Malignant Drosophila tumors interrupt insulin signaling to induce cachexia-like wasting

SUMMARY

Tumors kill patients not only through well-characterized perturbations to their local environment, but also through poorly understood pathophysiological interactions with distant tissues. Here we use a Drosophila tumor model to investigate the elusive mechanisms underlying such long-range interactions. Transplantation of tumors into adults induces robust wasting of adipose, muscle and gonadal tissues that are distant from the tumor, phenotypes that resemble the cancer cachexia seen in human patients. Interestingly, malignant but not benign tumors induce peripheral wasting. We identify the Insulin Growth Factor Binding Protein (IGFBP) homolog ImpL2, an antagonist of insulin signaling, as a secreted factor mediating wasting. ImpL2 is sufficient to drive tissue loss, and insulin activity is reduced in peripheral tissues of tumor-bearing hosts. Importantly, knocking downs ImpL2 specifically in the tumor ameliorates wasting phenotypes. We propose that the tumor-secreted IGFBP creates insulin resistance in distant tissues and thus drives a systemic wasting response.

Graphical abstract

INTRODUCTION

Cancer is a leading cause of death in industrialized societies, yet the mechanisms by which a tumor claims the life of its host are not always clear. In some cases the growth of primary or secondary tumors disrupts the function of essential organs, but in other instances lethality is caused by physiological alterations at a distance from the tumor site. These distant influences, sometimes grouped as ‘paraneoplastic syndromes’, are major contributors to the morbidity and mortality of cancer patients.

A particularly debilitating distant tumor-host interaction is cancer cachexia, which is estimated to occur in >80% of patients with advanced cancers and to account for >20% of cancer deaths (Fearon et al., 2013; Tisdale, 2002). Cancer cachexia is a metabolic disorder that produces progressive tissue wasting, most evident in loss of adipose and muscle tissue. Cachectic patients show heightened risk of respiratory failure, increased susceptibility to chemotherapeutic toxicity, and other lethal sequelae. Unlike anorexia, in which caloric intake is reduced, wasting induced by cachexia is not reversed by supplemental nutrition. Available therapies for this clinically critical condition are notably limited in scope and effect.

Mediators of cancer cachexia remain mysterious. Cachexia is seen frequently with certain types of tumors and only rarely with others. Patient studies are complicated by heterogeneities in patient population, presentation, tumor pathology, comorbidities, and accompanying thereapeutic regimes. Most experimental investigations of cachexia rely on transplants of tumor cell lines into rodents (Bennani-Baiti and Walsh, 2011). These studies have implicated several factors such as IL-6, TNF-α, and metabolic products, but these reflect only a subset of human cachectic conditions (Fearon et al., 2012). Overall, the dearth of knowledge of mechanisms by which tumors induce cachexia has prompted the National Cancer Institute to highlight it as a ‘Provocative Question’ limiting progress in cancer treatment (provocativequestions.nci.nih.gov).

In recent years, studies in Drosophila have contributed significant insight into genetic factors driving human cancer. Analysis of fly ‘oncogenes and tumor suppressor genes’ have led for instance to the identification of the Hippo pathway (Harvey and Hariharan, 2012; Pan, 2010) and uncovered the cancer-relevant phenomenon of cell competition (Gonzalez, 2013; Patel and Edgar, 2014); flies have also been used to develop new cancer therapeutics (Dar et al., 2012). While most fly cancer models focus on autonomous tumor growth, fly tumors also show interactions with their host, including invasion of local tissues (Pagliarini and Xu, 2003; Woodhouse et al., 1998) and recruitment of innate immune cells (Cordero et al., 2010; Pastor-Pareja et al., 2008). The recent appreciation of parallels in physiological regulation between flies and humans (Leopold and Perrimon, 2007) creates an opportunity to use Drosophila as a simple system to study mechanisms by which cancer can perturb this homeostasis. Here we use a Drosophila model to demonstrate that fly tumors can induce cachexia-like phenotypes, and identify a tumor-secreted factor that drives wasting by preventing insulin signaling reception in peripheral tissues.

