Host autophagy mediates organ wasting and nutrient mobilization for tumor growth
2021; Springer Nature; Volume: 40; Issue: 18 Linguagem: Inglês
10.15252/embj.2020107336
ISSN1460-2075
AutoresRojyar Khezri, Petter Holland, Todd A. Schoborg, Ifat Abramovich, Szabolcs Takáts, Caroline Dillard, Ashish Jain, Fergal O’Farrell, Sebastian W. Schultz, William M. Hagopian, Eduardo Martín Quintana, Rachel Ng, Nadja Sandra Katheder, Mohammed Mahidur Rahman, José Teles-Reis, Andreas Brech, Heinrich Jasper, Nasser M. Rusan, A. Hope Jahren, Eyal Gottlieb, Tor Erik Rusten,
Tópico(s)Mesenchymal stem cell research
ResumoArticle26 July 2021Open Access Source DataTransparent process Host autophagy mediates organ wasting and nutrient mobilization for tumor growth Rojyar Khezri Rojyar Khezri Centre for Cancer Cell Reprogramming, Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway Department of Molecular Cell Biology, Institute for Cancer Research, Oslo University Hospital, Oslo, Norway Search for more papers by this author Petter Holland Petter Holland Centre for Cancer Cell Reprogramming, Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway Department of Molecular Cell Biology, Institute for Cancer Research, Oslo University Hospital, Oslo, Norway Search for more papers by this author Todd Andrew Schoborg Todd Andrew Schoborg Cell Biology and Physiology Center, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD, USA Search for more papers by this author Ifat Abramovich Ifat Abramovich The Ruth and Bruce Rappaport Faculty of Medicine, Technion - Israel Institute of Technology, Bat Galim, Haifa, Israel Search for more papers by this author Szabolcs Takáts Szabolcs Takáts Centre for Cancer Cell Reprogramming, Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway Department of Molecular Cell Biology, Institute for Cancer Research, Oslo University Hospital, Oslo, Norway Search for more papers by this author Caroline Dillard Caroline Dillard Centre for Cancer Cell Reprogramming, Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway Department of Molecular Cell Biology, Institute for Cancer Research, Oslo University Hospital, Oslo, Norway Search for more papers by this author Ashish Jain Ashish Jain orcid.org/0000-0001-6549-2788 Centre for Cancer Cell Reprogramming, Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway Department of Molecular Cell Biology, Institute for Cancer Research, Oslo University Hospital, Oslo, Norway Search for more papers by this author Fergal O'Farrell Fergal O'Farrell Centre for Cancer Cell Reprogramming, Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway Department of Molecular Cell Biology, Institute for Cancer Research, Oslo University Hospital, Oslo, Norway Search for more papers by this author Sebastian Wolfgang Schultz Sebastian Wolfgang Schultz orcid.org/0000-0002-3661-2178 Centre for Cancer Cell Reprogramming, Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway Department of Molecular Cell Biology, Institute for Cancer Research, Oslo University Hospital, Oslo, Norway Search for more papers by this author William M Hagopian William M Hagopian Centre for Earth Evolution and Dynamics, University of Oslo, Oslo, Norway Search for more papers by this author Eduardo Martin Quintana Eduardo Martin Quintana Centre for Cancer Cell Reprogramming, Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway Department of Molecular Cell Biology, Institute for Cancer Research, Oslo University Hospital, Oslo, Norway Search for more papers by this author Rachel Ng Rachel Ng orcid.org/0000-0001-8969-9628 Cell Biology and Physiology Center, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD, USA Search for more papers by this author Nadja Sandra Katheder Nadja Sandra Katheder Department of Molecular Cell Biology, Institute for Cancer Research, Oslo University Hospital, Oslo, Norway Immunology Discovery, Genentech, Inc., South San Francisco, CA, USA Search for more papers