respiratory capacity. Upon hetIL-15 treatment, tumor-infiltrating CD8+T cells showed elevated extracellular acidification rate (ECAR), resulting in a pronounced shift in the OCR/ECAR ratio in comparison to control, confirming their increased proliferating status. These tumor-infiltrating CD8+T cells also showed increased mitochondrial potential and/or mass and fatty acid (FA) uptake, as evidenced by increased MitoTracker and Bodipy staining, respectively. Transcriptomic analysis revealed increased expression of genes involved in several metabolic pathways such as oxidative phosphorylation, FA oxidation and glycolysis. Monotherapy with FF had no effect on tumor growth delay, whereas the FF- hetIL-15 combination resulted in complete eradication of the tumors in 85%. Combination therapy reshaped the tumor microenvironment, resulting in higher IFN-γ and in differentially expressed metabolites as shown by tumor proteomics and metabolomics, respectively. The combination treatment increased the mitochondrial function, FA uptake and OCR, revealing a more metabolically active phenotype. We conclude that hetIL-15 supports a favorable metabolic profile of intratumoral effector lymphocytes, important for their function. Our data suggest that metabolic reprogramming of tumor specific CD8+T cells using FF is a promising strategy to overcome T cell exhaustion and to promote survival in a metabolically hostile tumor microenvironment. Citation Format: Dimitris Stellas, Sevasti Karaliota, Vasiliki Stravokefalou, Katherine C. Goldfarbmuren, Breana Myers, George N. Pavlakis, Barbara K. Felber. hetIL-15 and Fenofibrate combination alters the metabolic fitness of tumor-infiltrating CD8+T cells leading to TNBC tumor eradication in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4064.

Abstract 599: Combination of CDK4/6 inhibitors with biguanides for treatment of triple negative breast cancer

Abstract Triple negative breast cancer (TNBC) is the most aggressive subtype of breast cancer (BC) and is associated with early relapse and high frequency of metastasis. This has been attributed in part to the lack of targeted therapies. TNBC occurs more frequently in younger women of African and Hispanic ancestry and with a higher risk of mortality than women of European ancestry. Emerging data shows activation of the AKT/mTOR pathway by insulin may occur in aggressive TNBC and inhibition of mTOR signaling by rapamycin, a potent mTOR inhibitor, induces cell cycle arrest and downregulation of cyclin D1 in TNBC cells. CDK4/6 inhibitors have demonstrated to be an effective therapeutic for estrogen receptor positive BC and although their use in TNBC patients remains uncertain their use in combination with other targeted therapies may be promising. We have developed a set of newly designed biguanides with potent anticancer action and safety doses in vivo that in combination with CDK4/6 inhibitors block TNBC cell growth in vitro and in vivo. Using TNBC cell proliferation in vitro assays, new biguanides show dose-dependent inhibition of cell proliferation (P<0.01). Further, combination of biguanides with ribocliclib, an orally bioavailable CDK4/6 inhibitor, produced additive and synergistic effects in TNBC cell lines in vitro (P<0.01). Biguanides are known to activate de LKB1-AMPK pathway. Western immunoblots showed our biguanide analogues also induce AMPK phosphorylation and significantly reduce phosphorylation of downstream mTOR signaling pathway components including S6 ribosomal protein and 4E-BP1, an effect that is potentiated by combination treatment with ribociblib. Further, cyclin D1 is known to be overexpressed in about 50% of BCs and binding to CDK 4/6 induces phosphorylation of Rb and cell cycle progression. Treatment with biguanides downregulated cyclin D1 expression in TNBC cells and combination treatment of ribociclib with biguanide JD006 was more effective at decreasing Rb phosphorylation than each drug alone. In addition, seahorse analysis evaluated mitochondrial respiratory function. Treatment with biguanides inhibited oxidative phosphorylation in a dose dependent manner, an important metabolic pathway that can drive drug resistance to therapy in cancer cells. In vivo, combination treatment of ribociclib with biguanide JD006 was more effective than each agent alone in stopping human TNBC xenograft progression in nude mouse models (P<0.05). Our preliminary data offer evidence that human TNBC are highly vulnerable to metabolic reprogramming by new biguanides. This work can yield insight on unanticipated mechanisms of biguanide action and rationale for new combination therapies with CDK 4/6 inhibitors as a new option for treatment of TNBC. Funding: CBCRP B27IB3869, JCCC Breast Cancer Award, Hickey Family Foundation, NCI U54 CA143930, Team Research Grant, UCLA TDG. Citation Format: Mario Morales Martinez, Eduardo Mauricio Gonzalez, Gang Deng, Gaoyuan Ma, Hyunsoo Kim, Linsey Stiles, Nalo Hamilton, Michael E. Jung, Richard J. Pietras, Diana C. Marquez-Garban. Combination of CDK4/6 inhibitors with biguanides for treatment of triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 599.

