Protocol to analyze Drosophila intestinal tumor cellular heterogeneity using immunofluorescence imaging and nuclear size quantification.

Drosophila intestinal tumors show an extended cellular heterogeneity. We devise a protocol to assess tumor cell heterogeneity by employing nuclear size measurement and immunofluorescence-based cell lineage analysis. We describe steps for intestinal dissection, staining, and imaging, followed by detailed procedures for nuclear size analysis. This approach detects overall heterogeneity across the entire tumor cell population and deviations within specific cell populations. The procedure is also applicable for analyzing the heterogeneity of wild-type intestinal cells in various contexts. For complete details on the use and execution of this protocol, please refer to Pranoto et al.1.

The roles of the native cell differentiation program aberrantly recapitulated in Drosophila intestinal tumors.

Many tumors recapitulate the developmental and differentiation program of their tissue of origin, a basis for tumor cell heterogeneity. Although stem-cell-like tumor cells are well studied, the roles of tumor cells undergoing differentiation remain to be elucidated. We employ Drosophila genetics to demonstrate that the differentiation program of intestinal stem cells is crucial for enabling intestinal tumors to invade and induce non-tumor-autonomous phenotypes. The differentiation program that generates absorptive cells is aberrantly recapitulated in the intestinal tumors generated by activation of the Yap1 ortholog Yorkie. Inhibiting it allows stem-cell-like tumor cells to grow but suppresses invasiveness and reshapes various phenotypes associated with cachexia-like wasting by altering the expression of tumor-derived factors. Our study provides insight into how a native differentiation program determines a tumor's capacity to induce advanced cancer phenotypes and suggests that manipulating the differentiation programs co-opted in tumors might alleviate complications of cancer, including cachexia.

Remodeling of E-cadherin subcellular localization during cell dissemination.

Given the role of E-cadherin (E-cad) in holding epithelial cells together, an inverse relationship between E-cad levels and cell invasion during the epithelial-mesenchymal transition and cancer metastasis has been well recognized. Here we report that E-cad is necessary for the invasiveness of RasV12-transformed intestinal epithelial cells in Drosophila. E-cad/ß-catenin disassembles at adherens junctions and assembles at invasive protrusions--the actin- and cortactin-rich invadopodium-like protrusions associated with the breach of the extracellular matrix (ECM)--during dissemination of RasV12-transformed intestinal epithelial cells. Loss of E-cad impairs the elongation of invasive protrusions and attenuates the ability of RasV12-transformed cells to compromise the ECM. Notably, E-cad and cortactin affect each other's localization to invasive protrusions. Given the essential roles of cortactin in cell invasion, our observations indicate that E-cad plays a role in the invasiveness of RasV12-transformed intestinal epithelial cells by controlling cortactin localization to invasive protrusions. Thus our study demonstrates that E-cad is a component of invasive protrusions and provides molecular insights into the unconventional role of E-cad in cell dissemination in vivo.

Wear and tear of the intestinal visceral musculature by intrinsic and extrinsic factors.

BACKGROUND: The gut visceral musculature plays essential roles in not only moving substances through the lumen but also maintaining the function and physiology of the gut. Although the development of the visceral musculature has been studied in multiple model organisms, how it degenerates is poorly understood. RESULTS: Here, we employ the Drosophila midgut as a model to demonstrate that the visceral musculature is disrupted by intrinsic and extrinsic factors, such as aging, feeding, chemical-induced tissue damage, and oncogenic transformation in the epithelium. Notably, we define four prominent visceral musculature disruption phenotypes, which we refer as "sprout," "discontinuity," "furcation," and "crossover" of the longitudinal muscle. Given that the occurrence of these phenotypes is increased during aging and under various stresses, we propose that these phenotypes can be used as quantitative readouts of deterioration of the visceral musculature. Intriguingly, administration of a tissue-damaging chemical dextran sulfate sodium (DSS) induced similar visceral musculature disruption phenotypes in zebrafish larvae, indicating that ingestion of a tissue-damaging chemical can disrupt the visceral musculature in a vertebrate as well. CONCLUSIONS: Our study provides insights into the deterioration of the gut visceral musculature and lays a groundwork for investigating the underlying mechanisms in Drosophila as well as other animals.

Tumors overcome the action of the wasting factor ImpL2 by locally elevating Wnt/Wingless.

Tumors often secrete wasting factors associated with atrophy and the degeneration of host tissues. If tumors were to be affected by the wasting factors, mechanisms allowing tumors to evade the adverse effects of the wasting factors must exist, and impairing such mechanisms may attenuate tumors. We use Drosophila midgut tumor models to show that tumors up-regulate Wingless (Wg) to oppose the growth-impeding effects caused by the wasting factor, ImpL2 (insulin-like growth factor binding protein [IGFBP]-related protein). Growth of Yorkie (Yki)-induced tumors is dependent on Wg while either elimination of ImpL2 or elevation of insulin/insulin-like growth factor signaling in tumors revokes this dependency. Notably, Wg augmentation could be a general mechanism for supporting the growth of tumors with elevated ImpL2 and exploited to attenuate muscle degeneration during wasting. Our study elucidates the mechanism by which tumors negate the action of ImpL2 to uphold their growth during cachexia-like wasting and implies that targeting the Wnt/Wg pathway might be an efficient treatment strategy for cancers with elevated IGFBPs.

LGK974 suppresses lipopolysaccharide-induced endotoxemia in mice by modulating the crosstalk between the Wnt/ß-catenin and NF-κB pathways.