by this author Mohammed Mahidur Rahman Mohammed Mahidur Rahman orcid.org/0000-0001-5327-4193 Centre for Cancer Cell Reprogramming, Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway Department of Molecular Cell Biology, Institute for Cancer Research, Oslo University Hospital, Oslo, Norway Search for more papers by this author José Gerardo Teles Reis José Gerardo Teles Reis Centre for Cancer Cell Reprogramming, Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway Department of Molecular Cell Biology, Institute for Cancer Research, Oslo University Hospital, Oslo, Norway Search for more papers by this author Andreas Brech Andreas Brech Centre for Cancer Cell Reprogramming, Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway Department of Molecular Cell Biology, Institute for Cancer Research, Oslo University Hospital, Oslo, Norway Search for more papers by this author Heinrich Jasper Heinrich Jasper Immunology Discovery, Genentech, Inc., South San Francisco, CA, USA Buck Institute for Research on Aging, Novato, CA, USA Search for more papers by this author Nasser M Rusan Nasser M Rusan Cell Biology and Physiology Center, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD, USA Search for more papers by this author Anne Hope Jahren Anne Hope Jahren Centre for Earth Evolution and Dynamics, University of Oslo, Oslo, Norway Search for more papers by this author Eyal Gottlieb Eyal Gottlieb orcid.org/0000-0002-9770-0956 The Ruth and Bruce Rappaport Faculty of Medicine, Technion - Israel Institute of Technology, Bat Galim, Haifa, Israel Search for more papers by this author Tor Erik Rusten Corresponding Author Tor Erik Rusten [email protected] orcid.org/0000-0002-9150-2676 Centre for Cancer Cell Reprogramming, Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway Department of Molecular Cell Biology, Institute for Cancer Research, Oslo University Hospital, Oslo, Norway Search for more papers by this author Rojyar Khezri Rojyar Khezri Centre for Cancer Cell Reprogramming, Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway Department of Molecular Cell Biology, Institute for Cancer Research, Oslo University Hospital, Oslo, Norway Search for more papers by this author Petter Holland Petter Holland Centre for Cancer Cell Reprogramming, Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway Department of Molecular Cell Biology, Institute for Cancer Research, Oslo University Hospital, Oslo, Norway Search for more papers by this author Todd Andrew Schoborg Todd Andrew Schoborg Cell Biology and Physiology Center, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD, USA Search for more papers by this author Ifat Abramovich Ifat Abramovich The Ruth and Bruce Rappaport Faculty of Medicine, Technion - Israel Institute of Technology, Bat Galim, Haifa, Israel Search for more papers by this author Szabolcs Takáts Szabolcs Takáts Centre for Cancer Cell Reprogramming, Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway Department of Molecular Cell Biology, Institute for Cancer Research, Oslo University Hospital, Oslo, Norway Search for more papers by this author Caroline Dillard Caroline Dillard Centre for Cancer Cell Reprogramming, Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway Department of Molecular Cell Biology, Institute for Cancer Research, Oslo University Hospital, Oslo, Norway Search for more papers by this author Ashish Jain Ashish Jain orcid.org/0000-0001-6549-2788 Centre for Cancer Cell Reprogramming, Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway Department of Molecular Cell Biology, Institute for Cancer Research, Oslo University Hospital, Oslo, Norway Search for more papers by this author Fergal O'Farrell Fergal O'Farrell Centre for Cancer Cell Reprogramming, Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway Department of Molecular Cell Biology, Institute for Cancer Research, Oslo University Hospital, Oslo, Norway Search for more papers by this author