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fondue: Biological Overview | References

Malignant tumors trigger a complex network of inflammatory and wound repair responses, prompting Dvorak's characterization of tumors as "wounds that never heal." Some of these responses lead to profound defects in blood clotting, such as disseminated intravascular coagulopathy (DIC), which correlate with poor prognoses. This study demonstrates that a new tumor model in Drosophila provokes phenotypes that resemble coagulopathies observed in patients. Fly ovarian tumors overproduce multiple secreted components of the clotting cascade and trigger hypercoagulation of fly blood (hemolymph). Hypercoagulation occurs shortly after tumor induction and is transient; it is followed by a hypocoagulative state that is defective in wound healing. Cellular clotting regulators accumulate on the tumor over time and are depleted from the body, suggesting that hypocoagulation is caused by exhaustion of host clotting components. Rescuing coagulopathy by depleting a tumor-produced clotting factor improves survival of tumor-bearing flies, despite the fact that flies have an open (non-vascular) circulatory system. As clinical studies suggest that lethality in patients with high serum levels of clotting components can be independent of thrombotic events, this work establishes a platform for identifying alternative mechanisms by which tumor-driven coagulopathy triggers early mortality. Moreover, it opens up exploration of other conserved mechanisms of host responses to chronic wounds (Hsi, 2023).

Tumor-host interactions, as well as autonomous growth of the tumor itself, play central roles in cancer progression, morbidity, and mortality.Drosophila has recently emerged as a valuable system to study these interactions, elucidating mechanisms that can be conserved with mammals. However, current systems have key limitations. In larval models, tumor-associated pupation defects prevent lifespan analysis, whereas in adult allograft models, wounding from transplantation confounds the study of the response to the tumor alone. In adult transgenic gut tumor models, perturbation of this essential organ may directly impact host metabolism, physiology, and the microbiome. An alternative genetic model was developed, wherein a tumor is induced to grow in the non-essential11 ovarian epithelium of an adult female fly. In the ovarian carcinoma (OC) model, tumorigenesis is driven by expression of the oncogenes Ras^V12 and aPKC^ΔN,directed to the follicle epithelium via traffic jam-Gal4 (tj-GAL4) and restricted to adulthood via a ubiquitously expressed temperature-sensitive GAL4 repressor (tubGAL80ts). This spatiotemporal control facilitates the study of both tumor and adult host from the initial stages of transformation to a fully formed malignancy (Hsi, 2023).

Defining characteristics shared by mammalian and fly carcinomas include overproliferation, loss of cell polarity, and defective differentiation. Phosphohistone H3 staining revealed elevated mitotic rates in OC cells. Epithelial organization was strongly disrupted, as was the localization of apically and basolaterally polarized proteins (Figures 1E, 1F, and S1C). Tumor cells failed to upregulate Hindsight (Hnt), a marker of mature differentiated follicle cells, and exhibited prolonged expression of the early follicle cell marker FasIII (Figures 1G and 1H).14 Thus, fly OC cells are transformed into malignant epithelial-derived tumors (Hsi, 2023).

An additional characteristic of malignant tumors is their potential to kill hosts. Importantly, OC-bearing flies show dramatically accelerated mortality compared with controls, with median survival reduced by ~50%. GAL80-mediated silencing of neural tj-GAL4 suggests that this limited expression is not responsible for lethality. GFP labeling of oncogene-expressing cells revealed no dissemination, indicating a lack of metastasis. Because female flies do not require ovaries to live, lethality appears to arise from systemic effects of the tumor on the host, often called paraneoplasias. Similar to other adult tumor models, OC-bearing flies exhibit bloating resulting from the accumulation of fluid in the hemocoel. Over the course of tumorigenesis, OCs also induce reduction in fat body lipid storage. Finally, tumor-bearing hosts displayed breakdown of the blood-brain barrier (BBB). The OC model therefore recapitulates previously documented Drosophila paraneoplasias (Hsi, 2023).

