scholarly journals Generation of a double binary transgenic zebrafish model to study myeloid gene regulation in response to melanocyte transformation

2017 ◽  
Author(s):  
Amy Kenyon ◽  
Daria Gavriouchkina ◽  
Giorgio Napolitani ◽  
Vincenzo Cerundolo ◽  
Tatjana Sauka-Spengler
Keyword(s):  
Immune Response ◽  
Cancer Cells ◽  
Cell Transformation ◽  
Transcriptional Profiling ◽  
Cathepsin L ◽  
Transgenic Zebrafish ◽  
Zebrafish Model ◽  
Galectin 3 ◽  
Underlying Mechanisms

ABSTRACTA complex network of inflammation succeeds somatic cell transformation and malignant disease. Immune cells and their associated molecules are responsible for detecting and eliminating cancer cells as they establish themselves as the precursors of a tumour. By the time a patient has a detectable solid tumour, cancer cells have escaped the initial immune response mechanisms. To date, no model exists for studying the underlying mechanisms that govern the initial phase of the immune response when transformed cells become precursors of cancer. Here we describe the development of a double binary zebrafish model designed for exploring regulatory programming of the myeloid cells as they respond to oncogenic transformed melanocytes. A hormone-inducible binary system allows for temporal control of different Ras-oncogenes (NRasK61Q, HRasG12V, KRasG12V) expression in melanocytes, enabling analysis of melanocyte transformation and melanoma initiation. This model was coupled to binary cell-specific biotagging models allowing in vivo biotinylation and subsequent isolation of macrophage or neutrophil nuclei for regulatory profiling of their active transcriptomes. Nuclear transcriptional profiling of neutrophils, performed for the first time as they respond to the earliest precursors of melanoma in vivo, revealed an intricate landscape of regulatory factors that may promote progression to melanoma including fgf1, fgf6, cathepsin H, cathepsin L, galectin 1 and galectin 3. The model presented here provides a powerful platform to study the myeloid response to the earliest precursors of melanoma.Summary StatementWe present an innovative double binary zebrafish model for exploring the underlying regulatory mechanisms that govern the myeloid response mechanisms at the onset of melanoma.

scholarly journals Live cell imaging of Salmonella Typhimurium interaction with zebrafish larvae after injection and immersion delivery methods

2016 ◽  
Author(s):  
Macarena A Varas ◽  
Alonso Fariña ◽  
Francisco Díaz-Pascual ◽  
Javiera Ortíz-Severín ◽  
Andrés E Marcoleta ◽  
...  
Keyword(s):  
Immune Response ◽  
Bacterial Infections ◽  
Inoculum Size ◽  
Live Cell ◽  
Transgenic Zebrafish ◽  
Human Pathogens ◽  
Zebrafish Model ◽  
Zebrafish Larvae ◽  
Serovar Typhimurium

Surrogate host models have been employed to study bacterial virulence mechanisms of important human pathogens. Particularly, zebrafish (Danio rerio) has been used to determine the role of vertebrate innate immunity during bacterial infections. The easy-to-obtain large number of embryos and optical transparency of larvae allow live cell imaging of the infection progress and the major cellular types of the innate immune system that develop during the first days of embryogenesis. In zebrafish model, microinjecting bacteria into embryos and/or larvae can cause infection. Alternatively, an infection can be generated by static immersion of larvae on a microbial suspension. Both methods differ in the mode and time of infection, inoculum size and host response. In this work, we compare the in vivo immune response induced by Salmonella enterica serovar Typhimurium (S. Typhimurium) inoculated by immersion and microinjection in zebrafish larvae. To this end, an immersion protocol using transgenic zebrafish larvae was developed for in vivo monitoring of GFP-tagged S. Typhimurium infection progress and immune response during 72 h. The infection progress was compared to that of zebrafish larvae inoculated by microinjection. Our results in zebrafish corroborate previous Salmonella virulence studies in murine models and reveal that host-pathogen interaction not only depends on the virulence of the strain, but also on the inoculation method and host conditions.