RESULTS

Tumor Induces Cachexia-Like Wasting in Host Tissues

Transplantation of imaginal discs from larvae into the hemocoel, the open body cavity of adults, is a classical technique for evaluating tissue growth (Hadorn, 1963). We transplanted GFP-labeled eye discs that were either WT or else mutant for the tumor suppressor gene scrib and overexpressing oncogenic Ras^V12, an established genetic cooperation system that induces malignant Drosophila tumors (Brumby and Richardson, 2003; Pagliarini and Xu, 2003). As shown by Pagliarini and Xu (2003), initially ~70 mm² scrib Ras^V12 tumor fragments grew continuously and induced a distinctive bloating of the abdomen before killing the host, when the tumor reached ~1,000 mm², whereas WT discs grew only a limited amount before ceasing and the transplanted host survived for weeks (Fig. 1A,B; Fig. S1A-E). We observed small numbers of cells disseminating from the tumor and invading other tissues, but these events were rare. At 5 days after transplantation, when the tumor is ~ 600 mm² (Fig. S1C,E), metastatic-like events were seen in <5% of the hosts.

Figure 1. Drosophila tumors can induce peripheral wasting.

(A, B). GFP-labeled scrib Ras^V12 tumor transplanted into WT adult host, after 1 and 5 days. (C-N) Phenotypes of peripheral tissues in control and tumor-hosting females. Fat body-specific reporter indicates depletion of this tissue in the abdomen of hosts in the presence of the tumor (C, D; green=Yolk-GFP). Lipid droplets, which are the storage vesicles of the adipose tissue, mobilize and aggregate into enlarged units (E, F; red=Nile red). Mitochondria-localized reporter reveals abnormal structure in the thoracic muscle of tumor-bearing hosts (G, H; green=Mito-GFP). Reduced ATP levels (M) and defects in both climbing ability (K) and climbing speed (L) indicate compromised muscle function. Ovaries are severely shrunken (I,J; F-actin=magenta; nuclei=cyan) in tumor-bearing compared to control hosts. N quantifies ovarian health as the percentage of non-apoptotic stage 9-10 ovarioles (see Materials and Methods). Scale bars: C=250μm; E=25μm; G=5μm; I=500μm. **p<0.01, ***p<0.001, Student's t-test; standard deviation is indicated. p and N values: Table S1.

While autonomous features of the tumor have been well-documented, we asked whether the tumor also had non-autonomous effects on the host. By contrast to the rare metastatic events, we discovered that on day 5, 100% of tumor-bearing hosts showed robust wasting phenotypes in tissues distant from the transplant. No phenotypes were seen in control hosts transplanted in parallel with WT disc fragments, indicating that wasting is not due to surgery nor any potential microbial infection. We first examined adipose tissues. Transplantation into hosts carrying an adipose reporter revealed a marked reduction in the fat body throughout the abdomen, irrespective of proximity to the tumor (Fig. 1C,D). In addition to the reduction of tissue mass, analysis of individual fat body cells with the lipophilic dye Nile Red demonstrated enlargement of lipid droplets, a phenotype associated with resource depletion (Fig. 1E,F) (Gutierrez et al., 2007). We next analyzed muscle. Microscopic examination of mitochondrially-imported GFP reveals that whereas mitochondria are regularly spaced between indirect flight muscle fibers of WT adults, packing in tumor-bearing hosts is irregular with a distinctly abnormal morphology (Fig. 1G, H). This phenotype is also seen in flies with degenerating muscle and mitochondrial fragmentation (Clark et al., 2006; Deng et al., 2008; Park et al., 2006), and consistent with this interpretation, muscle ATP levels were strongly reduced in tumor-bearing hosts (Fig. 1M). Functional tests revealed muscle weakness phenotypes specifically in tumor-bearing hosts. In climbing assays, both ability and speed (Fig. 1K, L) were strongly reduced, suggesting deteriorating muscle function (Demontis et al., 2013). Muscle defects were progressive, being evident at 3 days and increasing in severity with time after transplant.

