Abstract
BackgroundRadioactive or stable isotopic labeling of metabolites is a strategy that is routinely used to map the cellular fate of a selected labeled metabolite after it is added to cell culture or to the circulation of an animal. However, a labeled metabolite can be enzymatically changed in cellular metabolism, complicating the use of this experimental strategy to understand how a labeled metabolite moves between organs. These methods are also technically demanding, expensive and potentially toxic. To allow quantification of the bulk movement of metabolites between organs, we have developed a novel application of stable isotope ratio mass spectrometry (IRMS).ResultsWe exploit natural differences in 13C/12C ratios of plant nutrients for a low-cost and non-toxic carbon labeling, allowing a measurement of bulk carbon transfer between organs in vivo. IRMS measurements were found to be sufficiently sensitive to measure organs from individual Drosophila melanogaster larvae, giving robust measurements down to 2.5 μg per sample. We apply the method to determine if carbon incorporated into a growing solid tumor is ultimately derived from food or host tissues.ConclusionMeasuring tumor growth in a D. melanogaster larvae tumor model reveals that these tumors derive a majority of carbon from host sources. We believe the low cost and non-toxic nature of this methodology gives it broad applicability to study carbon flows between organs also in other animals and for a range of other biological questions.
Highlights
Radioactive or stable isotopic labeling of metabolites is a strategy that is routinely used to map the cellular fate of a selected labeled metabolite after it is added to cell culture or to the circulation of an animal
We set out to determine if carbon incorporated by an expanding tumor is sourced from ingested food or existing host tissues using a well-established Drosophila melanogaster malignant tumor model driven by clonal expression of oncogenic RasV12 and loss of the tumor suppressor scribble [3, 4]
Labeled metabolites are expensive and only allow relative measurements between samples for one metabolite per experiment, not absolute measurements of the mass transfer of carbon. To allow this type of measurement, we developed an experimental methodology that we have named CArbon Transfer measured by Stable Isotope Ratios (CATSIR), which exploits differences in the abundance of 13C/12C of biomolecules in edible plants to allow low-cost and non-toxic tracking of the carbon in metabolites
Summary
Radioactive or stable isotopic labeling of metabolites is a strategy that is routinely used to map the cellular fate of a selected labeled metabolite after it is added to cell culture or to the circulation of an animal. Existing methods like radioactive 14C-tracing or 13C detection through mass spectrometry could allow us to follow a selected metabolite by adding it to an experimental system and looking for the label in the tumor This methodology has been essential to establish the fundamentals of tumor metabolism, by infusing labeled metabolites into cell culture media or the blood of an animal [5]. Labeled lactate has recently been described to be integrated by tumor metabolism in vivo [6], possibly secreted by poorly oxygenated regions of a tumor and oxidized in well-oxygenated regions [7, 8] Existing applications of these methods allow a measurement of tumor uptake of a labeled metabolite, but there is no demonstrated application of this methodology to study the transfer of metabolites from other host organs to a tumor in a living animal. Labeled metabolites are expensive and only allow relative measurements between samples for one metabolite per experiment, not absolute measurements of the mass transfer of carbon
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