Introduction: Chitinase-like proteins (CLPs) are associated with tissue-remodeling and inflammation but also with several disorders, including fibrosis, atherosclerosis, allergies, and cancer. However, CLP’s role in tumors is far from clear.

Methods: Here, we utilize Drosophila melanogaster and molecular genetics to investigate the function of CLPs (imaginal disc growth factors; Idgf’s) in Ras^V12 dysplastic salivary glands.

Results and discussion: We find one of the Idgf’s members, Idgf3, is transcriptionally induced in a JNK-dependent manner via a positive feedback loop mediated by reactive oxygen species (ROS). Moreover, Idgf3 accumulates in enlarged endosomal vesicles (EnVs) that promote tumor progression by disrupting cytoskeletal organization. The process is mediated via the downstream component, aSpectrin, which localizes to the EnVs. Our data provide new insight into CLP function in tumors and identifies specific targets for tumor control.

Introduction: Tumor-associated macrophages may act to either limit or promote tumor growth, yet the molecular basis for either path is poorly characterized.

Methods: We use a larval Drosophila model that expresses a dominant-active version of the Ras-oncogene (Ras^V12) to study dysplastic growth during early tumor progression. We performed single-cell RNA-sequencing of macrophage-like hemocytes to characterize these cells in tumor- compared to wild-type larvae. Hemocytes included manually extracted tumor-associated- and circulating cells.

Results and discussion: We identified five distinct hemocyte clusters. In addition to Ras^V12 larvae, we included a tumor model where the activation of effector caspases was inhibited, mimicking an apoptosis-resistant setting. Circulating hemocytes from both tumor models differ qualitatively from control wild-type cells—they display an enrichment for genes involved in cell division, which was confirmed using proliferation assays. Split analysis of the tumor models further reveals that proliferation is strongest in the caspase-deficient setting. Similarly, depending on the tumor model, hemocytes that attach to tumors activate different sets of immune effectors—antimicrobial peptides dominate the response against the tumor alone, while caspase inhibition induces a shift toward members of proteolytic cascades. Finally, we provide evidence for transcript transfer between hemocytes and possibly other tissues. Taken together, our data support the usefulness of Drosophila to study the response against tumors at the organismic level.

Fibrotic lesions accompany several pathological conditions, including tumors. We show that expression of a dominant-active form of the Ras oncogene in Drosophila salivary glands (SGs) leads to redistribution of components of the basement membrane (BM) and fibrotic lesions. Similar to several types of mammalian fibrosis, the disturbed BM attracts clot components, including insect transglutaminase and phenoloxidase. SG epithelial cells show reduced apicobasal polarity accompanied by a loss of secretory activity. Both the fibrotic lesions and the reduced cell polarity are alleviated by ectopic expression of the antimicrobial peptide drosomycin (Drs), which also restores the secretory activity of the SGs. In addition to extracellular matrix components, both Drs and F-actin localize to fibrotic lesions.

Cancer cells contain multiple biological alterations that allow them to escape from host surveillance mechanisms. One of the mechanisms that play an essential role in host protection against tumor growth is immunity. However, the immune system may act as a double-edged sword with the potential to both promote and limit tumor growth in a context-dependent manner. This involves both internal and external signaling events such as stress signaling pathways but also communication between cells and/or between cells and the extracellular matrix (ECM). In this thesis, Drosophila melanogaster (the fruit fly) was used to understand the role of two immune-related components, namely the antimicrobial peptide Drosomycin (Drs) and a chitinase-like protein (Idgf3), in a tumor model that involves a tubular organ, namely the salivary glands.

In Paper I we investigated Drs function and regulation upon expression of the oncogene Ras^V12. Initially, Drs was upregulated in the whole SG upon Ras^V12 expression. However, at the later stage of the tumor, Drs expression was restricted to the proximal region. In contrast, at the distal region, the hallmarks of cancer phenotypes, such as activation of the pro-tumorogenic JNK pathway, adhering immune cells and production of reactive oxygen species (ROS), were elevated. By overexpressing Drs in the distal region, we found that Drs interferes with most cancer hallmarks, including the JNK-pathway, recruitment of immune cells, and ROS production.

In Paper II we further characterized the hallmarks of cancer in our model system by addressing external and internal changes and whether Drs may influence them. At the extracellular compartment, we demonstrate the redistribution of the ECM in tumors, recruitment of immune components, including prophenoloxidases (PPOs) and Drs, and identified F-actin as a part of the ECM. Intracellularly, the organs' primary function, secretion, is lost, and the cell’s epithelial organization is disturbed. Drs reversed the majority of these changes.