To better understand OC progression and its impact on host survival, a system was developed to grade OC tumors based on distinct morphological characteristics. Assessing grade was more appropriate than mass because OC tumors, particularly at early grades, contain non-transformed germline cells that are polyploid and large. Trade 1 tumors are defined as exhibiting a loss of epithelial polarity and overproliferation, most evident at the poles of individual follicles, as well as beginning germline death. Although grade 1 tumors do not disrupt follicle organization and transformed cells remain in situ, grade 2 tumors show fusion between individual follicles as well as germline absence. At grade 3, the muscle sheath and its associated basement membrane breaks down, allowing fusion between neighboring ovarioles. Further disruption of ovariole morphology resulting from organ-wide basement membrane breakdown characterizes grade 4 tumors. Using this system, tumor grade reliably increased with time after oncogene induction and that most dying animals displayed grade 3 or 4 tumors regardless of that individual's time of death. The accelerated host mortality driven by tumor progression in a non-essential organ in the absence of metastasis place the OC model in a unique position to study tumor-host interactions (Hsi, 2023).

To identify factors regulating these interactions, bulk transcriptome analysis was performed comparing wild-type (WT), pre-vitellogenic follicle cells to OC follicle cells 20 days after tumor induction (ATI). Expression of matrix metalloproteinase 1 and puckered were increased, indicating high levels of JNK activity, as were midline fasciclin and SOCS36E, suggesting increased STAT activity Fluorescent reporters validated the increased activity of both pathways, similar to other Drosophila epithelial tumors. GstS1 and Zfh1, markers of follicle stem cells and early prefollicle cells, respectively, were upregulated alongside downregulation of Hnt, which marks more mature follicle cells, suggesting that some OC follicle cells retain a progenitor-like identity (Hsi, 2023).

Because tumor-released peptides are likely mediators of paraneoplasias, putative secreted proteins upregulated in the OC transcriptome were examined. Of 3,567 genes showing at least 2-fold increase, 246 are predicted to encode secreted factors. Several of these encode known signals overproduced by adult gut tumors as well as larval disc tumors, including the IL-6-like Unpaireds, PDGF/VEGF-related factors, and the insulin growth factor binding protein-like ImpL2 (Hsi, 2023).

Strikingly, among the upregulated genes predicted to encode secreted factors, multiple genes were noted that participate in the Drosophila clotting cascade (see OCs differentially express clotting factors and regulators), among a number of other secreted proteins. As in mammals, flies have an essential circulatory fluid whose loss following wounding must be prevented. In both mammals and flies, this happens through clotting in which soluble factors are polymerized and crosslinked to restore hemostasis. Genes whose products in larvae form the initial "soft clot" were overexpressed in tumor cells, including fondue (fon), hemolectin (hml), and fat body protein 1 (fbp1), which can be considered functionally analogous to human fibrin. Some of these gene products are substrates of the fly crosslinking enzyme encoded by transglutaminase (tg), which is homologous to mammalian clotting factor XIIIa31 and is also upregulated in OCs. The clotting pathway in flies includes an insect-specific reaction, called the melanization cascade. This reaction is responsible for clot maturation to form a "hard clot" and is regulated by the activity of pro-phenoloxidases (PPOs), which are released by specialized blood cells called crystal cells (CCs) following injury. Transcript levels of PPO1 and PPO2 were elevated in OC cells as well. Taken together, these data raise the possibility that fly tumors may interface with the host clotting cascade (Hsi, 2023).

It was therefore asked whether coagulation is altered by OC tumors. The capacity of hemolymph from tumor-bearing adult flies to form soft clots was measured. Adapting an assay previously used for larvae, inert Dynabeads were incubated with extracted hemolymph ex vivo. Hemolymph from control adults at day 5 and 10 induced clumping of beads, albeit less strongly than seen in larvae. In striking contrast, hemolymph from OC flies 5–10 days ATI readily generated large, macroscopic bead aggregates, indicating that these animals are in a hypercoagulable state. Aggregation activity subsequently decreases at days 15 and 20 ATI, demonstrating that the hypercoagulable state triggered by tumors is transient. The bead-clumping phenotype at day 5 represents the first demonstration of a hypercoagulation phenotype in Drosophila and the earliest paraneoplastic phenotype in an adult fly tumor model (Hsi, 2023).