SLC38A2 promotes cell proliferation and invasion by promoting glutamine metabolism in adenomyosis.

Adenomyosis is a benign uterine disorder that is associated with female infertility, a reduced clinical pregnancy rate and a high risk of miscarriage. Solute carrier family 38 member a2 (SLC38A2) is a glutamine (Gln) transporter that serves roles in various medical conditions. The present study aimed to reveal the role of SLC38A2 in adenomyosis. The mRNA expression levels of SLC38A2 in eutopic endometrial (EU) and ectopic endometrial (EC) tissues from adenomyotic patients were examined by reverse transcription-quantitative PCR. EU and EC cell proliferation and invasion were analyzed by Cell Counting Kit-8 and Transwell assays. Changes in the oxygen consumption rate (OCR) were determined to indicate the mitochondrial respiratory function and observed using a Seahorse analyzer. SLC38A2 expression in EC tissues was upregulated compared with that in normal endometrial tissues. SLC38A2 knockdown repressed EC cell proliferation and invasion. In addition, the Gln content and OCR were decreased in EC cells transfected with SLC38A2-knockdown lentivirus, whereas SLC38A2 overexpression had the opposite effect in EU cells. Furthermore, the increased proliferation and invasion rates and Gln level induced by SLC38A2 overexpression in EU cells were alleviated by CB-839, a glutaminase inhibitor. SLC38A2 overexpression promoted Gln metabolism and oxygen consumption rate, resulting in an increase in cell proliferation and invasion in the adenomyosis context. The present study indicated that reduction of SLC38A2 expression could be a novel target for adenomyosis therapy, and SLC38A2 may be a valuable clinical diagnostic molecule for adenomyosis.

The potential of baicalin to enhance neuroprotection and mitochondrial function in a human neuronal cell model

Baicalin is a flavone glycoside derived from flowering plants belonging to the Scutellaria genus. Previous studies have reported baicalin’s anti-inflammatory and neuroprotective properties in rodent models, indicating the potential of baicalin in neuropsychiatric disorders where alterations in numerous processes are observed. However, the extent of baicalin’s therapeutic effects remains undetermined in a human cell model, more specifically, neuronal cells to mimic the brain environment in vitro. As a proof of concept, we treated C8-B4 cells (murine cell model) with three different doses of baicalin (0.1, 1 and 5 μM) and vehicle control (DMSO) for 24 h after liposaccharide-induced inflammation and measured the levels of TNF-α in the medium by ELISA. NT2-N cells (human neuronal-like cell model) underwent identical baicalin treatment, followed by RNA extraction, genome-wide mRNA expression profiles and gene set enrichment analysis (GSEA). We also performed neurite outgrowth assays and mitochondrial flux bioanalysis (Seahorse) in NT2-N cells. We found that in C8-B4 cells, baicalin at ≥ 1 μM exhibited anti-inflammatory effects, lowering TNF-α levels in the cell culture media. In NT2-N cells, baicalin positively affected neurite outgrowth and transcriptionally up-regulated genes in the tricarboxylic acid cycle and the glycolysis pathway. Similarly, Seahorse analysis showed increased oxygen consumption rate in baicalin-treated NT2-N cells, an indicator of enhanced mitochondrial function. Together, our findings have confirmed the neuroprotective and mitochondria enhancing effects of baicalin in human-neuronal like cells. Given the increased prominence of mitochondrial mechanisms in diverse neuropsychiatric disorders and the paucity of mitochondrial therapeutics, this suggests the potential therapeutic application of baicalin in human neuropsychiatric disorders where these processes are altered.