Sebastian Wolfgang Schultz Sebastian Wolfgang Schultz orcid.org/0000-0002-3661-2178 Centre for Cancer Cell Reprogramming, Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway Department of Molecular Cell Biology, Institute for Cancer Research, Oslo University Hospital, Oslo, Norway Search for more papers by this author William M Hagopian William M Hagopian Centre for Earth Evolution and Dynamics, University of Oslo, Oslo, Norway Search for more papers by this author Eduardo Martin Quintana Eduardo Martin Quintana Centre for Cancer Cell Reprogramming, Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway Department of Molecular Cell Biology, Institute for Cancer Research, Oslo University Hospital, Oslo, Norway Search for more papers by this author Rachel Ng Rachel Ng orcid.org/0000-0001-8969-9628 Cell Biology and Physiology Center, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD, USA Search for more papers by this author Nadja Sandra Katheder Nadja Sandra Katheder Department of Molecular Cell Biology, Institute for Cancer Research, Oslo University Hospital, Oslo, Norway Immunology Discovery, Genentech, Inc., South San Francisco, CA, USA Search for more papers by this author Mohammed Mahidur Rahman Mohammed Mahidur Rahman orcid.org/0000-0001-5327-4193 Centre for Cancer Cell Reprogramming, Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway Department of Molecular Cell Biology, Institute for Cancer Research, Oslo University Hospital, Oslo, Norway Search for more papers by this author José Gerardo Teles Reis José Gerardo Teles Reis Centre for Cancer Cell Reprogramming, Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway Department of Molecular Cell Biology, Institute for Cancer Research, Oslo University Hospital, Oslo, Norway Search for more papers by this author Andreas Brech Andreas Brech Centre for Cancer Cell Reprogramming, Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway Department of Molecular Cell Biology, Institute for Cancer Research, Oslo University Hospital, Oslo, Norway Search for more papers by this author Heinrich Jasper Heinrich Jasper Immunology Discovery, Genentech, Inc., South San Francisco, CA, USA Buck Institute for Research on Aging, Novato, CA, USA Search for more papers by this author Nasser M Rusan Nasser M Rusan Cell Biology and Physiology Center, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD, USA Search for more papers by this author Anne Hope Jahren Anne Hope Jahren Centre for Earth Evolution and Dynamics, University of Oslo, Oslo, Norway Search for more papers by this author Eyal Gottlieb Eyal Gottlieb orcid.org/0000-0002-9770-0956 The Ruth and Bruce Rappaport Faculty of Medicine, Technion - Israel Institute of Technology, Bat Galim, Haifa, Israel Search for more papers by this author Tor Erik Rusten Corresponding Author Tor Erik Rusten [email protected] orcid.org/0000-0002-9150-2676 Centre for Cancer Cell Reprogramming, Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway Department of Molecular Cell Biology, Institute for Cancer Research, Oslo University Hospital, Oslo, Norway Search for more papers by this author Author Information Rojyar Khezri1,2, Petter Holland1,2,†, Todd Andrew Schoborg3,†, Ifat Abramovich4, Szabolcs Takáts1,2, Caroline Dillard1,2, Ashish Jain1,2, Fergal O'Farrell1,2, Sebastian Wolfgang Schultz1,2, William M Hagopian5, Eduardo Martin Quintana1,2, Rachel Ng3, Nadja Sandra Katheder2,6, Mohammed Mahidur Rahman1,2, José Gerardo Teles Reis1,2, Andreas Brech1,2, Heinrich Jasper6,7, Nasser M Rusan3, Anne Hope Jahren5, Eyal Gottlieb4 and Tor Erik Rusten *,1,2 1Centre for Cancer Cell Reprogramming, Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway 2Department of Molecular Cell Biology, Institute for Cancer Research, Oslo University Hospital, Oslo, Norway 3Cell Biology and Physiology Center, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD, USA 4The Ruth and Bruce Rappaport Faculty of Medicine, Technion - Israel Institute of Technology, Bat Galim, Haifa, Israel 5Centre for Earth Evolution and Dynamics, University of Oslo, Oslo, Norway 6Immunology Discovery, Genentech, Inc., South San Francisco, CA, USA 7Buck Institute for Research on Aging, Novato, CA, USA † These authors contributed equally to this work *Corresponding author. E-mail: [email protected] The EMBO Journal (2021)40:e107336https://doi.org/10.15252/embj.2020107336 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract 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. Synopsis Autophagy maintains mitochondrial health and nutrient recycling in tumor cells, and promotes the transfer of amino acids from microenvironmental to tumor cells, thereby sustaining tumor metabolism and growth. In this study, X-ray tomography, metabolomics and carbon tracing reveal that autophagy-mediated wasting of distal tissues provides amino acids and sugars that increase eye tumor biomass in Drosophila melanogaster. RasV12, scrib−/− tumors induce organ wasting and cause release of amino acids and sugar into circulation. Systemic autophagy mediates muscle wasting and nutrient release. Tumor biomass increasingly derive from host tissues as wasting ensues. Introduction Macroautophagy (referred to as autophagy herein) encapsulates cytoplasm in a double membrane vesicle that is subsequently degraded upon fusion with the lysosome. Through autophagy, cytoplasmic cargo, including glycogen, protein aggregates, and organelles such as mitochondria, are broken down and reused for energy production or as macromolecular building blocks (Galluzzi et al, 2015). Physiologically, autophagy is necessary to survive starvation in both unicellular organisms and animal models. Genetic studies have revealed that autophagy is required to sustain circulating amino acid levels in autophagy-deficient newborn mice (Kuma et al, 2004) and glucose during fasting in adult mice (Karsli-Uzunbas et al, 2014). As autophagy is essential for cellular metabolism and control systemic nutrient levels, a focused effort has been on understanding the relevance for autophagy during carcinogenesis (Kimmelman & White, 2017; Poillet-Perez & White, 2019). Indeed, during tumor growth, when nutrient and anabolic demands are high, findings in several genetically engineered mouse models have uncovered a tumor-supportive role of autophagy (Poillet-Perez & White, 2019). Autophagy deficiency in KrasG12D or BrafV600E lung cancer, or BrafV600E;PTEN−/− or KrasG12D, pancreatic cancer (PDAC) models decreased tumor progression (Guo et al, 2013; Rosenfeldt et al, 2013; Strohecker et al, 2013; Karsli-Uzunbas et al, 2014; Yang et al, 2014; Xie et al, 2015). Metabolic analyses in RAS-driven tumor cells and a KrasV12D; LKB1−/− lung cancer model showed that autophagy is required cell-autonomously to provide substrates to the TCA cycle for maintaining nucleotide pools and prevent energy crisis (Bhatt et al, 2019). Thus, autophagy is required to sustain metabolic functions in tumor cells in order to promote tumor progression. Autophagy is not however required to support tumor growth only from within the tumor cell itself. In flies and mice, Ras-driven tumors additionally invoke non-autonomous autophagy in cells of the microenvironment to support tumor growth, in part through transfer of amino acids from neighboring cells (Sousa et al, 2016; Katheder et al, 2017; Yang et al, 2018). In Drosophila, RasV12, scrib−/− tumors also induced a systemic autophagy stress response in muscle and adipose tissue, an observation akin to systemic effects in late-stage cancer patients suffering from cancer cachexia metabolic syndrome (Katheder et al, 2017). Cancer cachexia is a tumor-induced systemic cascade of events that leads to systemic inflammation, metabolic reprogramming, and organ degeneration, particularly of fat and skeletal and heart muscle whereas the liver increases in size (Baracos et al, 2018). Muscle samples