Then the ability of tumor-bearing hosts to create hard, melanized clots was tested. Two assays were used: first, measuring phenoloxidase (PO) activity levels of hemolymph ex vivo on a colorimetric substrate and second, by observing melanization of cuticular wounds in vivo. At day 15 ATI, PO activity was decreased in tumor-bearing flies in comparison with control, and this decrease became more severe at day 20. Tumor-bearing flies at days 15 and 20 ATI also showed a strong failure to melanize thoracic wounds compared with control flies. Indeed, 2 hours after wounding, bleeding was staunched in control flies, but hemolymph remained on the wounds of tumor-bearing flies 20 days ATI. The defects in hard clotting along with wound healing are consistent with a hypocoagulable state that follows the hypercoagulable phase (Hsi, 2023).

Given that hard clotting deficiencies temporally follow a period of increased soft clotting activity, it was hypothesized that, as with some human paraneoplastic coagulopathies, fly tumors overstimulate the clotting system, leading to an exhaustion of clot components within the host. A good candidate for such a factor are CCs, which lyse to release PPOs following activation and, though produced only in the larvae, persist into adulthood Counts of circulating CCs, labelled by BcF2-GFP, in OC hosts revealed a strong decrease 15 days ATI compared with control. This loss of CCs is consistent with the depletion of hemolymph PO activity and clotting failure. Further, many BcF2-GFP-positive cells were seen on OC tissue starting at day 10 ATI, while only a few are found on control ovaries. Transcriptional upregulation of PPO1 (which the BcF2-GFP transgene reports) within OC cells, along with the failure to identify conditions for specific antibody staining of CCs, prevented unambiguous counting of tumor-associated CCs, but bright BcF2-GFP signals of sizes consistent with CCs were seen at the OC anterior. Interestingly, when the major clotting component Fon tagged with GFP was overexpressed within the tumor, an anterior distribution of aggregated signal was seen that was distinct from the diffuse pattern seen in control ovaries. Overall, these results suggest a model in which the OC tumor activates elements of the clotting cascade that gradually consumes essential component (Hsi, 2023).

Finally, it was asked whether coagulopathy plays a role in the premature lethality of OC flies. This was tested by using RNAi to deplete Fon, which is overproduced >25-fold in the tumor and is required for clotting in larvae. Remarkably, knockdown of Fon in OC tumors extended median survival by ∼38% (Figure 4A). Lifespan extension was also seen with a second, independently generated RNAi construct. This extension was robust compared with control RNAis, and is of a magnitude comparable to the extension of tumor-bearing hosts seen when BBB disruption is prevented in a different tumor model. Increased lifespan correlated with reduced coagulopathy, as knocking down Fon attenuated early hypercoagulation as well as late hypocoagulation phenotypes. Quantitation revealed that hemolymph of flies bearing OC tumors depleted of Fon aggregated beads to a much lower extent than the hypercoagulative activity seen in control OC flies shortly ATI. At day 20 ATI, flies bearing OC tumors depleted of Fon also showed higher hemolymph PO activity and increased ability to melanize and seal wounds compared with control OC flies. Thus, Fon upregulation in the tumor regulates systemic coagulopathy and paraneoplastic lethality in the host (Hsi, 2023).

One explanation for coagulopathy accelerating tumor-bearing host mortality could be an autonomous effect on tumor progression. Yet, quantitation of OC grade revealed no change when Fon was depleted. A second possibility is that coagulopathy might reduce lifespan through enhancing wasting in tumor-bearing hosts. However, reducing wasting in such hosts by depletion of cachexia-inducing ImpL2 did not increase lifespan. Fon depletion in OC flies did reduce wasting, but without altering expression of either ImpL2 or other potential cachexia-inducing regulators Pvf2 and Pvf3 is not upregulated in OC tumors. A third possibility is that coagulopathy might accelerate host death through enhancing BBB permeability, which is a driver of tumor-associated lethality. BBB breakdown was present in OC flies even upon depletion of Fon. A fourth possibility was investigated-that coagulopathy causes an elevation of reactive oxygen species (ROS) levels through CC release of PPOs. ROS have well-documented deleterious effects on lifespan, but also have been suggested to be at times cytoprotective. OC flies heterozygous for the Bc mutation, which causes premature rupture of many CCs during the larval stage, showed no changes in lifespan, and OC flies carrying an engineered dominant PPO1 allele live only slightly longer than controls. Treatment with antioxidant N-acetyl cysteine or pro-oxidant paraquat did not change OC mortality substantially, providing limited support for a role for ROS in tumor-driven mortality . Finally, dying OC flies did not show increased intestinal permeability, as detected by the "smurf" assay. Overall, these data suggest that coagulopathy contributes to tumor-driven death via a currently unknown paraneoplastic mechanism (Hsi, 2023).

