Abstract Breast cancer metastasis occurs via blood and lymphatic vessels. Breast cancer cells ‘educate’ lymphatic endothelial cells (LECs) to support tumor vascularization and growth. However, despite known metabolic alterations in breast cancer, it remains unclear how lymphatic endothelial cell metabolism is altered in the tumor microenvironment and its effect in lymphangiogenic signaling in LECs. We analyzed metabolites inside LECs in co-culture with MCF-7, MDA-MB-231, and SK-BR-3 breast cancer cell lines using $$^1\hbox {H}$$ 1 H nuclear magnetic resonance (NMR) metabolomics, Seahorse, and the spatial distribution of metabolic co-enzymes using optical redox ratio imaging to describe breast cancer-LEC metabolic crosstalk. LECs co-cultured with breast cancer cells exhibited cell-line dependent altered metabolic profiles, including significant changes in lactate concentration in breast cancer co-culture. Cell metabolic phenotype analysis using Seahorse showed LECs in co-culture exhibited reduced mitochondrial respiration, increased reliance on glycolysis and reduced metabolic flexibility. Optical redox ratio measurements revealed reduced NAD(P)H levels in LECs potentially due to increased NAD(P)H utilization to maintain redox homeostasis. $$^{13}\hbox {C}$$ 13 C -labeled glucose experiments did not reveal lactate shuttling into LECs from breast cancer cells, yet showed other $$^{13}\hbox {C}$$ 13 C signals in LECs suggesting internalized metabolites and metabolic exchange between the two cell types. We also determined that breast cancer co-culture stimulated lymphangiogenic signaling in LECs, yet activation was not stimulated by lactate alone. Increased lymphangiogenic signaling suggests paracrine signaling between LECs and breast cancer cells which could have a pro-metastatic role.
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Induced pluripotent stem cells can utilize lactate as a metabolic substrate to support proliferation
Human‐induced pluripotent stem cells (iPSCs) hold the promise to improve cell‐based therapies. Yet, to meet rising demands and become clinically impactful, sufficient high‐quality iPSCs in quantity must be generated, a task that exceeds current capabilities. In this study, K3 iPSCs cultures were examined using parallel‐labeling metabolic flux analysis (13C‐MFA) to quantify intracellular fluxes at relevant bioprocessing stages: glucose concentrations representative of initial media concentrations and high lactate concentrations representative of fed‐batch culture conditions, prior to and after bolus glucose feeds. The glucose and lactate concentrations are also representative of concentrations that might be encountered at different locations within 3D cell aggregates. Furthermore, a novel method was developed to allow the isotopic tracer [U‐13C3] lactate to be used in the13C‐MFA model. The results indicated that high extracellular lactate concentrations decreased glucose consumption and lactate production, while glucose concentrations alone did not affect rates of aerobic glycolysis. Moreover, for the high lactate cultures, lactate was used as a metabolic substrate to support oxidative mitochondrial metabolism. These results demonstrate that iPSCs have metabolic flexibility and possess the capacity to metabolize lactate to support exponential growth, and that high lactate concentrations alone do not adversely impact iPSC proliferation.
more » « less- NSF-PAR ID:
- 10452045
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Biotechnology Progress
- Volume:
- 37
- Issue:
- 2
- ISSN:
- 8756-7938
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract Chinese hamster ovary (CHO) cells, predominant hosts for recombinant biotherapeutics production, generate lactate as a major glycolysis by‐product. High lactate levels adversely impact cell growth and productivity. The goal of this study was to reduce lactate in CHO cell cultures by adding chemical inhibitors to hexokinase‐2 (HK2), the enzyme catalyzing the conversion of glucose to glucose 6‐phosphate, and examine their impact on lactate accumulation, cell growth, protein titers, and
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Purpose To determine whether deuterated water (HDO) generated from the metabolism of [2H7]glucose is a sensitive biomarker of cerebral glycolysis and oxidative flux.
Methods A bolus of [2H7]glucose was injected through the tail vein at 1.95 g/kg into Sprague‐Dawley rats. A2H surface coil was placed on top of the head to record2H spectra of the brain every 1.3 minutes to measure glucose uptake and metabolism to HDO, lactate, and glutamate/glutamine. A two‐point Dixon method based on a gradient‐echo sequence was used to reconstruct deuterated glucose and water (HDO) images selectively.
Results The background HDO signal could be detected and imaged before glucose injection. The2H NMR spectra showed arrival of [2H7]glucose and its metabolism in a time‐dependent manner. A ratio of the HDO to glutamate/glutamine resonances demonstrates a pseudo–steady state following injection, in which cerebral metabolism dominates wash‐in of HDO generated by peripheral metabolism. Brain spectroscopy reveals that HDO generation is linear with lactate and glutamate/glutamine appearance in the appropriate pseudo–steady state window. Selective imaging of HDO and glucose is easily accomplished using a gradient‐echo method.
Conclusion Metabolic imaging of HDO, as a marker of glucose, lactate, and glutamate/glutamine metabolism, has been shown here for the first time. Cerebral glucose metabolism can be assessed efficiently using a standard gradient‐echo sequence that provides superior in‐plane resolution compared with CSI‐based techniques.
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Abstract Deuterated water (2H2O) is a widely used tracer of carbohydrate biosynthesis in both preclinical and clinical settings, but the significant kinetic isotope effects (KIE) of2H can distort metabolic information and mediate toxicity.18O‐water (H218O) has no significant KIE and is incorporated into specific carbohydrate oxygens via well‐defined mechanisms, but to date it has not been evaluated in any animal model. Mice were given H218O during overnight feeding and18O‐enrichments of liver glycogen, triglyceride glycerol (TG), and blood glucose were quantified by13C NMR and mass spectrometry (MS). Enrichment of oxygens 5 and 6 relative to body water informed indirect pathway contributions from the Krebs cycle and triose phosphate sources. Compared with mice fed normal chow (NC), mice whose NC was supplemented with a fructose/glucose mix (i.e., a high sugar [HS] diet) had significantly higher indirect pathway contributions from triose phosphate sources, consistent with fructose glycogenesis. Blood glucose and liver TG18O‐enrichments were quantified by MS. Blood glucose18O‐enrichment was significantly higher for HS versus NC mice and was consistent with gluconeogenic fructose metabolism. TG18O‐enrichment was extensive for both NC and HS mice, indicating a high turnover of liver triglyceride, independent of diet. Thus H218O informs hepatic carbohydrate biosynthesis in similar detail to2H2O but without KIE‐associated risks.