The largest tissue in the adult female is the ovaries, where tumor-induced tissue loss was particularly evident. Female hosts transplanted with WT discs contained ovaries filling a substantial portion of the abdomen, but ovaries in tumor-bearing flies were almost rudimentary (Fig. 1I,J). A fly ovary contains ~16-20 ovarioles, each of which produces a sequential series of egg chambers that develop into mature eggs (Spradling, 1993). We quantitated ovarian reduction by evaluating the health of each ovariole, defined as its ability to produce a late-stage egg chamber. Tumor-bearing hosts showed an 85% reduction in ovarian health (Fig. 1N); apoptosis of mid-stage egg chambers was evident (Fig. 2E,F), and there was a complete absence of late-stage and mature eggs. As with fat and muscle, ovarian phenotypes were highly penetrant and did not depend on physical contact with the tumor. Transplantation into male hosts also induced similar tissue wasting (Fig. S1F-O), indicating that these phenotypes are not sex-specific. Together, these results demonstrate that scrib Ras^V12 tumors influence distant tissues, including muscle, fat and gonads, to undergo a wasting-like phenotype reminiscent of human cachexia.

Figure 2. Tumor-bearing hosts are not starved.

Whole ovaries (A-D) and mid-stage follicles (E-H) of control hosts and tumor-bearing hosts, compared to those of WT fed and starved flies. Ovary wasting and follicle apoptosis in tumor-bearing hosts resemble that seen in starved WT flies, as evidenced by shrunken ovarian size and nuclear fragmentation. Assays measuring food ingestion, by scoring the presence of dyed-food in the intestinal tract (I; Scoring: 0= no food in abdomen; 1= some food detected; 2= gut and crop are full), prolonged capillary food consumption (CAFÉ) (J), and short-term feeding behavior by proboscis extension (K) show no significant differences between control hosts and tumor-bearing hosts. Scale bars: A=500μm; E=25μm. ns= p> 0.05, Student's t-test; standard deviation is indicated. p and N values: Table S1.

Tumor-Induced Wasting is not due to Starvation

Deterioration of adipose, gonadal and muscle tissue are also phenotypes seen in adult flies undergoing starvation (Fig. 2D,H) (Demontis and Perrimon, 2010; Drummond-Barbosa and Spradling, 2001; Scott et al., 2004). We therefore tested the possibility that tumor-bearing hosts were unable to consume normal amounts of food, leading to systemic malnutrition. We first used a qualitative test to measure ingestion (Edgecomb et al., 1994), and found no difference between hosts transplanted with a tumor and control hosts transplanted with a WT disc fragment (Fig. 2I). We then quantitated food consumption over a 24 hour period using the capillary feeding (CAFÉ) assay (Ja et al., 2007). Again, no difference was found between tumor-transplanted and control transplanted hosts (Fig. 2J). Finally, we used an acute consumption assay (Deak, 1976) that can distinguish feeding behavior: starved flies will consume larger amounts than fed flies in a short (5 minute) time period (Liming Wang, personal communication). In this feeding assay, as in the other two, tumor-bearing hosts did not differ from hosts transplanted with a WT disc fragment (Fig. 2K). Some human patients suffering from cancer exhibit reduced appetite, and such patients can benefit from supplementary caloric intake. We found that raising tumor-bearing fly hosts on high-calorie food did not rescue tissue wasting (data not shown), demonstrating that altered food intake (anorexia) is not responsible for tissue deterioration.

ImpL2 is Secreted by Malignant Tumors and Sufficient to Induce Wasting

As tumor-bearing flies feed normally but show wasting, we hypothesized that the tumor might interfere with the host's normal physiological response to food intake. To investigate the mechanism, we began by considering whether all fly tumor genotypes were capable of eliciting a similar response. We first tested the two components of the genetic cooperation system individually. Despite their slower growth, transplanted scrib tumors recapitulated the ovarian wasting phenotypes induced by scrib Ras^V12 tumors (Fig. 3E,F), even when scrib tumors were ~3 fold smaller (Fig. 3A,B). By contrast, transplanted Ras^V12 tumors had no effect (Fig. 3G), even though these tumors grew to be ~2 fold larger than scrib (Fig. 3B,C). In Drosophila, Ras^V12 expression alone induces benign ‘hyperplastic’ growth (Karim and Rubin, 1998) that does not disturb epithelial architecture, remains confined by a basement membrane, and retains differentiation potential; these are all characteristics distinct from the malignant ‘neoplastic’ growth of scrib tumors (Bilder, 2004; Elsum et al., 2012). To distinguish the effects of tumor burden from tumor character, we transplanted hyperplastic tumors induced by ectopic activation of the Hippo pathway transcription factor Yki (Yki^SA) (Dong et al., 2007). yki^SA-expressing tumors grow in adult hosts in an epithelial fashion and become much larger (~4-6 fold) than Ras^V12 as well as scrib Ras^V12 tumors (Fig. 3D). Nevertheless, even the largest such tumors did not induce ovarian degeneration, (Fig. 3H), nor defects in fat or climbing speed as did much smaller scrib tumors (Fig. S2A-H). These findings demonstrate that it is not the size, but rather the nature of the tumor that defines its ability to induce wasting.

