Toward Systems-Level Metabolic Analysis in Endocrine Disorders and Cancer

Metabolism is a dynamic network of biochemical reactions that support systemic homeostasis amidst changing nutritional, environmental, and physical activity factors. The circulatory system facilitates metabolite exchange among organs, while the endocrine system finely tunes metabolism through hormone release. Endocrine disorders like obesity, diabetes, and Cushing’s syndrome disrupt this balance, contributing to systemic inflammation and global health burdens. They accompany metabolic changes on multiple levels from molecular interactions to individual organs to the whole body. Understanding how metabolic fluxes relate to endocrine disorders illuminates the underlying dysregulation. Cancer is increasingly considered a systemic disorder because it not only affects cells in localized tumors but also the whole body, especially in metastasis. In tumorigenesis, cancer-specific mutations and nutrient availability in the tumor microenvironment reprogram cellular metabolism to meet increased energy and biosynthesis needs. Cancer cachexia results in metabolic changes to other organs like muscle, adipose tissue, and liver. This review explores the interplay between the endocrine system and systems-level metabolism in health and disease. We highlight metabolic fluxes in conditions like obesity, diabetes, Cushing’s syndrome, and cancers. Recent advances in metabolomics, fluxomics, and systems biology promise new insights into dynamic metabolism, offering potential biomarkers, therapeutic targets, and personalized medicine

Screening for Antibody Reduction at the Clone Selection Stage to Prevent Reduction During Manufacturing

Therapeutic monoclonal antibodies (mAbs) occupy a large part of the biopharmaceutical market; over 50 new mAbs are presently undergoing late-stage clinical trials, including human and humanized IgG1, 2 and 4 molecules, bispecifics, antibody-drug conjugates, and peptide-mAb fusion proteins.1 One of the associated challenges with the production of therapeutic mAbs is the reduction of the interchain disulfide bonds, which causes a loss of product, increased manufacturing complexity, and reduced drug product stability.5 Antibody reduction seems to be unpredictable and is typically not observed until the initial scale-up run. If left uncontrolled, reduction will be observed in all subsequent large-scale runs due to cell lysis caused by shear forces used to separate the supernatant from the cells during continuous centrifuge harvest operations. Small-scale centrifugation uses bench top centrifuges, which are more gentle than those used in the scale-up runs where reduction is most often observed. Cell lysis releases intracellular components, including reductases from the thioredoxin and glutathione enzymatic systems which have been shown to act on the antibody and reduce disulfide bonds.5 While we observe reduction in the large-scale manufacturing processes, small-scale reactors do not typically indicate disulfide bond reduction unless we artificially lyse the cells using a freeze-thaw method or a capillary shear device.16 However, freeze-thaw of the harvested cell culture fluid (HCCF), long-term storage at 5°C of the HCCF, or oxygen exposure from air surface transfer may cause the reducing components to lose activity. We investigated methods to propose a reduction assay to either identify clones that are unlikely to result in antibody reduction, or offer reduction mitigation strategies prior to the first scale-up run if a clone is likely to have reduced product. Thus, loss of product and delays to the project timeline can be prevented. Prior research suggests a correlation between reductase activity and antibody reduction. We continued this research and focused on identifying a correlation between antibody reduction and either the levels of expression or activities of the enzymes of the thioredoxin and glutathione systems. However, we did not find a correlation between either reductase expression or reductase activity and mAb reduction. NADPH is a cofactor for the glutathione reductase (GR) and the thioredoxin reductase (TrxR) and the total amount or ratio of NADP+/NADPH may correlate with mAb reduction but neither was quantified as part of this work. Interestingly, we found correlations between reductase expression and viability, VCD, titer, percent product aggregates, lactate, and pCO2. Additionally, we found a correlation between reductase activity and percent product aggregates. This data suggests that the intracellular redox environment is important for both cell growth and product quality. Future studies should focus on screening top clones from projects that do and do not have a history of reduction to determine variability in reductase expression and activity. Future studies should also look at NADPH

Screening for Antibody Reduction at the Clone Selection Stage to Prevent Reduction During Manufacturing