from cancer patients suffering from cachexia (Tardif et al, 2013; Aversa et al, 2016) and cancer cachexia Xenograft models in mice (Penna et al, 2013) show increased levels of molecular markers of autophagy. This has led to the idea that organ wasting may be in part executed by increased turnover of intracellular material by simultaneous elevated proteasomal and autophagic activity (Penna et al, 2014). Genetic experiments for whether autophagy is rate-limiting for organ wasting and how this may affect tumor growth in vivo remain limited. In a recent study, knockdown of the endocytosis and autophagy regulator BECN1 moderately reduced muscle wasting but not muscle morphological changes (Penna et al, 2019). Here we uncover that autophagy mediates systemic organ wasting and nutrient mobilization for tumor growth in a malignant tumor model in Drosophila melanogaster. Results Gradual organ atrophy and weight loss ensues during malignant tumor growth To assess the dynamics of organ atrophy at the whole-animal level, we adopted computed tomography (CT), the gold standard for evaluating adipose and muscle atrophy in cancer patients. Genetically induced GFP-labeled malignant RasV12; scrib−/− eye tumors grow and invade the neighboring central nervous system (CNS), extend the larval stage and kill the host by day 10–12 (Brumby & Richardson, 2003; Pagliarini & Xu, 2003). We optimized a fixation and staining protocol for high-resolution micro-CT (μ-CT) imaging of developmentally staged larvae (Schoborg et al, 2019). This enabled ready identification, segmentation, and calculation of tumor and organ volumes (Fig 1A–C; Movie EV1–7) (Schoborg et al, 2019) RasV12; scrib−/− tumors grow 10-fold in volume while invading and enveloping the brain from day 6 to 10 (Fig 1C, Movie EV7, quantified in 2A). Conversely, total larval muscle volume is initially similar to control animals carrying benign RasV12 tumors, and progressively shrink by approximately 50% from day 6 to day 10 (Fig 1B, quantified in 2B). The fat body, which perform adipose and liver functions, displayed a striking increase in translucency and lipid droplet size (Figs 2E and 3C) in Ras; scrib−/− tumor-carrying animals from day 8 (Figueroa-Clarevega & Bilder, 2015), although fat body volume remained unaltered (Fig 2D). Organ wasting and tumor growth were accompanied by approximately 35% dry weight loss and a progressive loss of motility and feeding from day 8 (Fig 2F–H). We established that muscle and fat body changes can be imaged and quantified in intact heat-killed whole larvae using a myosin heavy chain-GFP muscle reporter and backlight microscopy (Figs 3A–C and EV1A–C). RasV12; scrib−/− tumors inhibit ecdysone synthesis and offset the normal pupation at day 6 due to dilp8 secretion from tumor tissue (Colombani et al, 2012; Garelli et al, 2012). No muscle or adipose tissue atrophy was observed in day 10 animals where secretion of the molting hormone ecdysone was specifically obliterated in ecdysoneless (ecd1−/−) mutants, or by genetic elimination of the ecdysone-producing prothoracic gland cells (Fig EV1-EV4). Thus, the onset of muscle and adipose tissue wasting RasV12, scrib−/− larvae precede the reduction of feeding and reduced motility, arguing together that the wasting responses are not a simple function of reduced food intake or extended larval stage. Figure 1. Tumor-induced organ wasting A. Representative 2D μ-CT scans of RasV12; ctrl at day 6, RasV12; scrib−/− larvae at day 6 and day 10. Muscle (green), fat body (blue) and eye-antennal discs/tumor (red) are outlined. Scale bar: 1 mm. Anterior (A), Right (R). B. Representative 3D rendering of genotypes indicated in (A). Anterior (A), Dorsal (D), Left (L). C. Representative 3D rendering of RasV12; scrib−/− tumors (red) and central nervous system (yellow), over time. Download figure Download PowerPoint Figure 2. Tumor-induced organ wasting A. Quantification of tumor growth of