Endotoxemia, a type of sepsis caused by gram-negative bacterial endotoxin [i.e., lipopolysaccharide (LPS)], is associated with manifestations such as cytokine storm; failure of multiple organs, including the liver; and a high mortality rate. We investigated the effect and mechanism of action of LGK974, a Wnt signaling inhibitor, in mice with LPS-induced endotoxemia, an animal model of sepsis. LGK974 significantly and dose-dependently increased the survival rate and reduced plasma cytokine levels in mice with LPS-induced endotoxemia. Transcriptome analysis of liver tissues revealed significant changes in the expression of genes associated with the Wnt pathway as well as cytokine and NF-κB signaling during endotoxemia. LGK974 treatment suppressed the activation of NF-κB signaling and cytokine expression as well as the Wnt/ß-catenin pathway in the livers of endotoxemic mice. Coimmunoprecipitation of phospho-IκB and ß-transducin repeat-containing protein (ß-TrCP) was increased in the livers of endotoxemic mice but was reduced by LGK974 treatment. Moreover, LGK974 treatment decreased the coimmunoprecipitation and colocalization of ß-catenin and NF-κB, which were elevated in the livers of endotoxemic mice. Our results reveal crosstalk between the Wnt/ß-catenin and NF-κB pathways via interactions between ß-TrCP and phospho-IκB and between ß-catenin and NF-κB during endotoxemia. The results of this study strongly suggest that the crosstalk between the Wnt/ß-catenin and NF-κB pathways contributes to the mutual activation of these two pathways during endotoxemia, which results in amplified cytokine production, liver damage and death, and that LGK974 suppresses this vicious amplification cycle by reducing the crosstalk between these two pathways.

Paeonia lactiflora extract suppresses cisplatin-induced muscle wasting via downregulation of muscle-specific ubiquitin E3 ligases, NF-κB signaling, and cytokine levels.

ETHNOPHARMACOLOGICAL RELEVANCE: The dried root of Paeonia lactiflora Pall. (Radix Paeoniae) has been traditionally used to treat various inflammatory diseases in many Asian countries. AIM OF THE STUDY: Cisplatin is a broad-spectrum anticancer drug used in diverse types of cancer. However, muscle wasting is a common side effect of cisplatin chemotherapy. This study aimed to elucidate the effects of an ethanol extract of the root of Paeonia lactiflora Pall. (Radix Paeoniae, RP) on cisplatin-induced muscle wasting along with its molecular mechanism. MATERIAL AND METHODS: C57BL/6 mice were intraperitoneally injected with cisplatin and orally treated with RP. Megestrol acetate was used as a comparator drug. Skeletal muscle mass was measured as the weight of gastrocnemius and quadriceps muscles, and skeletal muscle function was measured by treadmill running time and grip strength. Skeletal muscle tissues were analyzed by RNAseq, western blotting, ELISA, and immunofluorescence microscopy. RESULTS: In mice treated with cisplatin, skeletal muscle mass and skeletal muscle function were significantly reduced. However, oral administration of RP significantly restored skeletal muscle mass and function in the cisplatin-treated mice. In the skeletal muscle tissues of the cisplatin-treated mice, RP downregulated NF-κB signaling and cytokine levels. RP also downregulated muscle-specific ubiquitin E3 ligases, resulting in the restoration of myosin heavy chain (MyHC) and myoblast determination protein (MyoD), which play crucial roles in muscle contraction and muscle differentiation, respectively. CONCLUSION: RP restored skeletal muscle function and mass in cisplatin-treated mice by restoring the muscle levels of MyHC and MyoD proteins via downregulation of muscle-specific ubiquitin E3 ligases as well as muscle NF-κB signaling and cytokine levels.

Dissemination of RasV12-transformed cells requires the mechanosensitive channel Piezo.

Dissemination of transformed cells is a key process in metastasis. Despite its importance, how transformed cells disseminate from an intact tissue and enter the circulation is poorly understood. Here, we use a fully developed tissue, Drosophila midgut, and describe the morphologically distinct steps and the cellular events occurring over the course of RasV12-transformed cell dissemination. Notably, RasV12-transformed cells formed the Actin- and Cortactin-rich invasive protrusions that were important for breaching the extracellular matrix (ECM) and visceral muscle. Furthermore, we uncovered the essential roles of the mechanosensory channel Piezo in orchestrating dissemination of RasV12-transformed cells. Collectively, our study establishes an in vivo model for studying how transformed cells migrate out from a complex tissue and provides unique insights into the roles of Piezo in invasive cell behavior.

The role of translationally controlled tumor protein in proliferation of Drosophila intestinal stem cells.

Translationally controlled tumor protein (TCTP) is a highly conserved protein functioning in multiple cellular processes, ranging from growth to immune responses. To explore the role of TCTP in tissue maintenance and regeneration, we employed the adult Drosophila midgut, where multiple signaling pathways interact to precisely regulate stem cell division for tissue homeostasis. Tctp levels were significantly increased in stem cells and enteroblasts upon tissue damage or activation of the Hippo pathway that promotes regeneration of intestinal epithelium. Stem cells with reduced Tctp levels failed to proliferate during normal tissue homeostasis and regeneration. Mechanistically, Tctp forms a complex with multiple proteins involved in translation and genetically interacts with ribosomal subunits. In addition, Tctp increases both Akt1 protein abundance and phosphorylation in vivo. Altogether, Tctp regulates stem cell proliferation by interacting with key growth regulatory signaling pathways and the translation process in vivo.