Therapeutic monoclonal antibodies (mAbs) occupy a large part of the biopharmaceutical market; over 50 new mAbs are presently undergoing late-stage clinical trials, including human and humanized IgG1, 2 and 4 molecules, bispecifics, antibody-drug conjugates, and peptide-mAb fusion proteins.1 One of the associated challenges with the production of therapeutic mAbs is the reduction of the interchain disulfide bonds, which causes a loss of product, increased manufacturing complexity, and reduced drug product stability.5 Antibody reduction seems to be unpredictable and is typically not observed until the initial scale-up run. If left uncontrolled, reduction will be observed in all subsequent large-scale runs due to cell lysis caused by shear forces used to separate the supernatant from the cells during continuous centrifuge harvest operations. Small-scale centrifugation uses bench top centrifuges, which are more gentle than those used in the scale-up runs where reduction is most often observed. Cell lysis releases intracellular components, including reductases from the thioredoxin and glutathione enzymatic systems which have been shown to act on the antibody and reduce disulfide bonds.5 While we observe reduction in the large-scale manufacturing processes, small-scale reactors do not typically indicate disulfide bond reduction unless we artificially lyse the cells using a freeze-thaw method or a capillary shear device.16 However, freeze-thaw of the harvested cell culture fluid (HCCF), long-term storage at 5°C of the HCCF, or oxygen exposure from air surface transfer may cause the reducing components to lose activity. We investigated methods to propose a reduction assay to either identify clones that are unlikely to result in antibody reduction, or offer reduction mitigation strategies prior to the first scale-up run if a clone is likely to have reduced product. Thus, loss of product and delays to the project timeline can be prevented. Prior research suggests a correlation between reductase activity and antibody reduction. We continued this research and focused on identifying a correlation between antibody reduction and either the levels of expression or activities of the enzymes of the thioredoxin and glutathione systems. However, we did not find a correlation between either reductase expression or reductase activity and mAb reduction. NADPH is a cofactor for the glutathione reductase (GR) and the thioredoxin reductase (TrxR) and the total amount or ratio of NADP+/NADPH may correlate with mAb reduction but neither was quantified as part of this work. Interestingly, we found correlations between reductase expression and viability, VCD, titer, percent product aggregates, lactate, and pCO2. Additionally, we found a correlation between reductase activity and percent product aggregates. This data suggests that the intracellular redox environment is important for both cell growth and product quality. Future studies should focus on screening top clones from projects that do and do not have a history of reduction to determine variability in reductase expression and activity. Future studies should also look at NADPH

Toward Systems-Level Metabolic Analysis in Endocrine Disorders and Cancer.

Metabolism is a dynamic network of biochemical reactions that support systemic homeostasis amidst changing nutritional, environmental, and physical activity factors. The circulatory system facilitates metabolite exchange among organs, while the endocrine system finely tunes metabolism through hormone release. Endocrine disorders like obesity, diabetes, and Cushings syndrome disrupt this balance, contributing to systemic inflammation and global health burdens. They accompany metabolic changes on multiple levels from molecular interactions to individual organs to the whole body. Understanding how metabolic fluxes relate to endocrine disorders illuminates the underlying dysregulation. Cancer is increasingly considered a systemic disorder because it not only affects cells in localized tumors but also the whole body, especially in metastasis. In tumorigenesis, cancer-specific mutations and nutrient availability in the tumor microenvironment reprogram cellular metabolism to meet increased energy and biosynthesis needs. Cancer cachexia results in metabolic changes to other organs like muscle, adipose tissue, and liver. This review explores the interplay between the endocrine system and systems-level metabolism in health and disease. We highlight metabolic fluxes in conditions like obesity, diabetes, Cushings syndrome, and cancers. Recent advances in metabolomics, fluxomics, and systems biology promise new insights into dynamic metabolism, offering potential biomarkers, therapeutic targets, and personalized medicine

Integrative metabolic flux analysis reveals an indispensable dimension of phenotypes.

Complete understanding of a biological system requires quantitation of metabolic fluxes that reflect its dynamic state. Various analytical chemistry tools, enzyme-based probes, and microscopy enable flux measurement. However, any method alone falls short of comprehensive flux quantitation. Here we show that integrating these techniques results in a systems-level quantitative map of absolute metabolic fluxes that constitute an indispensable dimension of characterizing phenotypes. Stable isotopes, mass spectrometry, and NMR spectroscopy reveal relative pathway fluxes. Biochemical probes reveal the physical rate of environmental changes. FRET-based and SRS-based microscopy reveal targeted metabolite and chemical bond formation. These techniques are complementary and can be computationally integrated to reveal actionable information on metabolism. Integrative metabolic flux analysis using various quantitative techniques advances biotechnology and medicine

MR-detectable metabolic biomarkers of response to mutant IDH inhibition in low-grade glioma.

