Transgenic Zebrafish Research Articles

A Review on Unlocking Toxicity: How Zebrafish embryos Help Ensure Safe Chemicals

Aquatic ecotoxicity testing has benefited greatly from zebrafish embryo toxicity testing (ZFET) because of its high throughput, low cost, sensitivity to a variety of toxicants, and ethical issues. This study highlights the benefits of ZFET over conventional fish toxicity testing, such as its capacity to detect teratogenic defects and possible long-term impacts. The limitations of ZFET are also addressed, notably its emphasis on acute dosages and difficulties with interspecies extrapolation. In addition to the generation of in vitro alternatives utilizing zebrafish cell lines, recent developments in ZFET technology are also highlighted, including the use of transgenic zebrafish lines and high-throughput screening techniques. The paper's conclusion includes a discussion of potential prospects for ZFET research. These include improving interspecies extrapolation methods, optimizing chronic toxicity assessment procedures, and integrating ZFET with other ecotoxicity testing strategies. ZFET is a great instrument for environmental protection and a crucial component in avoiding chemical contamination of aquatic ecosystems, so long as it is acknowledged for what it is and is open to future improvement

Characterization of a novel fast-growing zebrafish: a new approach to growth hormone transgenesis

The manipulation of the somatotropic axis, governing growth, has been a focus of numerous transgenic approaches aimed at developing fast-growing fish for research, medicine and aquaculture purposes. However, the excessively high growth hormone (GH) levels in these transgenic fish often result in deformities that impact both fish health and consumer acceptance. In an effort to mitigate these issues and synchronize exogenous GH expression with reproductive processes, we employed a novel transgenic construct driven by a tilapia luteinizing hormone (LH) promoter. This approach was anticipated to induce more localized and lower exogenous GH secretion. In this study, we characterized the growth and reproduction of these transgenic LHp-GH zebrafish using hormonal and physiological parameters. Our findings reveal that LHp-GH fish exhibited accelerated growth in both length and weight, along with a lower feed conversion ratio, indicating more efficient feed utilization, all while maintaining unchanged body proportions. These fish demonstrated higher expression levels of LH and GH in the pituitary and elevated IGF-1 levels in the liver compared to wild-type fish. An examination of reproductive function in LHp-GH fish unveiled lower pituitary LH and FSH contents, smaller follicle diameter in female gonads, and reduced relative fecundity. However, in transgenic males, neither the distribution of spermatogenesis stages nor sperm concentrations differed significantly between the fish lines. These results suggest that coupling exogenous GH expression with endogenous LH expression in females directs resource investment toward somatic growth at the expense of reproductive processes. Consequently, we conclude that incorporating GH under the LH promoter represents a suitable construct for the genetic engineering of commercial fish species, providing accelerated growth while preserving body proportions.

Design and Synthesis of Novel N-Benzylidene Derivatives of 3-Amino-4-imino-3,5-dihydro-4H-chromeno[2,3-d]pyrimidine under Microwave, In Silico ADME Predictions, In Vitro Antitumoral Activities and In Vivo Toxicity

The synthesis of a series of new N-benzylidene derivatives of 3-amino-4-imino-3,5-dihydro-4H-chromeno[2,3-d]pyrimidine 10(a-l) bearing two points of molecular diversity is reported. These new compounds were synthesized in five steps including two steps under microwave dielectric heating. They were fully characterized using 1H and 13C NMR, FTIR and HRMS. The in silico physicochemical properties of compounds 10(a-l) were determined according to Lipinski’s rules of five (RO5) associated with the prediction of their bioavailability. These new compounds 10(a-l) were tested for their antiproliferative activities in fibroblasts and eight representative human tumoral cell lines (Huh7 D12, Caco2, MDA-MB231, MDA-MB468, HCT116, PC3, MCF7 and PANC1). Among them, the compounds 10h and 10i showed sub-micromolar cytotoxic activity on tumor cell lines (0.23 < IC50 < 0.3 μM) and no toxicity on fibroblasts (IC50 > 25 μM). A dose-dependent inhibition of Store-Operated Ca+2 Entry (SOCE) was observed in the HEK293 cell line with 10h. In vitro embryotoxicity and angiogenesis on the mCherry transgenic zebrafish line showed that 10h presented no toxic effect and no angiogenic effect on embryos with a dose of 5 μM at 72 hpf.

Real-Time Observation of Germinal Vesicle Migration During Oocyte Meiotic Cell Division Using Ovarian Fluorescent Transgenic Zebrafish.

