Host autophagy mediates organ wasting and nutrient mobilization for tumor growth.

During tumor growth-when nutrient and anabolic demands are high-autophagy supports tumor metabolism and growth through lysosomal organelle turnover and nutrient recycling. Ras-driven tumors additionally invoke non-autonomous autophagy in the microenvironment to support tumor growth, in part through transfer of amino acids. Here we uncover a third critical role of autophagy in mediating systemic organ wasting and nutrient mobilization for tumor growth using a well-characterized malignant tumor model in Drosophila melanogaster. Micro-computed X-ray tomography and metabolic profiling reveal that RasV12 ; scrib-/- tumors grow 10-fold in volume, while systemic organ wasting unfolds with progressive muscle atrophy, loss of body mass, -motility, -feeding, and eventually death. Tissue wasting is found to be mediated by autophagy and results in host mobilization of amino acids and sugars into circulation. Natural abundance Carbon 13 tracing demonstrates that tumor biomass is increasingly derived from host tissues as a nutrient source as wasting progresses. We conclude that host autophagy mediates organ wasting and nutrient mobilization that is utilized for tumor growth.

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

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.

Plant growth chamber design for subambient pCO2 and δ13 C studies.

RATIONALE: Subambient pCO2 has persisted across the major Phanerozoic ice ages, including the entire late Cenozoic (ca 30 Ma to present). Stable isotope analysis of plant-derived organic matter is used to infer changes in pCO2 and climate in the geologic past, but a growth chamber that can precisely control environmental conditions, including pCO2 and δ13 C value of CO2 (δ13 CCO2 ) at subambient pCO2 , is lacking. METHODS: We designed and built five identical chambers specifically for plant growth under stable subambient pCO2 (ca 100 to 400 ppm) and δ13 CCO2 conditions. We tested the pCO2 and δ13 CCO2 stability of the chambers both with and without plants, across two 12-hour daytime experiments and two extended 9-day experiments. We also compared the temperature and relative humidity conditions among the chambers. RESULTS: The average δ13 CCO2 value within the five chambers ranged from -18.76 to -19.10‰; the standard deviation never exceeded 0.14‰ across any experiment. This represents better δ13 CCO2 stability than that achieved by all previous chamber designs, including superambient pCO2 chambers. Every pCO2 measurement (n = 1225) was within 5% of mean chamber values. The temperature and relative humidity conditions differed by no more than 0.4°C and 1.6%, respectively, across all chambers within each growth experiment. CONCLUSIONS: This growth chamber design extends the range of pCO2 conditions for which plants can be grown for δ13 C analysis of their tissues at subambient levels. This new capability allows for careful isolation of environmental effects on plant 13 C discrimination across the entire range of pCO2 experienced by terrestrial land plants.

Carbon and nitrogen stable isotopes in U.S. milk: Insight into production process.

RATIONALE: Stable isotope analysis (SIA), a potential method of verifying the geographic origin and production method of dairy products, has not been applied to United States (U.S.) dairy samples on a national scale. To determine the potential of carbon and nitrogen SIA in authenticity assessment of U.S. dairy products, we analyzed a geographically representative collection of conventional milk samples to determine isotopic variations with (1) Purchase Location and (2) Macronutrient Content. METHODS: A total of 136 milk samples spanning five commercially available varieties (3.25% [i.e., 'whole'], 2%, 1%, 0% [i.e., 'skim'] and 1% chocolate) were collected from randomly selected counties across the U.S. as part of the United States Department of Agriculture's (USDA's) National Food and Nutrient Analysis program. δ13 C and δ15 N values of bulk samples determined via elemental analysis/isotope ratio mass spectrometry (EA/IRMS) were used to assess the contribution of fat content, added sugar content and census-designated region of collection to isotopic variations within the dataset. RESULTS: There was a negative linear relationship between fat content and δ13 C values, with average milk δ13 C values decreasing by 0.33‰ for each 8.75% increase in dry weight (1% wet weight) fat content. The average δ13 C value of flavored 1% chocolate milk samples, which contain an additional 12 g of added sugar, was 2.05‰ higher than that of 1% unflavored milk (-16.47‰ for chocolate milk vs -18.52‰ for unflavored milk). When controlling for macronutrient content, milk samples collected in West region supermarkets possessed significantly lower δ13 C values than samples collected from Midwest, South, and Northeast regions. δ15 N values did not vary with macronutrient content or region of collection. CONCLUSIONS: Carbon stable isotope ratios in U.S. milk samples varied with macronutrient content and region of purchase, suggesting that SIA can provide insight into production processes within the U.S. dairy industry, with potential applications in national food adulteration and authentication efforts.

Large-scale plant growth chamber design for elevated pCO2 and δ13C studies.