The role of individual clotting proteins in Drosophila larval coagulation is not well understood, and as this work is the first to interrogate adult coagulation, how the tumor-produced factors participate in this process is not yet clear. For instance, depleting fbp1 within the tumor caused significant lifespan extensions, as did depleting fon, but depleting tg and hml did not. fbp1 expression is normally restricted to the larval stage, so knockdown in the tumor would strongly reduce its presence in the adult host, in contrast to tg and hml, which are produced by tumor-independent sources in the adult. However, fon is also transcribed in adult fat body, yet its knockdown in the tumor alone is able to extend host lifespan. Although lifespan extension of OC hosts by depletion of multiple clotting factors supports a role for coagulopathy in tumor-driven death, a mechanistic understanding of each protein's function will require further investigation (Hsi, 2023).

These data reveal an unexpected parallel between cancer patients and tumor-bearing flies: both can show widespread clotting defects that contribute to lethality. Systemic coagulopathies are common in cancer patients and have been studied since Trousseau's syndrome was described in 1865. One coagulopathogenic mechanism of human tumors is abnormal upregulation of tissue factor, which can trigger the clotting cascade. Although insects do not have a similar single coagulant-initiating factor, the broad cocktail of clot-regulating proteins secreted by OC tissue suggests an analogous response in fly tumors. Indeed the data shown in this study reveal the first hypercoagulative phenotype documented in the fly. This adds to compelling evidence that the similar physiological reaction of metazoans to wounds and tumors has quite an ancient origin, with a deeper level of conservation than previously appreciated (Hsi, 2023).

Clotting complications are the second leading cause of death for cancer patients, primarily through venous thromboembolisms (VTEs), which are detected in 10%–15% of all malignancies. However, coagulopathy may also impact mortality through non-thrombotic pathways as well. Elevated markers of clotting factors in circulation are strongly correlated with poor survival, yet thrombosis is often not observed in patients. There is also some evidence that prophylactic treatment with anticoagulants can improve cancer patient outcomes beyond prevention of thrombosis. Thus, there may be unappreciated mechanisms through which altered clotting behavior contributes to morbidity and mortality. The fly, with its open circulatory system, is unlikely to be dying from tumor-induced thrombosis, and thus can be used as a discovery system for potential alternative mechanisms. This work haves not determined the exact reason why coagulopathy promotes the death of tumor-bearing flies, but the data argue against several prima facie feasible possibilities (Hsi, 2023).

The initial hypercoagulative phenotype of OC flies, followed by hypocoagulation, echoes features of patient conditions such as disseminated intravascular coagulopathy (DIC). DIC is considered a consumptive coagulopathy, where ectopic activation of pro-coagulation pathways can paradoxically lead to excessive bleeding through local depletion of hemostatic components.2 The data point to fly CCs, which lyse to provide PO clot-hardening activity, as one consumed regulator, but do not currently distinguish the relative contributions of hyper- versus hypo-coagulation to the detrimental consequences for mortality of adult tumor-bearing hosts. Future studies will explore how this simple fly system reacts to the tumor-induced danger response, perhaps by triggering inflammatory, immune, or metabolic responses that have a negative impact on host survival (Hsi, 2023).

