Natural abundance isotope ratios to differentiate sources of carbon used during tumor growth in vivo

Abstract

Background 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. 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). Results We 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. Conclusion Measuring 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.

Host autophagy mediates organ wasting and nutrient mobilization for tumor growth

“… Sources of carbon incorporated into tumor biomass were differentiated by changing the isotopic carbon content of the food source 25 h before measuring the total carbon content and isotopic ratio of the tumor. A similar, but independent experiment is reported in Holland et al ( 2021 ). …”

“…To test this hypothesis, we sought to differentiate whether biomass of the growing tumor was derived from ingested food or nutrients released from host organ stores. As both sugars and amino acids were mobilized as a group, we chose to follow transfer of carbon using natural abundance stable isotope ratio mass spectrometry (SIRMS) using a recently developed protocol (Holland et al , 2021 ). We generated two types of fly food containing different 13 C/ 12 C ratios derived from plants with differences in 13 C vs. 12 C incorporation from CO 2 during photosynthesis.…”

“…We generated two types of fly food containing different 13 C/ 12 C ratios derived from plants with differences in 13 C vs. 12 C incorporation from CO 2 during photosynthesis. C4–type carbon fixation (e.g., corn, sugar cane) incorporates relatively more 13 C‐containing CO 2 than does C3‐type plants (e.g., potato, sugar beet) (Jahren et al , 2006 ; Chartrand & Mester, 2019 ; Holland et al , 2021 ). Larvae raised on C4‐based vs. C3‐based food revealed segregated 13 C/ 12 C tissue and hemolymph values in keeping with the isotopic composition of the food they were raised on (Fig EV4A ).…”

“…To determine how much carbon is derived from the food or host tissues after moving the larvae from one food to the other, δ 13 C measurement of any given day was compared to a set of measurements of larval cephalic complexes growing on only C3 or C4 food. This comparison is required because the δ 13 C of the cephalic complex with a growing tumor gradually changes as the tumor grows, as is described in more detail in the method publication (Holland et al , 2021 ). First, the relative ratio of carbon derived from food and host was determined by comparing the measured δ 13 C to the C3‐ and C4‐only samples.…”

RasV12; scrib−/− Tumors: A Cooperative Oncogenesis Model Fueled by Tumor/Host Interactions

“…A recently described stable carbon isotope tracing technique has allowed to distinguish whether tumor biomass is derived from food or host tissues. Using this technique, it was demonstrated that Ras V12 ; scrib −/− larval tumors first grow mainly on nutrients from food and progressively incorporates more nutrients from host tissues [115,140]. Autophagy is aberrantly activated in muscles and fat body of Ras V12 ; scrib −/− tumor-bearing larvae suggesting that wasted organs might provide nutrients to the tumor through "organ self-digestion" [54].…”