The transgenic (TG) zebrafish allows researchers to bio-image specific biological phenomena in cells and tissues in vivo. We established TG lines to monitor changes in the ovaries of live fish. The original TG line with ovarian fluorescence was occasionally established. Although the cDNA integrated into the line was constructed for the expression of enhanced green fluorescent protein (EGFP) driven by the medaka β-actin promoter, the expression of EGFP is restricted to the oocytes and gills in adult fish. Furthermore, we found that germinal vesicles (GVs) in oocytes of the established line can be observed by relatively strong fluorescence around the GV. In this study, we tried to capture the dynamic processes of germinal vesicle breakdown (GVBD) during meiotic cell division using the GV fluorescent oocytes. As a result, GV migration and GVBD could be monitored in real time. We also succeeded in observing actin filaments involved in the migration of GV to the animal pole. This strain can be used for education in the process of oocyte meiotic cell division.

Development of an Undergraduate Cell Biology Laboratory to Assess Pigmentation and Cell Size in a Zebrafish Model of Uveal Melanoma.

This study outlines a 2-week laboratory module for an authentic cell biology undergraduate research experience that uses zebrafish (Danio rerio), a popular model organism for research. Previous research has indicated that course-based undergraduate research experiences such as this one increase student confidence, active learning, and retention. During this research experience, students investigate variations in pigmentation in the caudal fins of wild type (WT) and transgenic fish [Tg(mitfa:GNAQQ209L)]. The transgenic fish express a hyperactive Gα protein, GNAQQ209L, under the melanocyte-specific mitfa promoter, offering insights into uveal melanoma, a common eye cancer. Students specifically analyze the black pigmented cells, melanophores, within the caudal fin. We determined that the transgenic zebrafish have increased pigmentation in their caudal fins, but smaller melanophores. These results suggest there are more melanophores in the Tg(mitfa:GNAQQ209L) fish compared to the WT. Future undergraduate research could investigate these cellular differences. This research experience imparts microscopy and image analysis skills and instills the ability to grapple with large datasets, statistical tests, and data interpretation in alignment with biology education principles. Post-laboratory surveys reveal students attain confidence in the above skills and in handling animals, along with a deeper appreciation for model organism research and its relevance to cancer cell biology.

Rapid identifying of COX-2 inhibitors from turmeric (Curcuma longa) by bioaffinity ultrafiltration coupled with UPLC-Q Exactive-Orbitrap-MS and zebrafish-based in vivo validation

Turmeric (Curcuma longa), a typical source with recognized anti-inflammatory activity, is one such medicine-food homology source, yet its anti-inflammatory mechanisms and specific component combinations remain unclear. In this study, a net fishing method combining bio-affinity ultrafiltration and ultra-high performance liquid chromatography-mass spectrometry (AUF-LC/MS) was employed and 13 potential COX-2 inhibitors were screened out from C. longa. 5 of them (C1, 17, 20, 22, 25) were accurately isolated and identified. Initially, their IC50 values were measured (IC50 of C1, 17, 20, 22 and 25 is 55.08, 48.26, 29.13, 111.28 and 150.48 μM, respectively), and their downregulation of COX-2 under safe concentrations (400, 40, 120, 50 and 400 μM for C1, 17, 20, 22 and 25, respectively) was confirmed on RAW 264.7 cells. Further, in transgenic zebrafish (Danio rerio), significant anti-inflammatory activity at safe concentrations (15, 3, 1.5, 1.5 and 3 μg/mL for C1, 17, 20, 22 and 25, respectively) were observed in a dose-dependent manner. More importantly, molecular docking analysis further revealed the mode of interaction between them and the key active site residues of COX-2. This study screened out and verified unreported COX-2 ligands, potentially accelerating the discovery of new bioactive compounds in other functional foods.

An antitumor fungal polysaccharide from Fomitopsis officinalis by activating immunity and inhibiting angiogenesis

Macrofungi, a class of unique natural resources, are gaining popularity owing to their potential therapeutic benefits and edibility. From Fomitopsis officinalis, a medicinal macrofungus with anticancer activity, a homogeneous heteropolysaccharide (FOBP50–1) with a molecular weight of 2.21 × 104 g/mol has been extracted and purified. FOBP50–1 was found to be composed of 3-O-methylfucose, fucose, mannose, glucose, and galactose with a ratio of 1: 6.5: 4.4: 8.1: 18.2. The sugar fragments and structure of FOBP50–1 were investigated, which included →6)-α-d-Galp-(1→, →2,6)-α-d-Galp-(1→, →3)-α-l-Fucp-(1→, α-d-Glcp-(1→, →3)-β-d-Manp-(1→, →6)-β-d-Manp-(1→, 3-O-Me-α-l-Fucp-(1→, according to the UV, FT-IR, GC–MS, and NMR data. Besides the structure elucidation, FOBP50–1 showed promising antitumor activity in the zebrafish assays. The following mechanism examination discovered that FOBP50–1 interacted with TLR-4, PD-1, and VEGF to activate immunity and inhibit angiogenesis according to a series of cell, transgenic zebrafish, and surface plasmon resonance (SPR) experiments. The KD values indicating the association of FOBP50–1 with TLR-4, PD-1, and VEGF, were 4.69 × 10−5, 7.98 × 10−6, 3.04 × 10−6 M, respectively, in the SPR experiments. All investigations have demonstrated that the homogenous fungal polysaccharide FOBP50–1 has the potential to be turned into a tumor immunotherapy agent.

