Rise In Oxygen Consumption Rate Research Articles

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.

Triazophos-induced Respiratory and Behavioral Effects and Development of Adverse Outcome Pathway (AOP) for short-term Exposed Freshwater Snail, Bellamya Bengalensis.

The physiological effects of triazophos were examined using respiratory and behavioral endpoints in Bellamya bengalensis under a 96-hour acute exposure regime. Physiological manifestation of respiratory stress was measured using the rate of oxygen consumption while behavioral toxicity was measured using crawling reflexes, touch response, and mucus production. The threshold effect values for LOEC (Lowest Observed Effect Concentration), NOEC (No Observed Effect Concentration), and MATC (Maximum Acceptable Toxicant Concentration) at 96h were 0.40, 0.60, and 0.075mg/l, respectively. Definitive 96h acute exposures for both respiratory and behavioral endpoints tests were determined using a control group and concentrations ranging from 0.40 to 1.60mg/l monitored for 24, 48, 72, and 96h. Test organisms irrespective of exposure concentration demonstrated an initial rise in oxygen consumption rate after 24h, followed by a progressive decrease in toxicant concentration and exposure period. The in silico structural analysis presents triazophos as having an electrophilic toxic structure similar to choline esterase inhibitors, and also capable of inducing oxidative stress. The AOP highlighted neurotoxicity and oxidative stress as plausible pathways of triazophos toxicity in mollusk species.

Synergy of heterodimeric IL-15 immunotherapy and a Fatty Acid Metabolism Modulator enhances intratumoral lymphocyte metabolism and eradicates EO771 murine breast tumors

Abstract Tumor-infiltrating cytotoxic CD8 +T cells frequently acquire an altered state of differentiation referred to as “exhaustion”, which prevents control of tumor growth. We study hetIL-15, a cytokine that stimulates the generation, proliferation and cytotoxic function of CD8 +T and NK cells. The objectives of this study was to assess the effects of hetIL-15 immunotherapy in TNBC tumors, to evaluate the metabolic profile of the tumor-infiltrating T cells and to study any potential synergy with a Fatty Acid Metabolism Modulator (FAMM). Using the EO771 orthotopic breast cancer model we monitored the efficacy of locoregional administration of hetIL-15 in combination with a FAMM. We monitored the effect of treatment on tumor growth, survival of mice and the status of tumor-infiltrating immune cells using Seahorse and flow cytometry. hetIL-15 locoregional administration resulted in complete tumor regression in 40% of the treated animals and increased survival. The infiltrating cytotoxic CD8 +T and NK cells were increased in hetIL-15 treated tumors and showed enhanced activation and proliferation. Their metabolic flux analysis confirmed a robust rise in Oxygen Consumption Rate (OCR) with substantial increase of spare respiratory capacity. Combined treatment of hetIL-15 immunotherapy and FAMM resulted in increased mitochondrial function, FA uptake and OCR. In addition, combination therapy resulted in statistically significant tumor growth delay and complete eradication of the tumors in 85% of mice. Our results indicate that metabolic reprogramming of tumor-specific CD8 +T cells might represent a strategy to promote survival in the metabolically hostile Tumor Microenvironment (TME), enhancing the clinical efficacy of immunotherapy.

Abstract 5082: Heterodimeric IL-15 (hetIL-15) immunotherapy synergizes with Fatty Acid Metabolism Modulator (FAMM) to eradicate TNBC EO771 murine tumors