scholarly journals Live cell imaging of Salmonella Typhimurium interaction with zebrafish larvae after injection and immersion delivery methods

2016 ◽  
Author(s):  
Macarena A Varas ◽  
Alonso Fariña ◽  
Francisco Díaz-Pascual ◽  
Javiera Ortíz-Severín ◽  
Andrés E Marcoleta ◽  
...  
Keyword(s):  
Immune Response ◽  
Bacterial Infections ◽  
Inoculum Size ◽  
Live Cell ◽  
Transgenic Zebrafish ◽  
Human Pathogens ◽  
Zebrafish Model ◽  
Zebrafish Larvae ◽  
Serovar Typhimurium

Surrogate host models have been employed to study bacterial virulence mechanisms of important human pathogens. Particularly, zebrafish (Danio rerio) has been used to determine the role of vertebrate innate immunity during bacterial infections. The easy-to-obtain large number of embryos and optical transparency of larvae allow live cell imaging of the infection progress and the major cellular types of the innate immune system that develop during the first days of embryogenesis. In zebrafish model, microinjecting bacteria into embryos and/or larvae can cause infection. Alternatively, an infection can be generated by static immersion of larvae on a microbial suspension. Both methods differ in the mode and time of infection, inoculum size and host response. In this work, we compare the in vivo immune response induced by Salmonella enterica serovar Typhimurium (S. Typhimurium) inoculated by immersion and microinjection in zebrafish larvae. To this end, an immersion protocol using transgenic zebrafish larvae was developed for in vivo monitoring of GFP-tagged S. Typhimurium infection progress and immune response during 72 h. The infection progress was compared to that of zebrafish larvae inoculated by microinjection. Our results in zebrafish corroborate previous Salmonella virulence studies in murine models and reveal that host-pathogen interaction not only depends on the virulence of the strain, but also on the inoculation method and host conditions.

scholarly journals Development of BCR-ABL1 Transgenic Zebrafish Model Reproducing Chronic Myeloid Leukemia (CML) Like-Disease and Providing a New Insight into CML Mechanisms

Cells ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 445
Author(s):  
Daniela Zizioli ◽  
Simona Bernardi ◽  
Marco Varinelli ◽  
Mirko Farina ◽  
Luca Mignani ◽  
...  
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Fusion Gene ◽  
Philadelphia Chromosome ◽  
Leukemic Cells ◽  
Transgenic Zebrafish ◽  
Hematopoietic Tissue ◽  
Zebrafish Model ◽  
Altered Expression

Zebrafish has proven to be a versatile and reliable experimental in vivo tool to study human hematopoiesis and model hematological malignancies. Transgenic technologies enable the generation of specific leukemia types by the expression of human oncogenes under specific promoters. Using this technology, a variety of myeloid and lymphoid malignancies zebrafish models have been described. Chronic myeloid leukemia (CML) is a clonal myeloproliferative neoplasia characterized by the BCR-ABL1 fusion gene, derived from the t (9;22) translocation causing the Philadelphia Chromosome (Ph). The BCR-ABL1 protein is a constitutively activated tyrosine kinas inducing the leukemogenesis and resulting in an accumulation of immature leukemic cells into bone marrow and peripheral blood. To model Ph+ CML, a transgenic zebrafish line expressing the human BCR-ABL1 was generated by the Gal4/UAS system, and then crossed with the hsp70-Gal4 transgenic line. The new line named (BCR-ABL1pUAS:CFP/hsp70-Gal4), presented altered expression of hematopoietic markers during embryonic development compared to controls and transgenic larvae showed proliferating hematopoietic cells in the caudal hematopoietic tissue (CHT). The present transgenic zebrafish would be a robust CML model and a high-throughput drug screening tool.