Figure 3. Neoplastic but not hyperplastic tumors induce wasting.

Growth of tumors (A-D) of different genotypes at 5 days post-transplantation, along with associated ovarian phenotypes (E-H). scrib Ras^V12 and scrib tumors induce wasting, while Ras^V12 and yki^SA tumors do not; wasting is independent of tumor burden. (I) Quantitative RT-PCR measurement of levels of transcripts (log₂ scale) encoding candidate secreted factors in scrib Ras^V12 vs. yki^SA tumors compared to controls. (J-L) Hindgut-driven ectopic expression demonstrates that ImpL2 but not Upd2 is sufficient to drive ovarian wasting. Scale bars: 500μm. *p<0.05, **p<0.01, Student's t-test; standard deviation is indicated. p and N values: Table S1.

Since scrib Ras^V12 and scrib tumors trigger wasting in tissues distant from their location, we hypothesized that the effect could be mediated by a secreted factor. We therefore searched transcriptome datasets (Bunker et al., 2015) for candidate factors upregulated specifically in malignant tumors but not benign tumors or WT discs. Two secreted factors that are upregulated >10-fold in scrib and scrib Ras^V12 discs are the IL-6-like cytokine Upd2 (Arbouzova and Zeidler, 2006), and ImpL2 (Honegger et al., 2008), a homolog of IGF-binding proteins; we also considered the insulin-like peptide dILP8 (Colombani et al., 2012; Garelli et al., 2012) which is >100-fold upregulated in scrib, scrib Ras^V12, and yki^SA (Fig. 3I). To test these candidates, we expressed each using a hindgut-specific GAL4 driver and examined whether wasting of gonadal and adipose tissue was induced. Interestingly, only ImpL2 expression resulted in a substantial reduction in ovarian size, which was accompanied by apoptotic egg chambers, loss of mature eggs and reduced fecundity (Fig. 3J-L). Hindgut-driven ImpL2 expression also reduced abdominal fat body and induced lipid droplet enlargement (Fig. S2O-Q). Ectopically-driven ImpL2 levels in hindgut were comparable to those expressed by scrib Ras^V12 tumors (Fig. S2S), and similar ovarian and adipose wasting phenotypes were observed when expressing ImpL2 using a muscle-specific GAL4 driver (Fig. S2I-N). Thus, excess ImpL2 production alone, independent of tumor growth, is sufficient to induce wasting in distant adult tissues.

Tumor Reduces Insulin Signaling Pathway Activity in Host Tissues

ImpL2 has been demonstrated to bind insulin in solution and to antagonize the insulin pathway in vitro (Honegger et al., 2008) and in vivo (Okamoto et al., 2013). If ImpL2 secreted from scrib Ras^V12 tumors plays a role in peripheral wasting, then tumor-bearing host tissues should show reduced insulin signaling. To assess this, we transplanted into hosts expressing an insulin pathway reporter. tGPH produces a GFP protein fused to the PH domain of GRP1, which is recruited to the plasma membrane upon insulin-stimulated PI3-Kinase activity (Britton et al., 2002). We found that in tumor-bearing but not control hosts, tGPH remains substantially cytoplasmic in both the fat body (Fig. 4A,B) and in egg chambers (Fig. 4C,D), indicating defective insulin signaling reception. We also used qPCR to measure mRNA levels of 4E-BP, a downstream FOXO target that is elevated when insulin signaling is low. In both muscle and ovary, 4E-BP levels are increased in tumor-bearing as compared to control hosts (Fig. 4E). Importantly, we compared levels of Drosophila Insulin-like Peptides (dILPs) in their neuroendocrine source cells between tumor-bearing and control hosts (Fig. S3A,B) and did not detect the retention seen for instance in nutrient-depleted flies (Rajan and Perrimon, 2012). Metabolic assays revealed elevated circulating trehalose levels (Fig. 4F), characteristic of hyperglycemia, but minor or no differences in triglycerides and glycogen, respectively (Fig. S3C-E). As defects in insulin secretion are not evident, but peripheral tissues nevertheless experience reduced insulin signaling and high circulating sugar levels, this suggests that the tumor induces insulin resistance.