Exposure to acifluorfen induces developmental toxicity in the early life stage of zebrafish

Acifluorfen, a selective herbicide from the diphenyl ether family, targets broad leaf weeds. Diphenyl ether inhibits chlorophyll production in green plants by inhibiting protoporphyrinogen oxidase (PPO), causing cellular damage. Despite its known impacts on plants, the influence of acifluorfen on zebrafish embryo development remains unclear. In this study, we explored the LC50 of acifluorfen in early-stage wild-type zebrafish, determining it to be 54.99 mg/L. Subsequent examinations revealed morphological changes in zebrafish, including reduced body length. Using the cmlc2:dsRED transgenic model, we observed heart dysfunction in acifluorfen-exposed zebrafish, marked by an enlarged heart area, edema, and decreased heart rate. In response to dose-dependent acifluorfen exposure, the inhibition of angiogenesis in the brain was observed in transgenic zebrafish models (fli1a:eGFP). Organ malformations, specifically in the liver and pancreas, were noted, in lfabp:dsRED;elastase:eGFP transgenic models, indicating reduced organ size in acifluorfen-exposed zebrafish. Furthermore, acifluorfen heightened the expression of apoptosis-related genes (casp8, casp9, and tp53) in zebrafish embryos. We then determined whether acifluorfen affected the viability of zebrafish liver (ZFL) cells based on its effects on liver development in vivo. The results indicated that the proliferation of ZFL cells decreased significantly in a dose-dependent manner. Additionally, acifluorfen-treated ZFL cells exhibited a slight increase in apoptotic cells stained with annexin V and propidium iodide. In summary, these findings establish a baseline concentration for acifluorfen's effects on aquatic ecosystems and non-target organisms.

Myclobutanil induces neurotoxicity by activating autophagy and apoptosis in zebrafish larvae (Danio rerio)

Myclobutanil (MYC), a typical broad-spectrum triazole fungicide, is often detected in surface water. This study aimed to explore the neurotoxicity of MYC and the underlying mechanisms in zebrafish and in PC12 cells. In this study, zebrafish embryos were exposed to 0, 0.5 and 1 mg/L of MYC from 4 to 96 hours post fertilization (hpf) and neurobehavior was evaluated. Our data showed that MYC decreased the survival rate, hatching rate and heart rate, but increased the malformation rate and spontaneous movement. MYC caused abnormal neurobehaviors characterized by decreased swimming distance and movement time. MYC impaired cerebral histopathological morphology and inhibited neurogenesis in HuC:egfp transgenic zebrafish. MYC also reduced the activities of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), and downregulated neurodevelopment related genes (gfap, syn2a, gap43 and mbp) in zebrafish and PC12 cells. Besides, MYC activated autophagy through enhanced expression of the LC3-II protein and suppressed expression of the p62 protein and autophagosome formation, subsequently triggering apoptosis by upregulating apoptotic genes (p53, bax, bcl-2 and caspase 3) and the cleaved caspase-3 protein in zebrafish and PC12 cells. These processes were restored by the autophagy inhibitor 3-methyladenine (3-MA) both in vivo and in vitro, indicating that MYC induces neurotoxicity by activating autophagy and apoptosis. Overall, this study revealed the potential autophagy and apoptosis mechanisms of MYC-induced neurotoxicity and provided novel strategies to counteract its toxicity.

Tributyltin-induced oxidative stress causes developmental damage in the cardiovascular system of zebrafish (Danio rerio)

