Cells In Zebrafish Embryos Research Articles

The effects of perfluorooctanoic acid on breast cancer metastasis depend on the phenotypes of the cancer cells: An in vivo study with zebrafish xenograft model

Per- and polyfluorinated substances (PFAS) have been associated with numerous human diseases. Recent in vitro studies have implicated the association of PFAS with an increased risk of breast cancer in humans. This study aimed to assess the toxic effects of PFAS during the development of human breast cancer using a zebrafish xenograft model. Perfluorooctanoic acid (PFOA) was used as a PFAS chemical of interest for this study. Two common breast cancer cell lines, MCF-7 and MDA-MB-231, were used to represent the diversity of breast cancer phenotypes. Human preadipocytes were co-implanted with the breast cancer cells into the zebrafish embryos to optimize the microenvironment for tumor cells in vivo. With this modified model, we evaluated the potential effects of the PFOA on the metastatic potential of the two types of breast cancer cells. The presence of human preadipocytes resulted in an enhancement to the metastasis progress of the two types of cells, including the promotion of cell in vivo migration and proliferation, and the increased expression levels of metastatic biomarkers. The enhancement of MCF-7 proliferation by preadipocytes was observed after 2 days post injection (dpi) while the increase of MDA-MB-231 proliferation was seen after 6 dpi. The breast cancer metastatic biomarkers, cadherin 1 (cdh1), and small breast epithelial mucin (sbem) genes demonstrated significant down- and upregulations respectively, by the co-injection of preadipocytes. In the optimized xenograft model, the PFOA consistently promoted cell proliferation and migration and altered the metastatic biomarker expression in MCF-7, which suggested a metastatic effect of PFOA on MCF-7. However, those effects were not consistently observed in MDA-MB-231. The presence of the preadipocytes in the xenograft model may provide a necessary microenvironment for the progress of tumor cells in zebrafish embryos. The finding suggested that the impacts of PFOA exposure on different phenotypes of breast cancers may differ.

Perfluorodecanoic acid induces the increase of innate cells in zebrafish embryos by upregulating oxidative stress levels

Several studies reported that the widespread use of perfluoroalkyl and polyfluoroalkyl substances (PFASs) causes increased environmental pollution, subsequently impacting aquatic organisms. Perfluoroalkyl substances such as perfluorooctane sulfonic acid (PFOS) and perfluorooctanoic acid (PFOA) reportedly cause cardiotoxicity, neurotoxicity, and developmental toxicity in different organisms. However, whether perfluorodecanoic acid (PFDA), a widely used perfluoroalkyl substance, induces animal embryos developmental toxicity remain unknown. Here, we explored the immunotoxicity and associated mechanisms of PFDA in zebrafish embryos via RNA sequencing, morphological assessment and behavioral alteration detection following exposure to 0.5, 1 and 2 mg/L of PFDA. Interestingly, We found that with the increase of PFDA to drug concentration, including neutrophils and macrophages, significantly increased the number of inherent cells, immune related genes expression. Furthermore, oxidative stress increased in the PFDA-treated embryos in a dose-dependent manner and inhibition of oxidative stress levels effectively rescued the number of neutrophils. Changes in embryonic behavior were observed after exposure to PFDA. Overall, our results suggest that PFDA may induce innate immune response by accumulation of oxidative stress in zebrafish at early developmental stages, and concern is needed about its environmental exposure risks for animals embryos development. Environmental implicationPerfluorinated and polyfluorinated alkyl substances (PFASs) are a class of synthetic organic compounds containing fluorine widely used as lubricants, surfactants, insecticides, etc. The PFDA, a typical perfluorinated compound, is often used as a wetting agent and flame retardant in industries.Several studies showed that PFASs can cause serious environmental pollution, leading to developmental toxicity to various animals, including reproductive toxicity, liver toxicity, heart toxicity, neurotoxicity, and immunotoxicity.However, there are still limited studies on the effects and mechanisms of PFDA on aquatic organisms. Therefore, there is a need to evaluate the ecological risks of PFDA in animals.

Adaptive optics in an oblique plane microscope.