In Paper III we addressed the role of Idgf3 and its effect on external and internal cues. Initially, we found that Idgf3 was induced in the Ras^V12 salivary glands. Upon knock-down of Idgf3, the cellular organization was restored, and tumor growth was limited. Moreover, Idgf3 expression was correlatively increasing with the progression of the tumor. In line with Paper I, we found a similar correlation with the JNK pathway. Through genetic experiments, we show JNK-mediated regulation of Idgf3 through ROS. By addressing the subcellular localization of Idgf3, we found the protein internalized within enlarged vesicles, which were coated with a cytoskeletal protein, Spectrin. Furthermore, the formation of enlarged vesicles promoted tumor progression through loss of cellular organization. Taken together, the findings presented here emphasize the complexity of the immune system and its function in tumor progression. Further studies are necessary to understand the potential for tumor therapy. 

Postmitotic tissues are incapable of replacing damaged cells through proliferation, but need to rely on buffering mechanisms to prevent tissue disintegration. By constitutively activating the Ras/MAPK-pathway via Ras(V12)-overexpression in the postmitotic salivary glands of Drosophila larvae, we overrode the glands adaptability to growth signals and induced hypertrophy. The accompanied loss of tissue integrity, recognition by cellular immunity and cell death are all buffered by blocking stress signalling through a genuine tissue-autonomous immune response. This novel, spatio-temporally tightly regulated mechanism relies on the inhibition of a feedback-loop in the JNK-pathway by the immune effector and antimicrobial peptide Drosomycin. While this interaction might allow growing salivary glands to cope with temporary stress, continuous Drosomycin expression in Ras(V12)-glands favors unrestricted hypertrophy. These findings indicate the necessity to refine therapeutic approaches that stimulate immune responses by acknowledging their possible, detrimental effects in damaged or stressed tissues.

Epithelial organ size and shape depend on cell shape changes, cell-matrix communication, and apical membrane growth. The Drosophila melanogaster embryonic tracheal network is an excellent model to study these processes. Here, we show that the transcriptional coactivator of the Hippo pathway, Yorkie (YAP/TAZ in vertebrates), plays distinct roles in the developing Drosophila airways. Yorkie exerts a cytoplasmic function by binding Drosophila Twinstar, the orthologue of the vertebrate actin-severing protein Cofilin, to regulate F-actin levels and apical cell membrane size, which are required for proper tracheal tube elongation. Second, Yorkie controls water tightness of tracheal tubes by transcriptional regulation of the d-aminolevulinate synthase gene (Alas). We conclude that Yorkie has a dual role in tracheal development to ensure proper tracheal growth and functionality.

Insects, including the fruit fly, Drosophila melanogaster are used to study a wide array of processes, many of which are known or are expected to be regulated by damage-associated molecular patterns (DAMPs). These include regenerative processes after wounding, replacement of cells by cell competition, induction of immunity and inflammation, responses against tumorous cells and neurodegeneration. Most, if not all of these processes have beneficial outcomes on organismal health but may also lead to pathologies, which often resemble those observed in humans. Drosophila offers unique opportunities to analyze and manipulate genes and pathways related to these immune consequences with high temporal and local resolution. Ultimately, such detailed analyses in the Drosophila model will aid in our understanding of the roles DAMPs play at the bifurcation between physiological and pathological outcomes in other animal species, including humans.

Chitinase-like proteins (CLPs) are associated with tissue-remodelling and inflammation but also with several disorders, including fibrosis, atherosclerosis, allergies, and cancer. However, the CLP's role in tumors is far from clear. Here, we utilize Drosophila melanogaster to investigate the function of CLPs (imaginal disc growth factors; Idgf's) in Ras^V12 dysplastic salivary glands. We find one of the Idgf's members, Idgf3, is transcriptionally induced tissue and cell-autonomously and in a non-canonical JNK-dependent manner via a positive feedback loop mediated by reactive oxygen species (ROS). Moreover, Idgf3 accumulates in enlarged endosomal vesicles (EVs) that promote tumor progression by disrupting cytoskeletal organization, independent of Rab5 and Rab11. The process is mediated via Rac1 and the downstream component, αSpectrin, localized to the EVs. Our data provide new insight into tissue-autonomous CLP function in tumors and identifies specific targets for tumor control.