scholarly journals Spironolactone, a Classic Potassium-Sparing Diuretic, Reduces Survivin Expression and Chemosensitizes Cancer Cells to Non-DNA-Damaging Anticancer Drugs

Cancers ◽  
2019 ◽  
Vol 11 (10) ◽  
pp. 1550 ◽  
Author(s):  
Tomomi Sanomachi ◽  
Shuhei Suzuki ◽  
Keita Togashi ◽  
Asuka Sugai ◽  
Shizuka Seino ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Kinase Inhibitors ◽  
Xenograft Model ◽  
Growth Factor Receptor ◽  
Survivin Expression ◽  
Mouse Xenograft Model ◽  
Anti Cancer ◽  
Underlying Mechanisms

Spironolactone, a classical diuretic drug, is used to treat tumor-associated complications in cancer patients. Spironolactone was recently reported to exert anti-cancer effects by suppressing DNA damage repair. However, it currently remains unclear whether spironolactone exerts combinational effects with non-DNA-damaging anti-cancer drugs, such as gemcitabine and epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs). Using the cancer cells of lung cancer, pancreatic cancer, and glioblastoma, the combinational effects of spironolactone with gemcitabine and osimertinib, a third-generation EGFR-TKI, were examined in vitro with cell viability assays. To elucidate the underlying mechanisms, we investigated alterations induced in survivin, an anti-apoptotic protein, by spironolactone as well as the chemosensitization effects of the suppression of survivin by YM155, an inhibitor of survivin, and siRNA. We also examined the combinational effects in a mouse xenograft model. The results obtained revealed that spironolactone augmented cell death and the suppression of cell growth by gemcitabine and osimertinib. Spironolactone also reduced the expression of survivin in these cells, and the pharmacological and genetic suppression of survivin sensitized cells to gemcitabine and osimertinib. This combination also significantly suppressed tumor growth without apparent adverse effects in vivo. In conclusion, spironolactone is a safe candidate drug that exerts anti-cancer effects in combination with non-DNA-damaging drugs, such as gemcitabine and osimertinib, most likely through the suppression of survivin.

scholarly journals Perturbations of BMP/TGF-β and VEGF/VEGFR signalling pathways in non-syndromic sporadic brain arteriovenous malformations (BAVM)

2018 ◽  
Vol 55 (10) ◽  
pp. 675-684 ◽  
Author(s):  
Kun Wang ◽  
Sen Zhao ◽  
Bowen Liu ◽  
Qianqian Zhang ◽  
Yaqi Li ◽  
...  
Keyword(s):  
Growth Factor ◽  
Transforming Growth Factor Beta ◽  
Arteriovenous Malformations ◽  
Transforming Growth Factor ◽  
Transgenic Zebrafish ◽  
Zebrafish Model ◽  
Brain Arteriovenous Malformations ◽  
Pathogenic Variants ◽  
Uncertain Significance

BackgroundBrain arteriovenous malformations (BAVM) represent a congenital anomaly of the cerebral vessels with a prevalence of 10–18/100 000. BAVM is the leading aetiology of intracranial haemorrhage in children. Our objective was to identify gene variants potentially contributing to disease and to better define the molecular aetiology underlying non-syndromic sporadic BAVM.MethodsWe performed whole-exome trio sequencing of 100 unrelated families with a clinically uniform BAVM phenotype. Pathogenic variants were then studied in vivo using a transgenic zebrafish model.ResultsWe identified four pathogenic heterozygous variants in four patients, including one in the established BAVM-related gene, ENG, and three damaging variants in novel candidate genes: PITPNM3, SARS and LEMD3, which we then functionally validated in zebrafish. In addition, eight likely pathogenic heterozygous variants (TIMP3, SCUBE2, MAP4K4, CDH2, IL17RD, PREX2, ZFYVE16 and EGFR) were identified in eight patients, and 16 patients carried one or more variants of uncertain significance. Potential oligogenic inheritance (MAP4K4 with ENG, RASA1 with TIMP3 and SCUBE2 with ENG) was identified in three patients. Regulation of sma- and mad-related proteins (SMADs) (involved in bone morphogenic protein (BMP)/transforming growth factor beta (TGF-β) signalling) and vascular endothelial growth factor (VEGF)/vascular endotheliual growth factor recepter 2 (VEGFR2) binding and activity (affecting the VEGF signalling pathway) were the most significantly affected biological process involved in the pathogenesis of BAVM.ConclusionsOur study highlights the specific role of BMP/TGF-β and VEGF/VEGFR signalling in the aetiology of BAVM and the efficiency of intensive parallel sequencing in the challenging context of genetically heterogeneous paradigm.