Tributyltin (TBT) can be used as an antifouling agent with anticorrosive, antiseptic and antifungal properties and is widely used in wood preservation and ship painting. However, it has recently been found that TBT can be harmful to aquatic organisms. In this study, to gain insight into the effects of TBT with respect to the development of the cardiovascular system in zebrafish embryos, zebrafish embryos were exposed to different concentrations of TBT solutions (0.2 μg/L, 1 μg/L, and 2 μg/L) at 2 h post-fertilization (hpf) TBT exposure resulted in decreased hatchability and heart rate, deformed features such as pericardial edema, yolk sac edema, and spinal curvature in zebrafish embryos, and impaired heart development. Expression of cardiac development-related genes (vmhc, myh6, nkx2.5, tbx5a, gata4, tbx2b, nppa) is dysregulated. Transgenic zebrafish Tg (fli1: EGFP) were used to explore the effects of TBT exposure on vascular development. It was found that TBT exposure could lead to impaired development of intersegmental vessels (ISVs), common cardinal vein (CCV), subintestinal vessels (SIVs) and cerebrovascular. The expression of vascular endothelial growth factor (VEGF) signaling pathway-related genes (flt1, flt4, kdr, vegfa) was downregulated. Biochemical indices showed that ROS and MDA levels were significantly elevated and that SOD and CAT activities were significantly reduced. The expression of key genes for prostacyclin synthesis (pla2, ptgs2a, ptgs2b, ptgis, ptgs1) is abnormal. Therefore, it is possible that oxidative stress induced by TBT exposure leads to the blockage of arachidonic acid (AA) production in zebrafish embryos, which affects prostacyclin synthesis and consequently the normal development of the heart and blood vessels in zebrafish embryos.

Involvement of Glucosamine 6 Phosphate Isomerase 2 (GNPDA2) Overproduction in β-Amyloid- and Tau P301L-Driven Pathomechanisms

Alzheimer’s disease (AD) is a neurodegenerative olfactory disorder affecting millions of people worldwide. Alterations in the hexosamine- or glucose-related pathways have been described through AD progression. Specifically, an alteration in glucosamine 6 phosphate isomerase 2 (GNPDA2) protein levels has been observed in olfactory areas of AD subjects. However, the biological role of GNPDA2 in neurodegeneration remains unknown. Using mass spectrometry, multiple GNPDA2 interactors were identified in human nasal epithelial cells (NECs) mainly involved in intraciliary transport. Moreover, GNPDA2 overexpression induced an increment in NEC proliferation rates, accompanied by transcriptomic alterations in Type II interferon signaling or cellular stress responses. In contrast, the presence of beta-amyloid or mutated Tau-P301L in GNPDA2-overexpressing NECs induced a slowdown in the proliferative capacity in parallel with a disruption in protein processing. The proteomic characterization of Tau-P301L transgenic zebrafish embryos demonstrated that GNPDA2 overexpression interfered with collagen biosynthesis and RNA/protein processing, without inducing additional changes in axonal outgrowth defects or neuronal cell death. In humans, a significant increase in serum GNPDA2 levels was observed across multiple neurological proteinopathies (AD, Lewy body dementia, progressive supranuclear palsy, mixed dementia and amyotrophic lateral sclerosis) (n = 215). These data shed new light on GNPDA2-dependent mechanisms associated with the neurodegenerative process beyond the hexosamine route.

Abstract 1432: Development of transparent transgenic zebrafish model to study CNS microglia and NF-kB activity in glioblastoma

Abstract Microglia are resident macrophages of the central nervous system (CNS) and have crucial roles in normal brain homeostasis, function, and immune response. Microglia play key roles in the healthy brain, ranging from phagocytosis of dying neurons and CNS debris to modulating adaptability. The role of microglia has been well documented but their specific contributions to the development and progression of cancers, such as glioblastoma, have recently garnered wide attention. The zebrafish have proven novel tool for modeling innate immune responses. A macrophage-specific marker mpeg1 and its promoter are used in mpeg1-driven transgenes. mpeg1-driven transgenes mpeg1:mCherryFP are expressed in macrophage-lineage cells that do not express neutrophil-marking transgenes. These zebrafish transgenes contribute to the fields of inflammation, infection, and leukocyte biology. We have recently developed a novel transparent transgenic homozygous strain Tg(6xNF-kB:EGFP); Casper(roy−/−, nacre−/−) of zebrafish to the in-vivo study of NF-kB activity under normal and inflammation stage at the cellular, organ-specific and whole organism level. To perform an in-vivo study of microglial function, we are developing Casper;mpeg1:mCherryFP microglia specific strain and integrating with newly developed Tg(6xNF-kB:EGFP); Casper(roy−/−, nacre−/−) strain through crossbreeding of Casper and transgenic zebrafish. We have generated F1 generation of newly developed strains of Casper;mpeg1:mCherryFP and Tg(6xNF-kB:EGFP);Casper. The presence of fluorescence mCherry and GFP, in crossbred progeny was identified through the screening of larvae at 72 HPF stage. The expression of these fluorescent proteins determines the presence of the reporter genes. F1 generation confirmed heterozygous and allowed them to grow into adults to breed transparent transgenic strain to get F2 generation. This process will be continued to F2, F3, F4, and F5 generation until achieving homozygous progeny. This model allows for in-vivo examination of microglia functional morphology by using fluorescent and confocal microscopy. This novel transgenic model will be applied to identify novel anti-inflammatory drug inhibitors in NF-kB pathophysiology for glioblastoma and cardio-oncology experimental therapeutics. Citation Format: Surendra K. Rajpurohit, Sai Nasanally, Simran Hotwani, Taitum Gossman, Keshu Bhatt, Aiden Van Derhei, Rohan Pasi, Devan-Anmol Manhiani, Kirk Kimmerling, Balakrishna L. Lokeshwar. Development of transparent transgenic zebrafish model to study CNS microglia and NF-kB activity in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1432.