Abstract Introduction: Metabolic fitness and T cell survival are crucial in anti-tumor responses because nutrients are often scarce and other regulatory molecules may be unfavorable in the tumor microenvironment leading to T cell dysfunction, stress, and apoptosis. Tumor-infiltrating cytotoxic CD8+T cells frequently acquire an altered state of differentiation referred to as “exhaustion” and, as a result, they fail to control tumor outgrowth. IL-15 cytokine stimulates the generation, proliferation and cytotoxic function of tumor specific CD8+ T cells and NK cells. The objective of this study was to assess the effects of hetIL-15 immunotherapy in triple negative breast cancer tumors (TNBC), to evaluate the metabolic profile of the tumor-infiltrating T cells and to study any potential synergy using a Fatty Acid Metabolism Modulator (FAMM) to enhance T cell metabolism. Study design and methods: We used the murine EO771 orthotopic breast cancer model to study the efficacy of locoregional administration of hetIL-15 immunotherapy in combination with a FAMM. We monitored the effect of treatment on number, metabolism and mitochondrial function of the tumor-infiltrating immune cells by flow cytometry, Seahorse flux analysis and Mitotracker, 2-NBDG and/or Bodipy staining. Results: hetIL-15 locoregional administration, as as single agent, resulted in complete regression in 40% of the treated animals and increased survival. Tumor-infiltrating cytotoxic CD8+T and NK cells increased in hetIL-15 treated tumors and showed enhanced activation and proliferation. Metabolic flux analysis of the tumor-infiltrating cytotoxic CD8+T cells from hetIL-15-treated mice confirmed a rise in oxygen consumption rate (OCR) with substantial increase of spare respiratory capacity, which supports an activated/non exhausted phenotype of these hetIL-15 treated effector cells. Since, further promoting fatty acid (FA) catabolism improves the tumor-infiltrated CD8+T cells’s ability to slow tumor progression, we combined hetIL-15 immunotherapy with a FAMM. Combination therapy resulted in increased mitochondrial function, FA uptake and OCR, revealing a more metabolically active phenotype compared to the tumor-infiltrating CD8+T cells from hetIL-15 monotherapy group. In addition, combined treatment of IL-15 immunotherapy and FAMM resulted in statistically significant EO771 tumor growth delay and complete eradication of the tumors in 85% of mice. Conclusions: Our results indicate that hetIL-15 synergizes with metabolic reprogramming of T cells to achieve superior antitumor efficacy and complete cures. We suggest that metabolic reprogramming of tumor-specific CD8+Tcells might represent a strategy to promote survival in the metabolically hostile TME as part of an approach to enhance the clinical efficacy of immunotherapy. Citation Format: Sevasti Karaliota, Dimitris Stellas, Vasiliki Stravokefalou, Breana Myers, Barbara K. Felber, George N. Pavlakis. Heterodimeric IL-15 (hetIL-15) immunotherapy synergizes with Fatty Acid Metabolism Modulator (FAMM) to eradicate TNBC EO771 murine tumors. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5082.

Abstract 5603: Heterodimeric IL-15 (hetIL-15) immunotherapy reverses CD8+T cell metabolic dysfunction in murine breast tumors