IL-37d Negatively Regulates NLRP3 Transcription via Receptor-mediated Pathway and Alleviates DSS-induced Colitis

2020 ◽  
Vol 27 (1) ◽  
pp. 84-93
Author(s):  
Yuan Li ◽  
Hongxia Chu ◽  
Mingsheng Zhao ◽  
Chaoze Li ◽  
Yetong Guan ◽  
...  
Keyword(s):  
Nlrp3 Inflammasome ◽  
Transcriptional Profiling ◽  
Inflammasome Activation ◽  
Downstream Effector ◽  
Nlrp3 Inflammasome Activation ◽  
Polymerase Chain ◽  
Underlying Mechanisms ◽  
And Control ◽  
Nlrp3 Expression

Abstract Background Interleukin-37 (IL-37) is a new negative immune regulator. It has 5 splicing forms, IL-37a–e, and most research mainly focuses on IL-37b functions in diverse diseases. Our previous research found that IL-37d inhibits lipopolysaccharide-induced inflammation in endotoxemia through a mechanism different from that of IL-37b. However, whether IL-37d plays a role in colitis and the underlying mechanisms is still obscure. Herein, we identified whether IL-37d regulates NLRP3 inflammasome activity and determined its effect on colitis. Methods NLRP3 inflammasome in macrophages from IL-37d transgenic (IL-37dtg) and control wild type (WT) mice were activated by lipopolysaccharide and adenosine 5′-triphosphate. The expression of NLRP3 inflammasome components and its downstream effector, IL-1β, were detected by real-time polymerase chain reaction, western blot, and ELISA. The models of alum-induced peritonitis and dextran sodium sulfate (DSS)-induced colitis were used to investigate the function of IL-37d on regulating the activity of NLRP3 inflammasome in vivo. Results Our results showed that the activation of NLRP3 inflammasome in macrophage and alum-induced peritonitis was inhibited by IL-37d. Strikingly, IL-37d suppressed NLRP3 expression at the priming step via inhibiting NF-κB activation by transcriptional profiling. Moreover, the recombinant protein IL-37d attenuated NLRP3 inflammasome activation and the production of IL-1β, which could be reversed by IL-1R8 knockdown. Finally, IL-37d transgenic mice resisted DSS-induced acute colitis and NLRP3 inflammasome activation. Conclusion Interleukin-37d inhibits overactivation of the NLRP3 inflammasome through regulating NLRP3 transcription in an IL-1R8 receptor-mediated signaling pathway.

scholarly journals Live imaging of neutrophil motility in a zebrafish model of WHIM syndrome

Blood ◽  
2010 ◽  
Vol 116 (15) ◽  
pp. 2803-2811 ◽  
Author(s):  
Kevin B. Walters ◽  
Julie M. Green ◽  
Jill C. Surfus ◽  
Sa Kan Yoo ◽  
Anna Huttenlocher
Keyword(s):  
Primary Immunodeficiency ◽  
Transgenic Zebrafish ◽  
Hematopoietic Tissue ◽  
Primary Immunodeficiency Disorder ◽  
Zebrafish Model ◽  
Whim Syndrome ◽  
Stromal Cell Derived Factor ◽  
Morpholino Oligonucleotides ◽  
Guanosine Triphosphatase