RATIONALE: Throughout at least the next century, CO(2) fertilization and environmental stresses (e.g. nutrient, moisture, insect herbivory) are predicted to affect yields of economically important crop species. Stable isotopes of carbon are used to study plant stresses, which affect yields, but a growth chamber design that can be used to isolate the effects of environmental stresses on crop-sized species through precise maintenance of pCO(2) levels and the δ(13)C values of atmospheric CO(2) (δ(13) C(CO2)) is lacking. METHODS: We designed and built low-cost plant growth chambers for growing staple crop species under precise pCO(2) and δ(13) C(CO2) conditions. Over the course of 14 hours, we assessed for pCO(2) stability at two targeted levels (ambient, ~400 ppm; and 2×, ~800 ppm) and measured the δ(13) C(CO2) value within the two chambers using a stable isotope ratio mass spectrometer. We also compared the temperature and relative humidity conditions within the two growth chambers, and in the ambient, outside air. RESULTS: Across our experimental period, we achieved δ(13) C(CO2) stability (ambient: -8.05 ± 0.17‰; elevated: -12.99 ± 0.29‰) that showed nearly half the variability of any previously reported values for other chamber designs. The stability of the pCO(2) conditions (ambient: 406 ± 3 ppm; elevated: 793 ± 54 ppm) was comparable with that in previous studies, but our design provided ~8 times more growing space than previous chamber designs. We also measured nearly identical temperature and relative humidity conditions for the two chambers throughout the experiment. CONCLUSIONS: Our growth chamber design marks a significant improvement in our ability to test for plant stress across a range of future pCO(2) scenarios. Through significant improvement in δ(13) C(CO2) stability and increased chamber size, small changes in carbon isotope fractionation can be used to assess stress in crop species under specific environmental (temperature, relative humidity, pCO(2)) conditions.

Elimination of nitrogen interference during online oxygen isotope analysis of nitrogen-doped organics using the "NiCat" nickel reduction system.

RATIONALE: Accurate online analysis of the δ(18)O values of nitrogen-bearing organic compounds is of interest to several emergent fields, including ecology, forensics and paleontology. During online analysis, high-temperature conversion (HTC) of nitrogen-bearing organics produces N(2) gas which creates isobaric interference with the isotopic measurement. Specifically, N(2) reacts with trace amounts of oxygen in the mass spectrometer source to form (14)N(16)O (m/z 30), which prevents accurate evaluation of the sample (12)C(18)O peak (m/z 30). METHODS: We present an alternative system to the conventional HTC, which uses a nickel-catalyzed ("NiCat") reduction furnace to convert HTC-produced CO into CO(2), allowing for δ(18)O measurement using signal intensities at m/z 44 and 46. RESULTS: This system yields identical δ(18)O values for nitrogen-doped and undoped sucrose and cellulose compounds up to molar yield ratios of N(2):CO = 0.22. In contrast, our conventional HTC system configured to factory recommendations with the stock gas chromatography (GC) column produced a discrepancy of ~5‰ between nitrogen-doped and undoped samples. CONCLUSIONS: Because of its ability to eliminate isobaric interference, the NiCat system is a viable alternative to conventional HTC for δ(18)O measurement, and can be constructed from relatively inexpensive and readily available materials. As an additional advantage, the CO(2) analyte produced by NiCat may be cryofocused, to allow for oxygen-isotope determinations on very small amounts of sample substrate.

Offline oxygen isotope analysis of organic compounds with high N:O.

Although the advantages of online δ(18)O analysis of organic compounds make its broad application desirable, researchers have encountered NO(+) isobaric interference with CO(+) at m/z 30 (e.g. (14)N(16)O(+), (12)C(18)O(+)) when analyzing nitrogenous substrates. If the δ(18)O value of inter-laboratory standards for substrates with high N:O value could be confirmed offline, these materials could be analyzed periodically and used to evaluate δ(18)O data produced online for nitrogenous unknowns. To this end, we present an offline method based on modifications of the methods of Schimmelmann and Deniro (Anal. Chem. 1985; 57: 2644) and Sauer and Sternberg (Anal. Chem. 1994; 66: 2409), whereby all the N(2) from the gas products of a chlorinated pyrolysis was eliminated, resulting in purified CO(2) for analysis via a dual-inlet isotope ratio mass spectrometry system. We evaluated our method by comparing observed δ(18)O values with previously published or inter-laboratory calibrated δ(18)O values for five nitrogen-free working reference materials; finding isotopic agreement to within ±0.2‰ for SIGMA® cellulose, IAEA-CH3 cellulose (C(6)H(10)O(5)) and IAEA-CH6 sucrose (C(12)H(22)O(11)), and within ±1.8‰ for IAEA-601 and IAEA-602 benzoic acids (C(7)H(6)O(2)). We also compared the δ(18)O values of IAEA-CH3 cellulose and IAEA-CH6 sucrose that was nitrogen-'doped' with adenine (C(5)H(5)N(5)), imidazole (C(3)H(4)N(2)) and 2-aminopyrimidine (C(4)H(5)N(3)) with the undoped δ(18)O values for the same substrates; yielding isotopic agreement to within ±0.7‰. Finally, we provide an independent analysis of the δ(18)O value of IAEA-600 caffeine (C(8)H(10)N(4)O(2)), previously characterized using online systems exclusively, and discuss the reasons for an average 1.4‰ enrichment in δ(18)O observed offline relative to the consensus online δ(18)O value.

Minimization of sample requirement for delta18O in benzoic acid.

The measurement of the oxygen stable isotope content in organic compounds has applications in many fields, ranging from paleoclimate reconstruction to forensics. Conventional High-Temperature Conversion (HTC) techniques require >20 microg of O for a single delta(18)O measurement. Here we describe a system that converts the CO produced by HTC into CO(2) via reduction within a Ni-furnace. This CO(2) is then concentrated cryogenically, and 'focused' into the isotope ratio mass spectrometry (IRMS) source using a low-flow He carrier gas (6-8 mL/min). We report analyses of benzoic acid (C(7)H(6)O(2)) reference materials that yielded precise delta(18)O measurement down to 1.3 microg of O, suggesting that our system could be used to decrease sample requirement for delta(18)O by more than an order of magnitude.