Abstract 1431: Generation of transparent transgenic zebrafish strain to study cardiovascular functionin translational cardio-oncology research

Abstract Transgenic zebrafish lines have been extensive use in biomedical research and their primary applications. The zebrafish has proven to be an ideal tool for direct In-vivo observations of developmental processes. In vertebrates, including zebrafish and humans, the ability to obtain high-resolution, in-vivo imaging is limited due to the natural opacification of skin and subdermal structures. To conduct in-vivo whole organism imaging studies, skin transparency is a primary requirement, necessitating blocking of pigmentation. Zebrafish casper mutant maintains skin transparency throughout its life, offering the ideal combination of sensitivity and resolution for in-vivo analyses and imaging. Our Laboratory is developing a novel transparent transgenic zebrafish model to address basic and translational cardio-oncology and experimental therapeutics. The fli1 promoter in zebrafish having an enhanced expression reporter gene of green fluorescent protein, GFP. This model allows for the in-vivo examination of the vascular system by using fluorescent and confocal microscopy. In this study, we developed transparent transgenic strain of Caspe/fli1:EGFP and integrating with recently developed casper/myl7/annexin-5:YFP and casper/fli1:EGFP strain through crossbreeding of casper and transgenic zebrafish. We have generated F1 generation of newly developed strains of Caspe/fli1:EGFP and casper/myl7/annexin-5:YFP and casper/fli1:EGFP. The presence of fluorescence expressions RFP, YFP and GFP, in crossbred progeny identified through the screening of larvae at 72 HPF stage. Expression of these fluorescent proteins determines the presence of the reporter genes. Positive transgenic larvae validated by confocal microscopy. The progenies of the F1 generation are heterozygous in nature and allow them to grow into adults to breed transparent transgenic strain to get F2 generation. This process will be continued to F2, F3, F4 and F5 generation until obtain homozygous progeny of newly developed strain. The development of casper/myl7/annexin-5:YFP x casper/fli1:EGFP will aid new dimensions in Cardio-oncology research for cardiac injury and Vasotoxicity induced by anticancer drug treatment in cancer patients. Citation Format: Surendra K. Rajpurohit, Taitum Gossman, Rohan Pasi, Sai Nasanally, Keshu Bhatt, Aiden Van Derhei, Simaran Hotwani, Devan-Anmol Manhiani, Kirk Kimmerling, Vishal Arora, Balakrishna L. Lokeshwar. Generation of transparent transgenic zebrafish strain to study cardiovascular functionin translational cardio-oncology research [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1431.

Abstract 149: Modeling patient-specific CHEK2 genetic variants associated with high-risk neuroblastoma

Abstract Background: Neuroblastoma (NB) is the most common extra-cranial solid tumor in children and accounts for approximately 10% of childhood cancer deaths. Recently, next-generation sequencing of patients enrolled in the SickKids Cancer Sequencing (KiCS) program revealed somatic and germline alterations in genes involved in the DNA damage response (DDR). Multiple germline and somatic pathogenic variants in CHEK2 were detected in patients with NB. Aims and Methods: To better understand potential roles for CHEK2 in NB pathogenesis, we are modeling patient-derived CHEK2 variants in an established transgenic zebrafish model of MYCN-driven NB. Using a CRISPR/Cas9 “knock-in” approach, we have generated stable zebrafish lines that are homozygous and heterozygous for variants of interest with either null, heterozygous, or wild-type tp53 functional status. We aim to assess tumor incidence, growth, and metastasis using direct visualization of disseminated EGFP+ tumor cells, as compared to MYCN over-expression only control fish. Given potential drug sensitivities, we also plan to assess the efficacy of various DDR pathway inhibitors in combination with frontline chemotherapies, which are effective in the treatment of DDR-deficient adult cancers. Results: Targeting chek2 in a tp53 null background had no significant effect on tumor incidence and growth compared to tp53 mutant control animals (p=0.1570 log-rank test). This contrasts with other patient-specific DDR models including gene targeting brca2 (p=0.0235, log-rank test), atm (p<0.001, log-rank test), and bard1 (p<0.001, log-rank test), potentially highlighting Tp53-dependent roles for Chek2 in NB formation in vivo. Through functional characterization of patient-specific CHEK2 variants in NB progression, our work will provide important preclinical information that will inform future diagnostics and therapeutic strategies for patients with high-risk NB. Citation Format: Xueting Xiong, Sarah Cohen-Gogo, Anita Villani, Adam Shlien, Meredith S. Irwin, Madeline Hayes. Modeling patient-specific CHEK2 genetic variants associated with high-risk neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 149.