Abstract Introduction: Metabolic fitness and T cell survival are crucial in anti-tumor responses because nutrients are often scarce and other regulatory molecules may be unfavorable in the tumor microenvironment leading to T cell dysfunction, stress, and apoptosis. Tumor-infiltrating cytotoxic CD8+T cells often acquire an altered state of differentiation referred to as “exhaustion” and, as a result, they fail to control tumor outgrowth. IL-15 cytokine stimulates the generation, the proliferation and the cytotoxic function of tumor specific CD8+ T cells and NK cells. The objective of this study was to assess the effects of hetIL-15 immunotherapy after locoregional administration in triple negative breast cancer tumors (TNBC) and to evaluate the metabolic profile of the tumor infiltrating T cells. Study design and methods: We studied the therapeutic efficacy of hetIL-15 immunotherapy in murine EO771 orthotopic breast cancer model. We monitored the effect of the treatment on tumor size and immune infiltration using flow cytometry and immunohistochemistry. We also evaluated hetIL-15 effects on the cell metabolism and the mitochondrial function of the tumor-infiltrating immune cells, using flow cytometry, Seahorse flux analysis, Mitotracker, 2-NBDG and/or Bodipy staining. Results: hetIL-15 peritumoral administration, as monotherapy resulted in complete regression in 40% of the treated animals and increased survival. We demonstrated that tumor infiltrating cytotoxic CD8+T and NK cells were increased in hetIL-15 treated tumors and showed enhanced activation and proliferation, resulting in enhanced intratumoral immunological killing. Metabolic flux analysis of the tumor-infiltrating CD8+T cells from treated mice confirmed a rise in oxygen consumption rate (OCR) with substantial increase of spare respiratory capacity, which supports an activated/non exhausted phenotype of these hetIL-15 treated effector cells. Consistent with the above finding, tumor infiltrated CD8+T cells from hetIL-15 treated mice showed increased mitochondrial potential and fatty acid uptake, as evidenced by increased Mitotracker and Bodipy staining, respectively. In addition, tumor infiltrating CD8+T cells from hetIL-15 treated mice presented elevated extracellular acidification rate (ECAR) and showed a pronounced shift in the OCR to ECAR ratio in comparison to control, confirming their increased proliferating status. Conclusions: Our results indicate that hetIL-15 not only increases the infiltration and the cytotoxic properties of the CD8+T cells inside the tumors, but also enhances the metabolic reprogramming of T cells to achieve superior antitumor efficacy. We suggest that metabolic reprogramming of tumor-specific CD8+T cells might represent a strategy to promote survival in the metabolically hostile TME as part of an approach to enhance the clinical efficacy of immunotherapy. Citation Format: Dimitris Stellas, Sevasti Karaliota, Vasiliki Stravokefalou, Breana Myers, Barbara K. Felber, George N. Pavlakis. Heterodimeric IL-15 (hetIL-15) immunotherapy reverses CD8+T cell metabolic dysfunction in murine breast tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5603.

Abstract 5694: Complete regression of murine breast tumors by hetIL-15FC monotherapy correlates with infiltration by T, NK, cDC1 cells and a novel population of dendritic cells

Abstract Introduction: IL-15 cytokine stimulates the proliferation and cytotoxic function of CD8+ T and NK cells. Several preclinical models have supported the anti-tumor activity of IL-15. We have produced the native heterodimeric IL-15 (hetIL-15) of human, macaque or mouse and also fusions to the Fc antibody fragments (hetIL-15FC). Experimental procedures: We studied the therapeutic efficacy of hetIL-15 or hetIL-15FC immunotherapy in the murine EO771 orthotopic breast cancer model in syngeneic C57BL/6 mice. The effects of hetIL-15FC on immune cells were compared in tumors, lymph nodes and spleen by flow cytometry, immunohistochemistry (IHC) and gene expression profiling (Nanostring). We also assessed the metabolic profile of tumor infiltrating T cells. Results: hetIL-15 therapy delayed the primary tumor growth and increased overall survival. hetIL-15 or hetIL-15FC peritumoral administration resulted in tumor regression in 40% of the treated animals and resistance to tumor re-challenge. Therefore, monotherapy induced long term immunological memory able to block tumor growth. Both CD8+ T and NK cells were increased in hetIL-15FC treated tumors; phenotypic analysis showed they were activated and in an active proliferation state. Transcriptomic analysis confirmed the activated state of the T and NK cells, whereas metabolic flux analysis of the infiltrated CD8+T cells from treated mice confirmed a rise in oxygen consumption rate (OCR) with substantial increase of spare respiratory capacity (as a measure of mitochondrial reserve), which supports an activated/non exhausted phenotype of these hetIL-15 treated effector cells. In addition, peritumoral hetIL-15FC administration resulted in an increase of infiltrating, conventional type 1 dendritic cells (cDC1s), suggesting mechanisms of DC-lymphocyte interactions in the tumor. We also observed a new population of intra-tumoral DCs induced by hetIL-15, CD24+ F4/80high cDC2s (F4/80high DCs). The new DC population was negatively correlated with the tumor size of EO771 tumor bearing mice. Phenotypic profiling of F4/80high DCs identified expression of several cDC1 specific markers, including CD103 and IRF8 and t-SNE analysis clustered F4/80high DCs close to cDC1s and macrophages. Conclusion: Our results show complete eradication of EO771 tumors by hetIL-15FC treatment, correlating with different tumor infiltrating immune cell types. We propose that the anti-cancer activity of hetIL-15 in primary tumors is orchestrated by the interplay of CD8+T cells, cDC1s and a novel subset of antigen presenting cells with a distinct phenotypic profile. hetIL-15 supported a favorable metabolic profile of intra-tumoral effector lymphocytes, important for their function. Effective hetIL-15 treatment leads to development of long-term anti-tumor memory. Citation Format: Sevasti Karaliota, Dimitris Stellas, Vasiliki Stravokefalou, Bethany Nagy, Cristina Bergamaschi, Barbara K. Felber, George N. Pavlakis. Complete regression of murine breast tumors by hetIL-15FC monotherapy correlates with infiltration by T, NK, cDC1 cells and a novel population of dendritic cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5694.