Rhodiola crenulata alleviates hypobaric hypoxia-induced brain injury by maintaining BBB integrity and balancing energy metabolism dysfunction

Background/Purpose: Rhodiola crenulata (Hook. f. et Thoms.) H. Ohba (R. crenulate), a famous and characteristic Tibetan medicine, has been demonstrated to exert an outstanding brain protection role in the treatment of high-altitude hypoxia disease. However, the metabolic effects of R. crenulate on high-altitude hypoxic brain injury (HHBI) are still incompletely understood. Herein, the anti-hypoxic effect and associated mechanisms of R. crenulate were explored through both in vivo and in vitro experiments. Study design/MethodsThe mice model of HHBI was established using an animal hypobaric and hypoxic chamber. R. crenulate extract (RCE, 0.5, 1.0 and 2.0 g/kg) and salidroside (Sal, 25, 50 and 100 mg/kg) was given by gavage for 7 days. Pathological changes and neuronal apoptosis of mice hippocampus and cortex were evaluated using H&E and TUNEL staining, respectively. The effects of RCE and Sal on the permeability of blood brain barrier (BBB) were detected by Evans blue staining and NIR-II fluorescence imaging. Meanwhile, the ultrastructural BBB and cerebrovascular damages were observed using a transmission electron microscope (TEM). The levels of tight junction proteins Claudin-1, ZO-1 and occludin were detected by immunofluorescence. Additionally, the metabolites in mice serum and brain were determined using UHPLC-MS and MALDI-MSI analysis. The cell viability of Sal on hypoxic HT22 cells induced by CoCl2 was investigated by cell counting kit-8. The contents of LDH, MDA, SOD, GSH-PX and SDH were detected by using commercial biochemical kits. Meanwhile, intracellular ROS, Ca2+ and mitochondrial membrane potential were determined by corresponding specific labeled probes. The intracellular metabolites of HT22 cells were performed by the targeted metabolomics analysis of the Q300 kit. The cell apoptosis and necrosis were examined by YO-PRO-1/PI, Annexin V/PI and TUNEL staining. In addition, mitochondrial morphology was tested by Mito-tracker red with confocal microscopy and TEM. Real-time ATP production, oxygen consumption rate, and proton efflux rate were measured using a Seahorse analyzer. Subsequently, MCU, OPA1, p-Drp1ser616, p-AMPKα, p-AMPKβ and Sirt1 were determined by immunofluorescent and western blot analyses. ResultsThe results demonstrated that R. crenulate and Sal exert anti-hypoxic brain protection from inhibiting neuronal apoptosis, maintaining BBB integrity, increasing tight junction protein Claudin-1, ZO-1 and occludin and improving mitochondrial morphology and function. Mechanistically, R. crenulate and Sal alleviated HHBI by enhancing the tricarboxylic acid cycle to meet the demand of energy of brain. Additionally, experiments in vitro confirmed that Sal could ameliorate the apoptosis of HT22 cells, improve mitochondrial morphology and energy metabolism by enhancing mitochondrial respiration and glycolysis. Meanwhile, Sal-mediated MCU inhibited the activation of Drp1 and enhanced the expression of OPA1 to maintain mitochondrial homeostasis, as well as activation of AMPK and Sirt1 to enhance ATP production. ConclusionCollectively, the findings suggested that RCE and Sal may afford a protective intervention in HHBI through maintaining BBB integrity and improving energy metabolism via balancing MCU-mediated mitochondrial homeostasis by activating the AMPK/Sirt1 signaling pathway.

Particulate matters 2.5 induce tumor progression in lung cancer by increasing the activity of hnRNPA2B1 resulting in retarding mRNA decay of oxidative phosphorylation.