Abstract 259: Zebrafish empowered large-scale drug screen to uncover novel targets of self-renewal in T-cell acute lymphoblastic leukemia initiating cells

Abstract T-cell Acute Lymphoblastic Leukemia (T-ALL) patients who experience relapse have a dismal prognosis, with 5-year overall survival rates of 10% for adults and 36% for children. Relapse is attributed to cytotoxic chemotherapy’s inability to target Leukemia Initiating Cells (LICs) that have the potential to re-populate the cancer. There is a need for novel therapeutic strategies to target LIC self-renewal in T-ALL with improved safety profiles and better chances of transition to clinical development. This project utilized a transgenic zebrafish model that accurately recapitulates the most aggressive form of the human T-ALL. This model offered several advantages, including a high frequency of LICs and suitability for drug testing. Over 770 FDA-approved drugs were screened, in vivo using >2,500 syngeneic CG1 zebrafish. Further secondary screening in human T-ALL cells narrowed down the potential hits into 4 compounds that specifically interfere with self-renewal. Finally, the hit drug Amiloride was confirmed by a limiting dilation assay (LDA), resulting in a more than 4-fold decrease in the LIC frequency (p=0.0026). The top hit, Amiloride, which reduced the frequency of LICs in zebrafish and human T-ALL cells, is an inhibitor of the Sodium Hydrogen Exachanger-1 (NHE1). NHE1 has been previously linked to the proliferation, migration, and drug resistance of different cancers. In addition, Amiloride treatment was found to impair mitochondrial function in cancer cells, offering a possible mechanism for the inhibition of LICs. Research has not yet investigated how NHE1, and the altered mitochondrial dynamics related to its inhibition affect the development and persistence of LICs in T-ALL. In our human T-ALL cells, we found that Amiloride significantly reduced mitochondrial basal respiration (p=0.0074), ATP production (p=0.0008) and increased the expression of genes associated with mitochondrial stress such as TFAM, PINK1, PARKIN and MT-CO1. Experiments using shRNA mediated knock down of the NHE1 in human T-ALL cells are underway and will be followed by investigating the impact of NHE1 inhibition on primary patient-derived T-ALL cells in a mouse model. Overall, this project showcases of the power of the zebrafish in cancer research in an efficient and accurate large-scale screen for drugs that impact LIC self-renewal. We have identified Amiloride as a top hit that reduces the frequency of LIC in vivo and in vitro. Finally, we are continuing to investigate the role of the NHE1 in self-renewal in human T-ALL and the associated mitochondrial alterations. Citation Format: Majd Al-Hamaly, Yelena Chernyavskaya, Emma Arvin, Logan Turner, Meghan Haney, Jessica Blackburn. Zebrafish empowered large-scale drug screen to uncover novel targets of self-renewal in T-cell acute lymphoblastic leukemia initiating cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 259.

Abstract 1430: Development of transparent transgenic zebrafish model to study anti-cancer drug induced in vivo cardiotoxicity

