EyaHOST, a modular genetic system for investigation of intercellular and tumor-host interactions in Drosophila melanogaster.

Cell biology and genetic analysis of intracellular, intercellular and inter-organ interaction studies in animal models are key for understanding development, physiology, and disease. The MARCM technique can emulate tumor development by simultaneous clonal tumor suppressor loss-of-function generation coupled with GAL4-UAS-driven oncogene and marker expression, but the utility is limited for studying tumor-host interactions due to genetic constraints. To overcome this, we introduce EyaHOST, a novel system that replaces MARCM with the QF2-QUAS binary gene expression system under the eya promoter control, unleashing the fly community genome-wide GAL4-UAS driven tools to manipulate any host cells or tissue at scale. EyaHOST generates epithelial clones in the eye epithelium similar to MARCM. EyaHOST-driven Ras V12 oncogene overexpression coupled with scribble tumor suppressor knockdown recapitulates key cancer features, including systemic catabolic switching and organ wasting. We demonstrate effective tissue-specific manipulation of host compartments such as neighbouring epithelial cells, immune cells, fat body, and muscle using fly avatars with tissue-specific GAL4 drivers. Organ-specific inhibition of autophagy or stimulation of growth-signaling through PTEN knockdown in fat body or muscle prevents cachexia-like wasting. Additionally, we show that Ras V12 , scrib RNAi tumors induce caspase-driven apoptosis in the epithelial microenvironment. Inhibition of apoptosis by p35 expression in the microenvironment promotes tumor growth. EyaHOST offers a versatile modular platform for dissecting tumor-host interactions and other mechanisms involving intercellular and inter-organ communication in Drosophila . Highlights: * eyes absent , eye disc-specific enhancer drives clonal KD recombinase flip-out activated QF2 expression in the larval eye epithelium for simultaneous QUAS-driven gain and loss-of-function analysis of gene function. *Clones are visualized by QUAS-tagBFP or QUAS-eGFP facilitating analysis of existing fluorescent reporters.*The GAL4-UAS system and existing genome-wide genetic tools are released to independently manipulate any cell population in the animal for cell biology, intercellular or inter-organ analysis for developmental, physiological, or disease model analysis.*Fly avatars for tumor-host interaction studies with multiple organs allow live monitoring and manipulation of tumors and organs in translucent larva.

Autophagy power expands: fuse those cells!

The lysosomal degradation pathway of autophagy depends on a set of evolutionarily conserved autophagy-related molecules (ATGs) bestowed with the power to direct membrane trafficking and biology. In this issue of EMBO Journal, Kakanj P et al reveal a surprising role for the autophagy machinery in cell fusion (Kakanj et al, 2022). Autophagy is physiologically required for cell syncytium formation through dismantling the lateral plasma membrane during wound healing, and unchecked autophagy can drive cell fusion in epithelial tissues without compromising epithelial integrity.

Computed tomography with segmentation and quantification of individual organs in a D. melanogaster tumor model.

Drosophila melanogaster tumor models are growing in popularity, driven by the high degree of genetic as well as functional conservation to humans. The most common method to measure the effects of a tumor on distant organs of a human cancer patient is to use computed tomography (CT), often used in diagnosing cachexia, a debilitating cancer-induced syndrome most visibly characterized by loss of muscle mass. Successful application of high resolution micro-CT scanning of D. melanogaster was recently reported and we here present the segmentation of all visible larval organs at several stages of tumor development. We previously showed the strong expected reduction in muscle mass as the tumor develops, and we here report a surprisingly strong reduction also in gut and Malpighian tubules (kidney) volume. Time-point of tumor development was found to have a stronger correlation to cachectic organ volume loss than tumor volume, giving support to the previously proposed idea that tumor size does not directly determine degree of cachexia.

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.

NAD+ augmentation restores mitophagy and limits accelerated aging in Werner syndrome.