Particulate matter 2.5 (PM2.5) has been implicated in lung injury and various cancers, yet its precise mechanistic role remains elusive. To elucidate the key signaling pathways underpinning PM2.5-induced lung cancer progression, we embarked on a study examining the impact of PM2.5 both in vitro and in vivo. Lung cancer cell lines, A549 and H157, were employed for the in vitro investigations. Overexpression or knockdown techniques targeting the hnRNPA2B1 protein were implemented. Lung cancer cells were treated with a medium containing PM2.5 and subsequently prepared for in vitro evaluations. Cell growth, invasion, and migration were gauged using transwell and CCK-8 assays. Apoptosis was ascertained through flow cytometry and western blotting of pertinent proteins. Seahorse analyses probed the influence of PM2.5 on lung cancer energy metabolism. The RNA stability assay was employed to discern the impact of PM2.5 on the stability of oxidative phosphorylation-related genes in lung cancer. Our findings revealed that PM2.5 augmented cell proliferation, migration, and invasion rates. Similarly, a diminished apoptosis rate was observed in PM2.5-treated cells. Elevated expression of hnRNPA2B1 was detected in lung cancer cells exposed to PM2.5. Moreover, in cells treated with PM2.5, hnRNPA2B1 knockdown markedly curtailed cell proliferation by inducing G1-S cell cycle arrest and bolstered lung cancer cell apoptosis in vitro; it also curbed xenograft tumor growth. Mechanistically, our data suggest that PM2.5 undermines the stability of mRNA transcripts associated with oxidative phosphorylation (OXPHOS) and augments the formation of processing bodies (P-bodies), leading to an upsurge in OXPHOS levels. In conclusion, PM2.5 appears to drive lung cancer progression and migration by modulating the energy metabolism of lung cancer in a hnRNPA2B1-dependent manner.

Abstract A045: Elevated PGC1α during abrupt mammary gland involution leads to long-term metabolic reprogramming and genomic instability; hallmarks of breast cancer

Abstract Epidemiological data links higher parity and lack of breastfeeding with increased risk of breast cancer, specifically aggressive triple negative breast cancer, and associated higher mortality rate. Long-term breastfeeding and gradual weaning of an infant leads to gradual involution (GI) of the breast, while lack of or abrupt discontinuation of breastfeeding after birth leads to abrupt involution (AI), when rapid and massive cell death takes place. Our studies show several precancerous changes, such as increased collagen deposition, inflammation, and hyperplasia in the mammary gland (MG) of mice after AI1. Recent studies indicate peroxisome proliferator-activated receptor-gamma coactivator 1-apha (PGC1α) as a regulator of involution and energy metabolism. As metabolic reprogramming is a hallmark of cancer, metabolic changes in the MG related to involution warrant investigation. Objective: Our objective was to evaluate the metabolic effects of AI in MG. We hypothesized that AI leads to marked alteration in mammary lipid metabolism, mitochondrial biogenesis, and oxidative stress through elevations of PGC1α, which cause long-term metabolic reprogramming and genomic instability. Methods: FVB/n mice were paired for breeding. At partum, dams were randomized to AI or GI cohort and standardized to 6 pups per dam. AI mice had pups removed on day 7 postpartum (PPM). For GI mice 3 pups each were removed on day 28 and 31ppm. Tissues were harvested on day 28, 56, and 120 PPM. MG were subjected to Affymetrix, Gene Set Enrichment Analysis (GSEA), Seahorse Analysis, and lipidomics. Results were validated by qPCR and Western Blot. Superoxide species were detected by flow cytometry. DNA damage was analyzed via 8-hydroxy-2’-deoxygnuanosine (8-OHdG) ELISA. Results: Day 28 AI glands had significantly higher PGC1α expression than GI glands (p=0.006). Affymetrix and GSEA data showed day 28 AI glands to have enriched pathways related to fatty acid metabolism (p=0.004) and oxidative phosphorylation (p<0.001). Lipidomics showed elevated levels of oxidized sphingolipids and production of prostaglandin J2 (PGJ2) in day 28 AI glands (all p<0.02). Day 56 AI glands had higher levels of mitochondria superoxide species (p<0.0001) and higher PGJ2 synthesis (p=0.01). Day 120 AI MG had upregulation of an oxidized lipid (p=0.0476). Day 120 AI glands had significantly higher 8-OHdG levels (p=0.02), higher reliance of fatty acid substrates for energy (p=0.0185), and elevated extracellular acidification rates (p=0.0194). Conclusion: Although histologically both GI and AI MG return to near pre-pregnancy state within a month, our data shows long-term metabolic reprogramming in the AI MG similar to what is shown in breast cancer cells. These metabolic changes link to early elevations in PGC1α. Addressing the metabolic changes by targeting PGC1α provide a potential option to reduce the risk of developing breast cancer if a woman is unable to breastfeed. PMID6637535 *Fellowship T32CA229114 Citation Format: Kate S Ormiston, Kirti Kaul, Neelam Shinde, Gautam Sarathy, Morgan Bauer, Djawed Bennouna, Rachel Kopec, Ramesh Ganju, Sarmila Majumder, Bhuvaneswari Ramaswamy. Elevated PGC1α during abrupt mammary gland involution leads to long-term metabolic reprogramming and genomic instability; hallmarks of breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A045.