Abstract Cardiotoxicity is the second leading cause of death among cancer survivors and can manifest as arrhythmia, acute coronary syndrome, acute heart failure, or pericarditis/myocarditis. Chronic myocyte damage and apoptosis can transition from an asymptomatic phase to adaptive myocardial hypertrophy, followed by decreased left ventricular ejection fraction, and eventual heart failure. Cardio-oncology is an emerging field that studies cancer therapy-associated cardiotoxicities. The development of reliable and effective preclinical models for cardiotoxicity prediction and prevention is essential to improving the long-term cardiac health of cancer patients. Zebrafish are a promising animal model for the field of cardio-oncology and have emerged as an invaluable tool for investigating genetic and molecular aspects of cardiovascular research. Zebrafish heart cells are like human heart cells at the molecular level, making them an ideal system for studying the genetic underpinning of cardiac function. Zebrafish myl7 gene, a myosin light chain-7 gene, is a regulatory gene of heart orthologs to human MYL7. In-vivo imaging is limited due to the opacification of skin and subdermal structures. To conduct in-vivo whole organism imaging studies, skin transparency is a primary requirement, necessitating the blocking of pigmentation. The Zebrafish Casper mutant maintains skin transparency throughout its life, offering the ideal combination of sensitivity and resolution for in-vivo analyses and imaging. In this study, we have developed a novel transparent transgenic zebrafish model and established time-lapse in-vivo confocal microscopy to study cellular phenotype/pathologies of cardiomyocytes. The newly developed homozygous transparent transgenic Casper/myl7:RFP; annexin-5:YFP/NF-kB: GFP/mpeg1:mcherryFP, allows us to study cell death process (Annexin5) and inflammatory activity (NF-kB) in microglia and cardiomyocytes. We have successfully established in vivo fluorescence and confocal microscopy in this newly developed strain, enabling the study of cellular morphology and function changes under various conditions. This will allow us to elucidate cell death patterns, and cardio-inflammatory and cardiotoxicity pathways. Citation Format: Surendra K. Rajpurohit, Taitum Gossman, Sai Nasanally, Keshu Bhatt, Aiden Van Derhei, Simran Hotwani, Devan-Anmol Manhiani, Rohan Pasi, Jiaqi Mi, Vishal Arora, Balakrishna Lokeshwar. Development of transparent transgenic zebrafish model to study anti-cancer drug induced in vivo cardiotoxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1430.

Abstract 1994: Drug-induced cancer cell secretome promotes resistance in colon cancer

Abstract Colorectal cancer (CRC) is the third most common cancer in the world, with a projection of increased lethality in the upcoming years. Adjuvant chemotherapy with biological agents is the most practiced standard of care today. However, patients with metastatic CRC (mCRC) do not benefit from the wide range of therapies available for CRC, which either owe to unresectability or co-morbidity or more importantly resistance to therapy. 5-fluorouracil (5-FU) combined with leucovorin, oxaliplatin, or irinotecan (FOLFOX or FOLFIRI) has been routinely employed as the first-line therapy in colon cancer, but patients often develop resistance to 5-FU. Additionally, tumor cell dormancy plays a major role in tumor relapse which leads to poor prognosis in the patients. Despite many advances, the understanding of therapy-induced resistance is still in its infancy. Here, we show that 5-FU-induced tumor cell secretome helps in the outgrowth and metastasis of 5-FU-resistant clones in colon cancer. We used parental and 5-FU-resistant colon cancer cell lines, and transgenic zebrafish embryos to test the hypothesis and perform the colon cancer cell-based functional assays. We observed that 5-FU resistant (5-FU-R) colon cancer cells showed an increase in proliferation and survival when cultured with the secretome of 5-FU exposed colon cancer cells compared to the DMSO-induced secretome. Interestingly, we also noted elevated migration and invasion in the 5-FU-R colon cancer cells exposed to 5-FU-induced secretome compared to the DMSO counterpart. Additionally, in zebrafish xenograft, we found that 5-FU-R colon cancer cells cultured with 5-FU-induced secretome showed higher tail-vein metastatic burden compared to the 5-FU-R cells cultured with DMSO-induced secretome. This provides a background to further study in detail the therapy-induced resistance as well as dormancy in colorectal cancer which will help to develop approaches to prevent or reverse chemoresistance in patients who receive systemic therapy for mCRC. Citation Format: Shakti Ranjan Satapathy, Souvik Ghatak, Anita Sjölander. Drug-induced cancer cell secretome promotes resistance in colon cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1994.

Bao Yuan decoction alleviates fatigue by restraining inflammation and oxidative stress via the AMPK/CRY2/PER1 signaling pathway