RasV12; ctrl day 6 (n = 15) RasV12; scrib−/− tumors, day 6 (n = 15), day 7 (n = 5), day 8 (n = 4), day 9 (n = 5), and day 10 (n = 5). B. Quantifications of muscle volume, of larvae carrying tumors of RasV12; ctrl (n = 15), and RasV12; scrib−/− at day 6(n = 15), day 7 (n = 5), day 8 (n = 5), day 9 (n = 5), and dat 10 (n = 5). C. Quantification of width of Dorsal Oblique 3 (DO3) muscle in larvae carrying tumors of RasV12; ctrl at day 6 (n = 29) and RasV12; scrib−/−at days 6 (n = 29) and day 10 (n = 31). D. Quantification of adipose tissue volume in larvae carrying tumors of RasV12; ctrl at day 6 and RasV12; scrib−/− at days 6 and day 10. E. Representative confocal images of adipose tissue in RasV12; ctrl and RasV12; scrib−/− tumors bearing animals at indicated ages. Lipid droplets are highlighted with Lipid Tox staining. Scale bar: 50 μm. F. Quantification of dry weight of RasV12; ctrl (n = 9), RasV12; scrib−/− day 6 (n = 15) and day 10 (n = 18) tumor-bearing larvae excluding the tumor weight. G. Quantification of larval motility measure by crawling distance and crawling pattern in RasV12; ctrl (n = 14), RasV12; scrib−/− day 6 (n = 15), day 8 (n = 16), and day 10 (n = 14), each colored line represents a single larva. H. Coomassie feeding assay to asses food intake of RasV12; scrib−/− over time, three repeated measurements of an average food intake of 20 larvae. Data information: Values depict mean ± s.e.m. of minimum three independent pooled experiments. ns, not significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.0001 and ****P < 0.0001, from unpaired, two-tailed test. Source data are available online for this figure. Source Data for Figure 2 [embj2020107336-sup-0011-SDataFig2.xlsx] Download figure Download PowerPoint Figure 3. Autophagy is required for systemic wasting A–E. Cartoon (top) illustrates the genotypes of the larvae, eye-antennal disc (EAD, circle: tumor cells in green and microenvironment cell in black), systemic cells are illustrated as a square (wild-type cells in light brown and atg13-mutant cells in light orange) at indicated ages. Cartoon (left) illustrates the structure of cephalic complex attached to mouth hook. Representative images of Larva (image of larva using the backlight of microscope), cephalic complex (green highlights the GFP-labeled tumor clones), muscle (Phalloidin in green stains actin and Hoechst in blue stains nucleus), and adipose tissue (Lipid Tox in red stains for lipid droplets, Hoechst stains nucleus) from top to bottom. (A) RasV12; ctrl tumor-bearing animal at day 6. (B) RasV12; scrib−/− tumor-bearing animal at day 6. (C) RasV12; scrib−/− tumor-bearing animal at day 8. (D) RasV12, scrib−/−, atg13−/−//atg13−/− at day 8. (E) ey3.5-atg13; RasV12; scrib−/−, atg13−/−//atg13−/− animal complemented with eye-specific transgenic atg13 expression, rescuing the tumor growth, at day 8. F. Quantification of area of space occupied with lobes of fat body within larval segments 4 to 8 (shown in yellow dashed line in 2A), of animals bearing tumors at day 6 RasV12; ctrl (n = 45), RasV12; scrib−/− (n = 30), RasV12, scrib−/−, atg13−/−//atg13−/− (n = 30) and ey3.5-atg13; RasV12, scrib−/−, atg13−/−//atg13−/− (n = 29) and at day 10 RasV12; scrib−/− (n = 40), RasV12, scrib−/−, atg13−/−//atg13−/− (n = 25) and ey3.5-atg13; RasV12, scrib−/−, atg13−/−//atg13−/− (n = 28). G. Quantification of tumor volumes day 6 RasV12; ctrl (n = 7), RasV12; scrib−/− (n = 11) and day 8 RasV12; scrib−/− (n = 13), RasV12, scrib−/−, atg13−/−//atg13−/− (n = 9) and ey3.5-atg13; RasV12, scrib−/−, atg13−/−//atg13−/− (n = 9). H. Quantification of Ventral Longitudinal muscle 4 (VL4) (shown in yellow dashed line in 2A) of third segment of larvae carrying at day 6 RasV12; ctrl (n = 15), RasV12; scrib−/− (n = 14), and at day8 RasV12; scrib−/− (n = 11), RasV12, scrib−/−, atg13−/−//atg13−/− (n = 12) and ey3.5-atg13; RasV12; scrib−/−, atg13−/−//atg13−/− (n = 14). I. Quantification of dry weight excluding