>A tissue communication network coordinating innate immune response during muscle stress

Complex tissue communication networks function throughout an organism's lifespan to maintain tissue homeostasis. Using the genetic model Drosophila melanogaster, this study has defined a network of immune responses activated following the induction of muscle stresses, including hypercontraction, detachment, and oxidative stress. Of these stressors, loss of genes causing muscle detachment produce the strongest levels of JAK-STAT activation. In one of these mutants, fondue (fon), hemocyte recruitment and the accumulation of melanin was observed at muscle attachment sites (MASs), indicating a broad involvement of innate immune responses upon muscle detachment. Loss of fon results in pathogen-independent Toll signaling in the fat body and increased expression of the Toll-dependent antimicrobial peptide (AMP) Drosomycin. Interestingly, genetic interactions between fon and various Toll pathway components enhance muscle detachment. Finally, this study shows that JAK-STAT and Toll signaling are capable of reciprocal activation in larval tissues. A model of tissue communication is proposed for the integration of immune responses at the local and systemic level in response to altered muscle physiology (Green, 2018).

A common suite of coagulation proteins function in Drosophila muscle attachment

The organization and stability of higher order structures that form in the extracellular matrix (ECM) to mediate the attachment of muscles are poorly understood. The surprising discovery was made that a subset of clotting factor proteins are also essential for muscle attachment in the model organism Drosophila melanogaster. One such coagulation protein, Fondue (Fon), was identified as a novel muscle mutant in a pupal lethal genetic screen. Fon accumulates at muscle attachment sites and removal of this protein results in decreased locomotor behavior and detached larval muscles. A sensitized genetic background assay reveals that fon functions with the known muscle attachment genes thrombospondin (Tsp) and tiggrin (Tig). Interestingly, Tig is also a component of the hemolymph clot. It was further demonstrated that an additional clotting protein, Larval serum protein 1γ (Lsp1γ), is also required for muscle attachment stability and accumulates where muscles attach to tendons. While the local biomechanical and organizational properties of the ECM vary greatly depending on the tissue microenvironment, it is proposed that shared extracellular protein-protein interactions influence the strength and elasticity of ECM proteins in both coagulation and muscle attachment (Green, 2016). <

Coagulation and survival in Drosophila melanogaster fondue mutants

Drosophila larval coagulation factors have been identified in vitro. Better understanding of insect hemolymph coagulation calls for experiments in vivo. this study has characterized a fondue (fon) mutation and null alleles isolated by imprecise excision of a transposable element. Loss of fon was pupal lethal, but adults could be recovered by expressing the UAS::fonGFP construct of Lindgren (2008). Despite their lethality, fon mutations did not affect larval survival after wounding either when tested alone or in combination with a mutation in the hemolectin clotting factor gene. This reinforces the idea of redundant hemostatic mechanisms in Drosophila larvae, and independent pleiotropic functions of the Fondue protein in coagulation and a vital process in metamorphosis (Bajzek, 2012).

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REFERENCES

Search PubMed for articles about Drosophila fondue

Bajzek, C., Rice, A. M., Andreazza, S., Dushay, M. S. (2012). Coagulation and survival in Drosophila melanogaster fondue mutants. J Insect Physiol, 58(10):1376-1381 PubMed ID: 22885376

Green, N., Odell, N., Zych, M., Clark, C., Wang, Z. H., Biersmith, B., Bajzek, C., Cook, K. R., Dushay, M. S. and Geisbrecht, E. (2016). A common suite of coagulation proteins function in Drosophila muscle attachment. Genetics [Epub ahead of print]. PubMed ID: 27585844

Hsi, T. C., Ong, K. L., Sepers, J. J., Kim, J. and Bilder, D. (2023). Systemic coagulopathy promotes host lethality in a new Drosophila tumor model. Curr Biol 33(14): 3002-3010. PubMed ID: 37354901

Lindgren, M., Riazi, R., Lesch, C., Wilhelmsson, C., Theopold, U., Dushay, M. S. (2008). Fondue and transglutaminase in the Drosophila larval clot. J Insect Physiol, 54(3):586-592 PubMed ID: 18222466

Scherfer, C., Qazi, M. R., Takahashi, K., Ueda, R., Dushay, M. S., Theopold, U., Lemaitre, B. (2006). The Toll immune-regulated Drosophila protein Fondue is involved in hemolymph clotting and puparium formation. Dev Biol, 295(1):156-163 PubMed ID: 16690050

date revised: 6 March, 2025

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