Abstract CXCR4 is a G protein–coupled chemokine receptor that has been implicated in the pathogenesis of primary immunodeficiency disorders and cancer. Autosomal dominant gain-of-function truncations of CXCR4 are associated with warts, hypo-gammaglobulinemia, infections, and myelokathexis (WHIM) syndrome, a primary immunodeficiency disorder characterized by neutropenia and recurrent infections. Recent progress has implicated CXCR4-SDF1 (stromal cell-derived factor 1) signaling in regulating neutrophil homeostasis, but the precise role of CXCR4-SDF1 interactions in regulating neutrophil motility in vivo is not known. Here, we use the optical transparency of zebrafish to visualize neutrophil trafficking in vivo in a zebrafish model of WHIM syndrome. We demonstrate that expression of WHIM mutations in zebrafish neutrophils induces neutrophil retention in hematopoietic tissue, impairing neutrophil motility and wound recruitment. The neutrophil retention signal induced by WHIM truncation mutations is SDF1 dependent, because depletion of SDF1 with the use of morpholino oligonucleotides restores neutrophil chemotaxis to wounds. Moreover, localized activation of a genetically encoded, photoactivatable Rac guanosine triphosphatase is sufficient to direct migration of neutrophils that express the WHIM mutation. The findings suggest that this transgenic zebrafish model of WHIM syndrome may provide a valuable tool to screen for agents that modify CXCR4-SDF1 retention signals.

scholarly journals Exosome-Derived ADAM17 Promotes Liver Metastasis in Colorectal Cancer

2021 ◽  
Vol 12 ◽  
Author(s):  
Jinbing Sun ◽  
Zhihua Lu ◽  
Wei Fu ◽  
Kuangyi Lu ◽  
Xiuwen Gu ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Metastatic Colorectal Cancer ◽  
Cancer Cells ◽  
Cancer Metastasis ◽  
Metastatic Niche ◽  
Colorectal Cancer Cells ◽  
Niche Formation ◽  
Underlying Mechanisms

Exosomes derived from cancer cells are deemed important drivers of pre-metastatic niche formation at distant organs, but the underlying mechanisms of their effects remain largely unknow. Although the role of ADAM17 in cancer cells has been well studied, the secreted ADAM17 effects transported via exosomes are less understood. Herein, we show that the level of exosome-derived ADAM17 is elevated in the serum of patients with metastatic colorectal cancer as well as in metastatic colorectal cancer cells. Furthermore, exosomal ADAM17 was shown to promote the migratory ability of colorectal cancer cells by cleaving the E-cadherin junction. Moreover, exosomal ADAM17 overexpression as well as RNA interference results highlighted its function as a tumor metastasis-promoting factor in colorectal cancer in vitro and in vivo. Taken together, our current work suggests that exosomal ADAM17 is involved in pre-metastatic niche formation and may be utilized as a blood-based biomarker of colorectal cancer metastasis.

scholarly journals Sildenafil triggers tumor lethality through altered expression of HSP90 and degradation of PKD2

2020 ◽  
Vol 41 (10) ◽  
pp. 1421-1431 ◽  
Author(s):  
Lu Chen ◽  
Yang Liu ◽  
Alexander Becher ◽  
Kristina Diepold ◽  
Evi Schmid ◽  
...  
Keyword(s):  
Tumor Growth ◽  
Cancer Cells ◽  
Hsp90 Inhibitor ◽  
Altered Expression ◽  
Cancer Subtypes ◽  
Low Concentrations ◽  
Underlying Mechanisms ◽  
Approved Drugs