138 FATTY ACID β-OXIDATION REGULATES BOVINE EMBRYO OXYGEN METABOLISM

Mammalian oocytes are rich in endogenous lipid, which provides a potential source of metabolic energy during oocyte maturation and early embryo development after fertilisation. Despite this, studies on early embryo metabolism have focussed on consumption of substrates from the culture medium, with comparatively little consideration for endogenous stores. Additionally, the interaction between fatty acid β-oxidation and oxygen consumption during pre-implantation development has not been investigated. This study has investigated the relationship between embryo fatty acid metabolism and oxygen consumption rate (OCR), a marker of overall oxidative metabolic activity. The amount of oxygen consumption by bovine blastocysts coupled to ATP synthesis was assessed by measuring embryo OCR following acute treatment with oligomycin, which inhibits ATP synthase. Bovine embryos were then produced in vitro and cultured from zygote to blastocyst stage (8 days) in the presence of β-mercaptoacetate (BMA), a competitive inhibitor of fatty acid β-oxidation, or L-carnitine, a cofactor that promotes β-oxidation. OCR of individual embryos was measured using a noninvasive, highly sensitive oxygen probe (Unisense), whereas OCR of embryos in groups was determined using the Oxygen Biosensor (OBS) fluorimetric assay (BD Biosciences, Erembodegem, Belgium). Mean OCR measured by each technique correlated well. Data are presented as means ± SEM. The effect of acute inhibition were compared by a paired t-test, with pre-treated embryos acting as their own control, whereas the effect of L-carnitine and BMA on OCR was compared across treatment groups using ANOVA with Dunn’s test for unequal groups post hoc, using Sigmaplot (Systat Software Inc., San Jose, CA, USA). Treatment with oligomycin caused blastocyst OCR to fall from 23.5 ± 3.3 to 7.9 ± 2.5 pmol/embryo/h (n = 3; P = 0.03). Inhibition of β-oxidation by BMA led to a modest, but consistent increased OCR in cleavage stage embryos: 4-cell: 14.8 ± 7.2 (n = 12) v. 8.9 ± 1.1 pmol/embryo/h (n = 3) control; 8-cell: 15.8 ± 2.2 (n = 6) v. 11.9 ± 2.3 pmol/embryo/h (n = 3) control. At the morula stage, BMA led to a significant rise in OCR (26.8 ± 3.6 (n = 7) v. 7.8 ± 1.7 pmol/embryo/h (n = 3) control; P = 0.003) and at the blastocyst stage, OCR was significantly elevated in the presence of BMA; 41.3 ± 2.6 (n = 19) v. 16.9 ± 1.3 pmol/embryo/h (n = 14) control (P = 0.01). By contrast, when groups of embryos were cultured in the presence of L-carnitine, blastocyst OCR was reduced by 45% from 24.7 ± 5.1 (group control) to 15.9 ± 3.7 pmol/embryo/h (n = 30; P < 0.06). We report that 66.5% of oxygen consumption by bovine blastocysts is oligomycin sensitive, meaning that 33.5% of oxygen consumption is used for processes other than ATP synthesis. Surprisingly, inhibition of fatty acid β-oxidation caused total bovine embryo OCR to rise, although the reasons for this are unclear. It is possible that inhibiting β-oxidation leads to an accumulation of free fatty acids in the mitochondrion, which can disrupt the inner membrane and lead decoupling of ATP synthesis from oxygen consumption. Thus, manipulating fatty acid oxidation in the early embryo dysregulates oxidative metabolism; the implications of this are under further investigation.