tumor of RasV12; scrib−/−, atg13−/−//atg13−/−at day 6 (n = 8) and day 10 (n = 7). J. Quantification of larval motility measured by crawling distance and crawling pattern for RasV12; scrib−/−, atg13−/−//atg13−/− at day 6 (n = 15) and at day 8 (n = 15), each colored line represents a single larva. Data information: Values depict mean±s.e.m. of minimum three independent pooled experiments. ns, not significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.0001 and ****P < 0.0001, from unpaired, two-tailed test. Scale bar: muscles 100 μm and adipose tissue 50 μm. Source data are available online for this figure. Source Data for Figure 3 [embj2020107336-sup-0012-SDataFig3.xlsx] Download figure Download PowerPoint Figure 4. Autophagy-driven wasting releases metabolites into circulation A. Changes in groups (carbohydrates, amino acids, and fatty acids) of storage metabolites measured in circulating hemolymph with progressing wasting, shown as log2 changes measured by LC-MS and calculated per larvae relative to RasV12; ctrl at day 6. B. Volcano plot showing autophagy-dependent changes to amounts of circulating metabolites at day 6. X-axis shows the log2 of fold change of RasV12; atg13 −/−scrib−/− // atg13 −/− vs. RasV12; scrib−/−, y-axis shows −log10 P-value, calculated by t-test. Metabolite names are shown for metabolites with log2 (FC) >± 1 and/or –log10(P) < 2. Green points indicate log2 (FC) >± 1, blue indicates −log10(P) < 2, and red indicates for above both thresholds. C. Autophagy-driven wasting releases metabolites into circulation of 113 reliably detected metabolites, those showing significant differences in any of the three comparisons are shown. Color indicates the log2 (fold change) difference and the numbers show the P-value of the comparison. The statistical test to define significance was FDR-adjusted t-test P-value < 0.05. Download figure Download PowerPoint Figure 5. Host-derived nutrients contribute to tumor biomass A. The amount of glycogen in the whole larvae measured by biochemical assay (n = 4), normalized to RasV12; ctrl at day 6, and per number of larvae. B–E. Representative confocal images of muscle and adipose tissue of larvae carrying RasV12; ctrl, RasV12; scrib−/− and RasV12; scrib−/−, atg13−/−//atg13−/− showing glycogen levels (white) at day 6 and day 8. F. Cartoon illustrating tumor growth (green) incorporation of molecules derived from food (in orange) or from host tissues (in blue). G. 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). Data information: Values depict mean ± s.e.m. of minimum three independent pooled experiments. ns, not significant, **P ≤ 0.01, from unpaired, two-tailed test. Scale bar: muscles 100 μm and adipose tissue 50 μm. Source data are available online for this figure. Source Data for Figure 5 [embj2020107336-sup-0013-SDataFig5.xlsx] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Tumor-induced organ wasting A–C. Representative images of larvae with GFP-labeled muscles at day 6 and 10. D–F. Cartoon (top) illustrates the genotypes of the larvae, prothoracic gland (gray oval), systemic cells are illustrated as a square (wild-type cells in light brown and ecdysone mutant cells in light red) at day 6 and day 10. Cartoon (left) illustrates the structure of cephalic complex attached to mouth hook. Larva (image of larva using the backlight of microscope), cephalic complex (no tumors), muscle (Phalloidin in green stains actin and Hoechst in blue stains muscle nuclei), and adipose tissue (Lipid Tox in red stains for lipid droplets, Hoechst stains cell nuclei) from top to bottom. (D) Wild type larva (w1118) control, (E) spok-Gal4,UAS-Dcr2.D;UAS-rpr larva that linger due to lack of cells expressing ecdysone hormone and (F) ecd1ts (ecdysoneless), larva that lingers due to ecdysone deficiency. Scale bar: muscle, 100 μm and adipose tissue, 50 μm. Download figure Download PowerPoint
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