Ethnopharmacological relevanceBaoyuan Decoction (BYD) was initially recorded in the classic of “Bo Ai Xin Jian” in the Ming dynasty. It is traditionally used for treating deficiency, labor and cowardice, deficiency of vital energy. In researches related to anti-fatigue effects, the reciprocal regulation of AMPK and circadian clocks likely plays an important role in anti-fatigue mechanism, while it has not yet been revealed. Therefore, we elucidated the anti-fatigue mechanism of BYD through AMPK/CRY2/PER1 pathway. Aim of the studyTo investigate the effect and mechanism of BYD in reducing fatigue, using pharmacodynamics, network pharmacology and transcriptomics through the AMPK/CRY2/PER1 signaling pathway. Materials and methodsFirstly, the chemical constituents of BYD were qualitatively identified by UHPLC-Q-Exactive Orbitrap/MS, establishing a comprehensive strategy with an in-house library, Xcalibur software and Pubchem combined. Secondly, a Na2SO3-induced fatigue model and 2,2′-Azobis (2-methylpropionamidine) dihydrochloride (AAPH)-induced oxidative stress model were developed to evaluate the anti-fatigue and anti-oxidant activities of BYD using AB zebrafish. The anti-inflammatory activity of BYD was evaluated using CuSO4-induced and tail cutting-induced Tg (lyz: dsRed) transgenic zebrafish inflammation models. Then, target screening was performed by Swiss ADME, GeneCards, OMIM and DrugBank databases, the network was constructed using Cytoscape 3.9.0. Transcriptome and network pharmacology technology were used to investigate the related signaling pathways and potential mechanisms after treatment with BYD, which were verified by real-time quantitative PCR (RT-qPCR). ResultsIn total, 114 compounds from the water extract of BYD were identified as major compounds. Na₂SO₃-induced fatigue model and AAPH-induced oxidative stress model indicated that BYD has significant anti-fatigue and antioxidant effects. Meanwhile, BYD showed significant anti-inflammatory effects on CuSO4-induced and tail cutting-induced zebrafish inflammation models. The KEGG result of network pharmacology showed that the anti-fatigue function of BYD was mainly effected through AMPK signaling pathways. Besides, transcriptome analysis indicated that the circadian rhythm, AMPK and IL-17 signaling pathways were recommended as one of the main pathways related to the anti-fatigue effect of BYD. The RT-qPCR results showed that compared with a model control group, the treatment of BYD significantly elevated the expression mRNA of AMPK, CRY2 and PER1. ConclusionHerein, we identified 114 chemical constituents of BYD, performed zebrafish activity validation, while demonstrated that BYD can relieve fatigue by AMPK/CRY2/PER1 signaling pathway through network pharmacology and transcriptome.

Spermidine treatment: induction of autophagy but also apoptosis?

Machado-Joseph disease (MJD), also known as spinocerebellar ataxia type 3, is a fatal neurodegenerative disease that causes loss of balance and motor co-ordination, eventually leading to paralysis. It is caused by the autosomal dominant inheritance of a long CAG trinucleotide repeat sequence within the ATXN3 gene, encoding for an expanded polyglutamine (polyQ) repeat sequence within the ataxin-3 protein. Ataxin-3 containing an expanded polyQ repeat is known to be highly prone to intraneuronal aggregation, and previous studies have demonstrated that protein quality control pathways, such as autophagy, are impaired in MJD patients and animal models of the disease. In this study, we tested the therapeutic potential of spermidine on zebrafish and rodent models of MJD to determine its capacity to induce autophagy and improve functional output. Spermidine treatment of transgenic MJD zebrafish induced autophagy and resulted in increased distances swum by the MJD zebrafish. Interestingly, treatment of the CMVMJD135 mouse model of MJD with spermidine added to drinking water did not produce any improvement in motor behaviour assays, neurological testing or neuropathology. In fact, wild type mice treated with spermidine were found to have decreased rotarod performance when compared to control animals. Immunoblot analysis of protein lysates extracted from mouse cerebellar tissue found little differences between the groups, except for an increased level of phospho-ULK1 in spermidine treated animals, suggesting that autophagy was indeed induced. As we detected decreased motor performance in wild type mice following treatment with spermidine, we conducted follow up studies into the effects of spermidine treatment in zebrafish. Interestingly, we found that in addition to inducing autophagy, spermidine treatment also induced apoptosis, particularly in wild type zebrafish. These findings suggest that spermidine treatment may not be therapeutically beneficial for the treatment of MJD, and in fact warrants caution due to the potential negative side effects caused by induction of apoptosis.

Understanding the toxicity mechanism of gelsemine in zebrafish

Gelsemium elegans (GE), also known as Duanchangcao, is a plant associated with toxic symptoms related to the abdomen; however, the toxicity caused by GE remains unknown. Gelsemine (GEL) is an alkaloid extracted from GE and is one of the most toxic alkaloids. This study used zebrafish as an animal model and employed high-throughput gene sequencing to identify genes and signaling pathways related to GEL toxicity. Exposure to GEL negatively impacted heart rate, swim bladder development, and activity in zebrafish larvae. Transcriptomics data revealed the enrichment of inflammatory and phagocyte signaling pathways. RT-PCR analysis revealed a decrease in the expression of pancreas-related genes, including the pancreatic coagulation protease (Ctr) family, such as Ctrl, Ctrb 1, and Ctrc, due to GEL exposure. Furthermore, GEL exposure significantly reduced Ctrb1 protein expression while elevating trypsin and serum amylase activities in zebrafish larvae. GEL also resulted in a decrease in pancreas-associated fluorescence area and an increase in neutrophil-related fluorescence area in transgenic zebrafish. This study revealed that GEL toxicity in zebrafish larvae is related to acute pancreatic inflammation.