Adaptive optics (AO) can restore diffraction-limited performance when imaging beyond superficial cell layers in vivo and in vitro, and as such, is of interest for advanced 3D microscopy methods such as light-sheet fluorescence microscopy (LSFM). In a typical LSFM system, the illumination and detection paths are separate and subject to different optical aberrations. To achieve optimal microscope performance, it is necessary to sense and correct these aberrations in both light paths, resulting in a complex microscope system. Here, we show that in an oblique plane microscope (OPM), a type of LSFM with a single primary objective lens, the same deformable mirror can correct both illumination and fluorescence detection. Besides reducing the complexity, we show that AO in OPM also restores the relative alignment of the light-sheet and focal plane, and that a projection imaging mode can stabilize and improve the wavefront correction in a sensorless AO format. We demonstrate OPM with AO on fluorescent nanospheres and by imaging the vasculature and cancer cells in zebrafish embryos embedded in a glass capillary, restoring diffraction limited resolution and improving the signal strength twofold.

Leukolectins support lifelong innate immunity in lower vertebrates, and reveal dichotomies of several leukocytic lineages

Innate immunity is vital for animal homeostasis and survival. First-line immuno-defense for fish larvae involves mucus enriched with leukolectin (LL) secreted by dermal lectocytes. Later during the critical transition from yolk-nutrition to feeding, additional larval immuno-protection in zebrafish (zF) is provided by macrophages containing LL (lectophages). This work investigated new LL-expression in embryos and in blood, structures of fish leukocytic LL and LL-genes, and LL-presence in chicken leukocytes. In zF-embryos, lectophages appear ∼10 hpf, while later, cells co-expressing myeloperoxidase- and LL-mRNA were detected (∼19 hpf). Furthermore, protein-extracts of Atlantic salmon (Ssal) leukocytes contained LL-proteins, compartmentalized in the cytosol. Cloning and sequencing revealed 94 % nt-sequence identity between variants of Ssal-leukolectins. Highly conserved LLs allowed production of epitope-specific anti-LL IgGs. Immuno-fluorescence-analysis demonstrated that most Ssal-bloodcells were LL-negative, but both some large cells with protrusions and some small, rounded cells did express LL. Immunoperoxidase-staining method confirmed LL-expression in some Ssal-leukocytes, identified as macrophages, PMN-leukocytes, thrombocytes and dendritic cells. However, closer examination revealed a dichotomy of these cell-categories into either LL-positive, or LL-negative variants. In situ hybridization demonstrated profuse LL-expression in Ssal head kidney interstitial tissue, while LL-transcripts were absent in large kidney tubules. Both hematopoietic (non-pigmented) marrow cells and melano-macrophages expressed LL-mRNA, implying that leukolectins provide lifelong innate immuno-protection. PCR-amplification using Ssal-leukocytic DNA as template, and direct sequencing yielded a leukocytic ll-gene. Some cells in salmon, cod, halibut, oikopleura and zebrafish embryos express LL-proteins and/or LL-mRNA, and LL-mRNA is detected in salmon, cod and chicken leukocytes. However, current genomes for these species lack recognizable LL-loci except the Ssal_v3.1 Genome-assembly. The data demonstrate an unexpected dichotomy of some leukocyte lineages into LL-positive or LL-negative cell-variants. Such dichotomies suggest exploring differential impacts from the duplicated leukocyte-lineages in health and disease.

Plexin B3 guides axons to cross the midline in vivo.

During the development of neural circuits, axons are guided by a variety of molecular cues to navigate through the brain and establish precise connections with correct partners at the right time and place. Many axon guidance cues have been identified and they play pleiotropic roles in not only axon guidance but also axon fasciculation, axon pruning, and synaptogenesis as well as cell migration, angiogenesis, and bone formation. In search of receptors for Sema3E in axon guidance, we unexpectedly found that Plexin B3 is highly expressed in retinal ganglion cells of zebrafish embryos when retinal axons are crossing the midline to form the chiasm. Plexin B3 has been characterized to be related to neurodevelopmental disorders. However, the investigation of its pathological mechanisms is hampered by the lack of appropriate animal model. We provide evidence that Plexin B3 is critical for axon guidance in vivo. Plexin B3 might function as a receptor for Sema3E while Neuropilin1 could be a co-receptor. The intracellular domain of Plexin B3 is required for Semaphorin signaling transduction. Our data suggest that zebrafish could be an ideal animal model for investigating the role and mechanisms of Sema3E and Plexin B3 in vivo.