DOP88 Lipids drive myofibroblast activation and the epithelial-mesenchymal transition process in Crohn’s Disease

Abstract Background Fibrostenosis represents one of the major clinical complications of Crohn’s Disease (CD). However, the molecular mechanisms underlying intestinal fibrogenesis remain poorly understood and anti-fibrotic therapy has not yet been developed. Growing evidences supports that oxidative stress and the epithelial-mesenchymal transition (EMT) process contribute to the development and progression of fibrosis. Here, we assessed the role of lipids in fibrogenesis and whether mitochondria dysfunction can support fibrosis formation. Methods Spatial metabolomics analysis and adipophilin (ADFP) staining were performed on surgical specimens from fibrotic ileum and the surrounding region (healthy) derived from CD patients. Fibroblasts isolated from fibrotic and healthy ileum and human induced pluripotent stem cell-derived intestinal organoids (iHOs) were treated for 1-3 weeks with lipid mix (2%) and analyzed by RNA sequencing. Pathways significantly modulated were validated by RT-PCR, WB, FACS analysis, and proliferative, migratory and contraction assays, and mitochondrial functions by Seahorse analyzer. Results Spatial metabolomics revealed a significant accumulation of lipids characterized by very long-chain fatty acids in the fibrotic area compared to surrounding healthy regions, which was further confirmed by ADFP staining. RNA-seq analysis showed overlapping dysregulated pathways between healthy fibroblasts stimulated with lipids and fibrotic fibroblasts, in particular genes related to cell cycle, cytoskeletal reorganization, migration, contractility and cell metabolism (oxidative stress) which were further confirmed by functional assays. Among the genes related to oxidative stress, lipid stimulation significantly decreased NFE2L2 and promoted an increase in CPT1A, gene encoding for a fatty oxidation mediator. Moreover, lipid-stimulated fibroblasts showed a reduction in mitochondrial respiration, particularly basal and maximal respiration, and spare respiratory capacity by Seahorse analysis. On the other hand, long exposure of iHOs to lipids promoted the EMT process through the upregulation of TWIST, ZEB, VIM, and S100a4. Conclusion Lipid stimulation induced myofibroblast activation through oxidative stress and alteration of mitochondrial metabolism, and increased the expression of EMT transcription factors in intestinal epithelial cells (iHOs) that may support fibroblast-mediated pro-fibrotic activity. These data indicate that regulators of fatty acid oxidation involved in the fibrotic process may represent new potential targets for the treatment of fibrostenotic CD patients.

Deficiency of TOP1MT enhances glycolysis through the stimulation of PDK4 expression in gastric cancer

BackgroundAbnormal glucose metabolism is one of the determinants of maintaining malignant characteristics of cancer. Targeting cancer metabolism is regarded as a new strategy for cancer treatment. Our previous studies have found that TOP1MT is a crucial gene that inhibits glycolysis and cell metastasis of gastric cancer (GC) cells, but the mechanism of its regulation of glycolysis remains unclear.MethodsTranscriptome sequencing data, clinic-pathologic features of GC from a variety of public databases, and WGCNA were used to identify novel targets of TOP1MT. Immunohistochemical results of 250 patients with GC were used to analyze the relative expression relationship between TOP1MT and PDK4. The function of TOP1MT was investigated by migration assays and sea-horse analysis in vitro.ResultsWe discovered a mitochondrial topoisomerase I, TOP1MT, which correlated with a higher risk of metastasis. Functional experiments revealed that TOP1MT deficiency promotes cell migration and glycolysis through increasing PDK4 expression. Additionally, the stimulating effect of TOP1MT on glycolysis may be effectively reversed by PDK4 inhibitor M77976.ConclusionsIn brief, our work demonstrated the critical function of TOP1MT in the regulation of glycolysis by PDK4 in gastric cancer. Inhibiting glycolysis and limiting tumor metastasis in GC may be accomplished by suppressing PDK4.