Abstract The repurposing of existing drugs has emerged as an attractive additional strategy to the development of novel compounds in the fight against cancerous diseases. Inhibition of phosphodiesterase 5 (PDE5) has been claimed as a potential approach to target various cancer subtypes in recent years. However, data on the treatment of tumors with PDE5 inhibitors as well as the underlying mechanisms are as yet very scarce. Here, we report that treatment of tumor cells with low concentrations of Sildenafil was associated with decreased cancer cell proliferation and augmented apoptosis in vitro and resulted in impaired tumor growth in vivo. Notably, incubation of cancer cells with Sildenafil was associated with altered expression of HSP90 chaperone followed by degradation of protein kinase D2, a client protein previously reported to be involved in tumor growth. Furthermore, the involvement of low doses of PU-H71, an HSP90 inhibitor currently under clinical evaluation, in combination with low concentrations of Sildenafil, synergistically and negatively impacted on the viability of cancer cells in vivo. Taken together, our study suggests that repurposing of already approved drugs, alone or in combination with oncology-dedicated compounds, may represent a novel cancer therapeutic strategy.

Fishing with a Transgenic Line: Using Zebrafish to Elucidate Mechanisms and Therapeutics in NUP98-NSD1 AML

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1638-1638
Author(s):  
Corey Filiaggi ◽  
Adam P Deveau ◽  
Sergey Prykhozhij ◽  
Graham Dellaire ◽  
Jason N. Berman
Keyword(s):  
Fluorescent Protein ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasm ◽  
Homeobox Gene ◽  
Transgenic Zebrafish ◽  
Gfp Expression ◽  
Zebrafish Model ◽  
Dismal Prognosis ◽  
Marker Expression

Abstract The NUP98-NSD1 (NND1) translocation is a fusion oncogene recently identified in pediatric acute myeloid leukemia (AML), where it occurs in approximately 16% of patients. NND1 predicts a dismal prognosis, with a 4-year event-free survival <10%. The mechanism of action of NND1 may be through the activation of the posterior homeobox gene, HOXA9. NND1 patients often harbour an internal tandem duplication of fms-like tyrosine kinase 3 (FLT3-ITD), another genetic lesion associated with poor prognosis. Co-expression of NND1 and FLT3-ITD results in worse survival than either aberration in isolation. NND1 may be sufficient to produce a myeloproliferative phenotype, but the interaction with FLT3-ITD activates essential downstream signaling pathways necessary for AML pathogenesis. A better understanding of the mechanisms by which NND1 dysregulates hematopoiesis and interacts with FLT3-ITD is fundamental to developing targeted therapies to improve the outcome in this disease. The zebrafish has been established as a robust and reliable model of hematologic malignancies, with conserved genetics and ease of genetic interrogation. Our group previously generated a transgenic zebrafish model expressing the related fusion oncogene, NUP98-HOXA9, in which embryos had anemia and expansion of myeloid cells, and adult fish exhibited a myeloproliferative neoplasm (MPN). Using this model, we discovered novel downstream epigenetic regulators that could be targeted therapeutically and restore normal embryonic hematopoiesis. Moreover, the up-regulated genes that we identified correlated with features of high-risk AML in human datasets, highlighting the translational relevance of this human disease model and justifying the employment of this approach to investigate NND1-driven AML (Deveau et al, Leukemia 2015). Plasmid constructs have been generated that incorporate human NND1 into the zebrafish using the Tol2 system, with detection by green fluorescent protein (GFP) expression. Injection of CMV-NND1-sGFP revealed strong GFP expression from 24-48 hours post fertilization (hpf) ubiquitously and in hematopoietic cells. Whole-mount in situ hybridization experiments of plasmid-injected embryos have shown that, similar to the NUP98-HOXA9 model, embryos expressing NND1 develop a pre-leukemic state, with a decrease in red blood cell marker expression (gata1) and an increase in myeloid marker expression (l-plastin). Currently no animal models exist for NND1 AML. Our initial studies have revealed a myeloproliferative phenotype in zebrafish embryos, providing an in vivo tool for further genetic and epigenetic interrogation, as well as a preclinical platform for novel drug discovery in this disease. Disclosures No relevant conflicts of interest to declare.

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