Mutations in isocitrate dehydrogenase 1 (IDH1mut) are reported in 70-90% of low-grade gliomas and secondary glioblastomas. IDH1mut catalyzes the reduction of α-ketoglutarate (α-KG) to 2-hydroxyglutarate (2-HG), an oncometabolite which drives tumorigenesis. Inhibition of IDH1mut is therefore an emerging therapeutic approach, and inhibitors such as AG-120 and AG-881 have shown promising results in phase 1 and 2 clinical studies. However, detection of response to these therapies prior to changes in tumor growth can be challenging. The goal of this study was to identify non-invasive clinically translatable metabolic imaging biomarkers of IDH1mut inhibition that can serve to assess response. Methods: IDH1mut inhibition was confirmed using an enzyme assay and 1H- and 13C- magnetic resonance spectroscopy (MRS) were used to investigate the metabolic effects of AG-120 and AG-881 on two genetically engineered IDH1mut-expressing cell lines, NHAIDH1mut and U87IDH1mut. Results: 1H-MRS indicated a significant decrease in steady-state 2-HG following treatment, as expected. This was accompanied by a significant 1H-MRS-detectable increase in glutamate. However, other metabolites previously linked to 2-HG were not altered. 13C-MRS also showed that the steady-state changes in glutamate were associated with a modulation in the flux of glutamine to both glutamate and 2-HG. Finally, hyperpolarized 13C-MRS was used to show that the flux of α-KG to both glutamate and 2-HG was modulated by treatment. Conclusion: In this study, we identified potential 1H- and 13C-MRS-detectable biomarkers of response to IDH1mut inhibition in gliomas. Although further studies are needed to evaluate the utility of these biomarkers in vivo, we expect that in addition to a 1H-MRS-detectable drop in 2-HG, a 1H-MRS-detectable increase in glutamate, as well as a hyperpolarized 13C-MRS-detectable change in [1-13C] α-KG flux, could serve as metabolic imaging biomarkers of response to treatment

MR-detectable metabolic biomarkers of response to mutant IDH inhibition in low-grade glioma.

Mutations in isocitrate dehydrogenase 1 (IDH1mut) are reported in 70-90% of low-grade gliomas and secondary glioblastomas. IDH1mut catalyzes the reduction of α-ketoglutarate (α-KG) to 2-hydroxyglutarate (2-HG), an oncometabolite which drives tumorigenesis. Inhibition of IDH1mut is therefore an emerging therapeutic approach, and inhibitors such as AG-120 and AG-881 have shown promising results in phase 1 and 2 clinical studies. However, detection of response to these therapies prior to changes in tumor growth can be challenging. The goal of this study was to identify non-invasive clinically translatable metabolic imaging biomarkers of IDH1mut inhibition that can serve to assess response. Methods: IDH1mut inhibition was confirmed using an enzyme assay and 1H- and 13C- magnetic resonance spectroscopy (MRS) were used to investigate the metabolic effects of AG-120 and AG-881 on two genetically engineered IDH1mut-expressing cell lines, NHAIDH1mut and U87IDH1mut. Results: 1H-MRS indicated a significant decrease in steady-state 2-HG following treatment, as expected. This was accompanied by a significant 1H-MRS-detectable increase in glutamate. However, other metabolites previously linked to 2-HG were not altered. 13C-MRS also showed that the steady-state changes in glutamate were associated with a modulation in the flux of glutamine to both glutamate and 2-HG. Finally, hyperpolarized 13C-MRS was used to show that the flux of α-KG to both glutamate and 2-HG was modulated by treatment. Conclusion: In this study, we identified potential 1H- and 13C-MRS-detectable biomarkers of response to IDH1mut inhibition in gliomas. Although further studies are needed to evaluate the utility of these biomarkers in vivo, we expect that in addition to a 1H-MRS-detectable drop in 2-HG, a 1H-MRS-detectable increase in glutamate, as well as a hyperpolarized 13C-MRS-detectable change in [1-13C] α-KG flux, could serve as metabolic imaging biomarkers of response to treatment

Neutrophil diversity is associated with T-cell immunity and clinical relevance in patients with thyroid cancer.

Neutrophil heterogeneity is involved in autoimmune diseases, sepsis, and several cancers. However, the link between neutrophil heterogeneity and T-cell immunity in thyroid cancer is incompletely understood. We investigated the circulating neutrophil heterogeneity in 3 undifferentiated thyroid cancer (UTC), 14 differentiated thyroid cancer (DTC) (4 Stage IV, 10 Stage I-II), and healthy controls (n = 10) by transcriptomic data and cytometry. Participants with UTC had a significantly higher proportion of immature high-density neutrophils (HDN) and lower proportion of mature HDN in peripheral blood compared to DTC. The proportion of circulating PD-L1+ immature neutrophils were significantly increased in advanced cancer patients. Unsupervised analysis of transcriptomics data from circulating HDN revealed downregulation of innate immune response and T-cell receptor signaling pathway in cancer patients. Moreover, UTC patients revealed the upregulation of glycolytic process and glutamate receptor signaling pathway. Comparative analysis across tumor types and stages revealed the downregulation of various T-cell-related pathways, such as T-cell receptor signaling pathway and T-cell proliferation in advanced cancer patients. Moreover, the proportions of CD8+ and CD4+ T effector memory CD45RA+ (TEMRA) cells from peripheral blood were significantly decreased in UTC patients compared to DTC patients. Finally, we demonstrated that proportions of tumor-infiltrated neutrophils were increased and related with poor prognosis in advanced thyroid cancer using data from our RNA-seq and TCGA (The Cancer Genome Atlas) data. In conclusion, observed prevalence of circulating immature high-density neutrophils and their immunosuppressive features in undifferentiated thyroid cancers underscore the importance of understanding neutrophil dynamics in the context of tumor progression in thyroid cancer