Figure 4. Tumor alters insulin signaling and metabolism in host.

Compared to control, scrib Ras^V12 tumor-bearing hosts show decreased plasma membrane recruitment of the tGPH reporter in fat body (A, B) and ovaries (C, D), illustrating decreased insulin signaling reception. (E) Increased transcription of the FoxO target 4E-BP by quantitative RT-PCR measurement in ovary and thorax also reveals decreased insulin signaling activity. Metabolic analysis (F) reveals elevated circulating trehalose levels in tumor-bearing hosts relative to controls; absolute values: Table S2. Scale bars: A, C=25μm. ns= p> 0.05, *p<0.05, **p<0.01, Student's t-test; standard deviation is indicated. p and N values: Table S1.

ImpL2 is Necessary for Robust Tumor-Induced Cachexia-Like Wasting

Finally, to test whether tumor-secreted ImpL2 in fact mediates the wasting phenotypes, we depleted its activity specifically within the malignant tumor. We knocked down ImpL2 via RNAi in an eyeless GAL4-driven dlg^RNAi Ras^V12 tumor model (Willecke et al., 2011) that causes both autonomous growth and non-autonomous wasting comparable to scrib Ras^V12 (Fig. 5C,E,G,I, J). Importantly, dlg^RNAi Ras^V12 ImpL2^RNAi tissue formed a tumorous mass within five days of transplantation, like dlg^RNAi Ras^V12 (Fig. 5A,B). Strikingly, however, reducing ImpL2 within the tumor itself significantly ameliorated each of the peripheral tissue phenotypes. Hosts bearing dlg^RNAi Ras^V12 ImpL2^RNAi tumors showed increased abdominal fat body mass (Fig. 5C,D); rescue of this tissue was also evident in the restoration of lipid droplet size (Fig. 5E,F). Muscle function assays further revealed improvements in both climbing ability and speed (Fig. 5I,J). Lastly, there is significant rescue of ovariole health, leading to a restoration of egg production (Fig. 5G,H,K). Rescue was only observed when ImpL2 was knocked down in the tumor; transplanting dlg^RNAi Ras^V12 tumors into ImpL2 null hosts did not ameliorate wasting (Fig. S4). The lack of full recovery with ImpL2-depleted tumors suggests that additional factors associated with malignancy may contribute. Nonetheless, the substantial rescue demonstrates that it is a central secreted factor driving tumor-induced tissue wasting.

Figure 5. Tumor-secreted ImpL2 is necessary and sufficient for robust wasting.

dlg^RNAi Ras^V12 ImpL2^RNAi and dlg^RNAi Ras^V12 tumors are comparable in size (A, B). Knockdown of ImpL2 in dlg^RNAi Ras^V12 tumors improves host abdominal fat body mass (C, D), reduces lipid droplet aggregation (E, F), and restores ovarian tissue size (G, H) and health (K) as compared to hosts bearing dlg^RNAi Ras^V12 tumor alone. Knockdown of ImpL2 in dlg^RNAi Ras^V12 tumors also restores host muscle function, as measured by climbing ability (I) and speed (J). Scale bars: A, C, G=500μm; E=25μm. **p<0.01, ***p<0.001, Student's t-test; standard deviation is indicated. p and N values: Table S1.

DISCUSSION

Cachexia remains a major obstacle to cancer treatment, in part because the molecular mechanisms that drive it remain uncertain. Here, we describe a fly model that mimics certain aspects of human cachexia, and utilize this model to identify a specific cachectic mediator. The tumor-induced wasting that we describe in flies resembles cancer cachexia in its independence from food consumption, its target tissues, its progressive nature, and its induction by certain but not all types of tumors. The fly model does not parallel all features associated with the human condition; for instance, we detect only slight upregulation of putative fly orthologs of mammalian regulators implicated in muscle catabolism (Fig. S3F) (Asp et al., 2010; Bonaldo and Sandri, 2013). Human cancer cachexia is clearly a heterogeneous and multifactorial condition (Fearon et al., 2012), and this complexity has impeded progress in its understanding. In this work we use a reductionist system to identify a single tumor-derived factor that can drive the robust deterioration of peripheral tissues.