Metabolic dysfunction is a primary feature of Werner syndrome (WS), a human premature aging disease caused by mutations in the gene encoding the Werner (WRN) DNA helicase. WS patients exhibit severe metabolic phenotypes, but the underlying mechanisms are not understood, and whether the metabolic deficit can be targeted for therapeutic intervention has not been determined. Here we report impaired mitophagy and depletion of NAD+, a fundamental ubiquitous molecule, in WS patient samples and WS invertebrate models. WRN regulates transcription of a key NAD+ biosynthetic enzyme nicotinamide nucleotide adenylyltransferase 1 (NMNAT1). NAD+ repletion restores NAD+ metabolic profiles and improves mitochondrial quality through DCT-1 and ULK-1-dependent mitophagy. At the organismal level, NAD+ repletion remarkably extends lifespan and delays accelerated aging, including stem cell dysfunction, in Caenorhabditis elegans and Drosophila melanogaster models of WS. Our findings suggest that accelerated aging in WS is mediated by impaired mitochondrial function and mitophagy, and that bolstering cellular NAD+ levels counteracts WS phenotypes.

Autophagy and Tumorigenesis in Drosophila.

The resurgence of Drosophila as a recognized model for carcinogenesis has contributed greatly to our conceptual advance and mechanistic understanding of tumor growth in vivo. With its powerful genetics, Drosophila has emerged as a prime model organism to study cell biology and physiological functions of autophagy. This has enabled exploration of the contributions of autophagy in several tumor models. Here we review the literature of autophagy related to tumorigenesis in Drosophila. Functional analysis of core autophagy components does not provide proof for a classical tumor suppression role for autophagy alone. Autophagy both serve to suppress or support tumor growth. These effects are context-specific, depending on cell type and oncogenic or tumor suppressive lesion. Future delineation of how autophagy impinges on tumorigenesis will demand to untangle in detail, the regulation and flux of autophagy in the respective tumor models. The downstream tumor-regulative roles of autophagy through organelle homeostasis, metabolism, selective autophagy or alternative mechanisms remain largely unexplored.

Microenvironmental autophagy promotes tumour growth.

As malignant tumours develop, they interact intimately with their microenvironment and can activate autophagy, a catabolic process which provides nutrients during starvation. How tumours regulate autophagy in vivo and whether autophagy affects tumour growth is controversial. Here we demonstrate, using a well characterized Drosophila melanogaster malignant tumour model, that non-cell-autonomous autophagy is induced both in the tumour microenvironment and systemically in distant tissues. Tumour growth can be pharmacologically restrained using autophagy inhibitors, and early-stage tumour growth and invasion are genetically dependent on autophagy within the local tumour microenvironment. Induction of autophagy is mediated by Drosophila tumour necrosis factor and interleukin-6-like signalling from metabolically stressed tumour cells, whereas tumour growth depends on active amino acid transport. We show that dormant growth-impaired tumours from autophagy-deficient animals reactivate tumorous growth when transplanted into autophagy-proficient hosts. We conclude that transformed cells engage surrounding normal cells as active and essential microenvironmental contributors to early tumour growth through nutrient-generating autophagy.

Contrasting phagosome pH regulation and maturation in human M1 and M2 macrophages.

Macrophages respond to changes in environmental stimuli by assuming distinct functional phenotypes, a phenomenon referred to as macrophage polarization. We generated classically (M1) and alternatively (M2) polarized macrophages--two extremes of the polarization spectrum--to compare the properties of their phagosomes. Specifically, we analyzed the regulation of the luminal pH after particle engulfment. The phagosomes of M1 macrophages had a similar buffering power and proton (equivalent) leakage permeability but significantly reduced proton-pumping activity compared with M2 phagosomes. As a result, only the latter underwent a rapid and profound acidification. By contrast, M1 phagosomes displayed alkaline pH oscillations, which were caused by proton consumption upon dismutation of superoxide, followed by activation of a voltage- and Zn(2+)-sensitive permeation pathway, likely HV1 channels. The paucity of V-ATPases in M1 phagosomes was associated with, and likely caused by, delayed fusion with late endosomes and lysosomes. The delayed kinetics of maturation was, in turn, promoted by the failure of M1 phagosomes to acidify. Thus, in M1 cells, elimination of pathogens through deployment of the microbicidal NADPH oxidase is given priority at the expense of delayed acidification. By contrast, M2 phagosomes proceed to acidify immediately in order to clear apoptotic bodies rapidly and effectively.