The effect of avoidance behavior on predicting fish passage rates through water diversion structures

Conserving fish populations within lotic ecosystems requires an understanding of the mechanisms that impede fish movement through a river. For rivers with diversions, negative fish population effects are ameliorated by barriers whose effectiveness is based on individual fish avoidance responses. Here, we evaluate two alternative avoidance models using new algorithms and videotape analyses. We tested the effects of a directed “danger-minimizing” behavior compared to “random avoidance” on predictions of fish passage rates at water diversion facilities using individual-based models. Validation data were collected using a two-dimensional analysis of fish swimming behavior in a small-scale channel with a louver diversion barrier. While the danger-minimizing model adequately captured the range of swimming behavior, it underestimated swimming exertion and the time exposed to the barrier. The random avoidance model produced significantly higher passage rates compared to the danger-minimizing model, yet it also resulted in a significant rise in oxygen consumption rate. Given departures between predicted and observed swimming behaviors we included an a posteriori evaluation of adding behavioral complexity to the movement rules. These evaluations demonstrated that positive system level predictions of high passage rates can obscure significant differences in individual level energetic costs. Our study emphasizes the value of examining multiple models at the individual level when extrapolating to the population level effects of fish encountering artificial barriers.

Metabolic effects of amoebic gill disease (AGD) and chloramine-T exposure in seawater-acclimated Atlantic salmon Salmo salar

Our aim was to determine possible metabolic effects amoebic gill disease (AGD) on Atlantic salmon Salmo salar. Standard (R(S)) and routine (R(ROU)) metabolic rates were evaluated by continually measuring oxygen consumption in 2 independent tanks of fish (18.69 +/- 1.01 kg m(-3), mean +/- SE). Active metabolic rate (R(ACT)) and metabolic scope (R(ACT) - R(S)) were assessed using a chasing protocol and determined at 3 time periods: (1) pre-infection, (2) 3 d post-infection, and (3) 2 d post-treatment. On Day 3 of the study, the fish were infected with amoebae isolated from the gills of AGD-affected salmon (2300 cells l(-1)). No significant elevations in R(ACT) or metabolic scope were detected 3 d post-infection and 2 d post-treatment; however, significant elevations in R(S) and R(ROU) were detected 3 d post-infection and 2 d post-treatment. Assessment of R(ROU) data, especially for the light period, also indicated a rise in oxygen consumption rate over the course of the experiment. Treatment of AGD-affected Atlantic salmon with chloramine-T (CL-T) appeared to briefly mitigate the rise in R(S), as there was a 30% drop (though non-significant) in R(S) following treatment. Despite this, R(S) continued the upward trend 1 d following treatment. These results suggest that over the course of AGD development, R(S) in Atlantic salmon increases. Therefore, considering the physical conditions which constrain R(ACT), we expect that metabolic scope would become compromised in fish more heavily affected with AGD. Treatment with CL-T shows promise for mitigating the respiratory effects of AGD and potentially minimising the loss of metabolic scope.

TEMPERATURE COEFFICIENTS OF RESPIRATION IN PSAMMECHINUS EGGS

With Cartesian diver micro-respiration technique the temperature characteristics of the respiration of Psammmechinus miliaris eggs were investigated:1. Between 10 and 21° C. the gradual rise in oxygen consumption rate of the unfertilized egg is best represented by Krogh's standard curve. Q10 at 18° C. is around 2.5, at 10° C. around 3.5.2. Fertilized eggs have the same temperature characteristics as unfertilized ones.3. In respect to temperature characteristics just before and just after fertilization, two classes are distinguishable among the sea-urchins: The Strongylocentrotus-Psammechinus group with equal Q10 values before and after fertilization, and the Arbacia group with higher Q10 values before fertilization.4. The significance of temperature characteristics for the biochemical processes involved in respiration of eggs before and after fertilization is discussed.