Insulin signaling is a central regulator of tissue mass in both flies and humans. Our data demonstrate that ImpL2, a secreted insulin antagonist produced by malignant tumors, is a major mediator that is both necessary and sufficient for wasting. In an accompanying paper, Kwon et al. show that ImpL2 is also a systemic wasting factor in a different fly tumor model. Reduced insulin signaling is further responsible for wasting induced by mycobacterial infection of flies (Dionne et al., 2006); whether ImpL2 is the relevant mediator in this case is not known. ImpL2 is the single fly homolog of mammalian IGFBPs, and can bind to systemic insulin-like ligands to antagonize insulin signaling. By this mechanism, the tumor effectively induces insulin resistance in peripheral tissues.

Insulin resistance is a frequent feature of both cachectic patients and rodent cachexia models (Honors and Kinzig, 2012; Tisdale, 2009); indeed some evidence suggests that exogenous insulin can ameliorate tissue loss in these contexts. The seven mammalian IGFBPs are variously upregulated or downregulated in different tumors, but have been evaluated in cancer primarily with respect to their affects on tumor growth (Baxter, 2014). Our data motivate assessments of whether highly cachectogenic human tumors, such as pancreatic and gastric cancers, display elevated expression of IGFBPs, and how therapies designed to correct insulin resistance might be used to treat such tumors.

ImpL2 joins the list of effectors induced by neoplastic transformation in fly tumors, including mitogens and pro-invasive factors. Recent work from our lab shows that the Upd3 mitogen is upregulated by dual activity of JNK and Hippo signaling (Bunker et al., 2015). The ImpL2 regulatory region, like that of Upd3, contains evolutionarily conserved binding sites for AP-1 and Sd transcription factors, suggesting that it may also be synergistically regulated by these pathways that monitor epithelial integrity. Despite the reduced insulin signaling in neoplastic tumors themselves (e.g. 4EBP levels are elevated ~21 fold (Bunker et al., 2015); they are hypersensitive to PI3K reduction (Willecke et al., 2011)), the tumors nevertheless robustly proliferate. How ImpL2-upregulating tumors escape insulin resistance remains an unanswered question, although metabolic changes suggested by transcriptome alterations may be a possible mechanism.

While tumor-specific inhibition of ImpL2 causes a significant amelioration of the wasting phenotype, rescue is not complete, suggesting that other aspects of tumor-host interaction remain to be uncovered. We found that a fly homolog of IL-6, a molecule implicated in several rodent cachexia models, was not sufficient to induce wasting, while partial ablation of host innate immune cells (Charroux and Royet, 2009) did not qualitatively alter wasting phenotypes (data not shown); however, contributing roles for these factors have not been ruled out. Future work will analyze other tumor-produced factors, including metabolites generated by anabolic and catabolic alterations in the tumor, to evaluate their involvement as well. The manipulability of the simple model developed here, including the ability to rapidly assess fully-defined combinations of host and tumor genotypes, opens the door to candidate as well as forward genetic approaches to identify additional factors mediating tumor-host interactions.

EXPERIMENTAL PROCEDURES

Genetics and Transplantation

scrib¹ Ras^V12 and Ras^V12 tumors were generated using the eyMARCM genetic system as in (Pagliarini and Xu, 2003). In knockdown experiments, dlg^RNAi Ras^V12 (Willecke et al., 2011) was used in combination with white^RNAi (Bloomington #28980) or ImpL2^RNAi (VDRC #30930); similar results were seen with an independent ImpL2^RNAi (NIG-FLY #1590-R3). Yki tumors were obtained from MS1096-GAL4 UAS Yki^S168A larvae and scrib tumors from scrib¹ homozygotes. Transplantation was adapted from (Woodhouse et al., 1998): WT or tumorous imaginal discs were dissected from wandering third instar larvae, fragmented and introduced through a pulled glass capillary needle into the abdomen of one day old virgin females. Host females were kept on a high-yeast diet, in the presence of males at 25° C, except for starved females who were kept on water only. Hosts were either OreR, Yolk GAL4 UAS-GFP, Mef2 GAL4 UAS-Mito-GFP, tGPH (Britton et al., 2002) or ImpL2^Def20 (Honegger et al., 2008). Ectopic expression experiments used Mef2GAL4 GAL80ts or bynGAL4 GAL80ts, raised at 18° and then shifted to 29° as adults to drive expression of UAS-s.ImpL2 (Honegger et al., 2008), UAS-dILP8 (Colombani et al., 2012) and UAS-Upd2 (Rajan and Perrimon, 2012).

Feeding, Locomotive, and Ovarian Assays

Ingestion was scored using a blue dye feeding assay adapted from (Edgecomb et al., 1994). Briefly, FD & C Blue No.1 food dye (2.5% w/v) was incorporated into yeast paste in order to visualize and score the presence of food in the crop and intestinal areas. CAFÉ (Ja et al., 2007) and PER (Deak, 1976) assays were perfomed in triplicate to measure consumption and feeding behavior, respectively (Liming Wang, personal communication). For CAFÉ, groups of 6 females were allowed to feed ad libitum for 24 hours on liquid food (5% yeast extract and 5% sucrose) dispensed from calibrated glass capillaries (World Precision Instruments); amounts consumed were measured and adjusted for evaporation. For PER, groups of 4 females were presented with a calibrated capillary (Drummond Scientific Company) containing liquid food and the amount consumed per feeding response was measured until fly was satiated. Presentation of water was used to control for thirst.

Climbing ability and speed assays were adapted from (Feany and Bender, 2000) and (Park et al., 2006). Groups of 10 females were placed in empty vials and after 1 hour of recovery were gently tapped to the bottom. Climbing ability was determined by the number of flies that reached an 8cm mark in 20 seconds; speed was calculated using the length of climbing time. For each group, 3 trials were recorded per assay; experiments were conducted in triplicate.

Ovarian health was quantified as the percentage of ovarioles that contained one or more non-apoptotic egg chamber at stage 9-10.

Microscopy and Image Analysis

All samples were fixed in 4% formaldehyde in phosphate buffered saline (PBS). Staining followed standard protocols, with anti-Dilp2 antibody (1:2,000; J. Veenstra), TRITC-phalloidin (1:400; Sigma), DAPI (1:1,000; Life Technologies) or Nile Red (1:5,000; Sigma). Images were captured on a Zeiss 700 confocal microscope. Figures were assembled using Adobe Illustrator.

Quantitative RT-PCR

Total RNA was prepared from groups of 15 tumors, 20-30 ovaries (RNeasy Mini Kit; QIAGEN), 20-25 thoraces and 12-16 whole flies (TRIzol reagent; Invitrogen and Direct-zol RNA MiniPrep Kit; Zymo Research). qPCR experiments were performed in triplicate using SYBR GreenER qPCR SuperMix. Relative quantification of mRNA levels was determined using the Comparative C_T method and normalized to alpha-tubulin (thoraces), rpl23 (ovaries) and gapdh (tumors and whole flies).

ATP Measurements

In triplicate, 5 thoraces were homogenized in 80 μl of extraction buffer (6 M Guanidine Hydrochloride, 4mM EDTA, 100mM Tris-HCl, pH 8.0), boiled for 5 minutes and centrifuged at 4° for 5 minutes. Supernatant was collected and diluted 1:50. ATP levels were quantified using an ATP Determination Kit (Life Technologies/Invitrogen) and normalized to total protein levels (Bradford method).

Metabolic Assays

For Glucose, Glycogen and TAG assays, 5 flies, in triplicate, were homogenized in PBST, heated to 70° for 10 minutes and centrifuged; supernatant was collected. Samples were processed and levels measured using manufacturer's protocols: Glucose (HK) Assay Kit (Sigma), Glycogen Colorimetric Assay Kit (BioVision) and Triglycerides LiquiColor Test (Stanbio Laboratory), respectively. Protein levels were determined with the BCA Protein Assay Kit (Thermo Fisher Scientific) and used for normalization.

For circulating trehalose assays, 1 μl of hemolymph, in triplicate, was collected by centrifugation and diluted in Trehalase Buffer. Samples were heated at 70° for 10 minutes and treated with porcine trehalase (Sigma). Levels were measured using the Glucose (HK) Assay Kit (Sigma) following manufacturer's protocol. Total levels were calculated after subtracting free glucose and normalized per fly.