Abstract 5425: MITF/TFE regulatory programs in neural crest migration are co-opted in melanoma metastasis

Abstract Introduction: Microphthalmia-associated transcription factor (MITF) is a critical regulator of melanocyte differentiation and melanoma oncogenesis. Melanoma expression profiling has revealed a strong trend between low MITF expression levels and invasive behavior. However, it is unclear how low levels of MITF are connected to invasive behavior. Preliminary data from our lab has identified transcription factor E3 (TFE3), a MITF paralog, as a transcriptional regulator in MITF-low melanoma states. We hypothesize that in melanoma cells with low MITF levels, TFE3 promotes invasion. To assess the possibility that the antagonistic regulatory interaction between MITF and a paralog was coopted from the embryo, we examine migratory behavior of neural crest cells in zebrafish embryos that are wild-type, mitfa loss-of-function (LOF) mutants, tfec (TF3 ortholog) LOF mutants, or mitfa/tfec double LOF embryos. Methods: We evaluated TFE3 levels by Western blot in a panel of melanoma cell lines known to express a range of MITF expression levels. We mutated TFE3 exon 4, which encodes the DNA binding domain, using a single guide RNA, and assessed cellular invasive potential using an in vitro transwell invasion assay. CUT&RUN sequencing was used to profile TFE3 genomic binding. Zebrafish embryos were generated in which the mitfa promoter drove expression of RNA encoding green fluorescent protein (GFP), and of the genotypes mentioned above. From transgenic wild-type and mitfa LOF mutant embryos we isolated GFP-expressing cells and conducted single cell-RNA sequencing. We assessed migration of GFP-labeled cells by fluorescent microscopy in 20-48 hour post fertilization zebrafish embryos of all genotypes mentioned above. Results: We found that melanoma cell lines with high levels of MITF (SKMEL3 and SKMEL28) had low levels of TFE3 protein, and those with low levels of MITF (A375 and RPMI 7952) had high levels of TFE3 protein. The TFE3-high cell lines were more invasive in vitro than the TFE3-low cell lines. TFE3-high cell lines depleted of TFE3 were less in invasive in vitro than parental cells. CUT&RUN profiling of TFE3 demonstrates that TFE3 binds mesenchymal-like enhancers in MITF-low cell lines. Single cell RNA sequencing revealed that in a melanocyte precursor cluster present in both wild types and in mitfa mutants, expression of tfec was higher in the latter. Finally, migration of GFP-expressing cells was higher in mitfa LOF mutant embryos than in mitfa/tfec double LOF mutant embryos. Conclusion: These data indicate that reduction of MITF expression up-regulates TFE3 expression in melanoma and tfec in zebrafish melanocytes, although the mechanism of this regulation is unclear. They also show that TFE3 and Tfec promote migration of metastatic melanoma and melanocyte precursors, respectively. Because regulatory mechanisms in cancer are often coopted from embryonic cells, studying the embryo may illuminate cancer pathogenesis. Citation Format: Jeremy Chang, Katelyn Campbell, Rachel Moore, Robert Cornell, Colin Kenny. MITF/TFE regulatory programs in neural crest migration are co-opted in melanoma metastasis [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 5425.

Single extract of Forsythia Suspense versus the prepared drug in pieces: comparison of their anti-inflammatory, antitumor and antibacterial effects in zebrafish

To compare the anti-inflammatory, antitumor and anti-bacterial effects of the single extract (in granules) and the prepared drug in pieces of Forsythia Suspense (Lianqiao, a traditional Chinese herbal medicine). In zebrafish embryo models of CuSO4 exposure, tail transection and LPS microinjection-induced inflammation, the anti-inflammatory effects of 10 μg/mL DEX, single extract of Forsythia Suspense, and the water extract of the prepared drug (400, 600, and 800 μg/mL) were evaluated by observing neutrophil counts, RT- qPCR, HE staining and survival analysis. Zebrafish embryo models bearing different human tumor cell xenografts were used to assess the anti-tumor effect of the drugs in different dosage forms by fluorescence staining and HE staining. The microbroth dilution method was used to evaluate the antibacterial efficacy of the drugs. In the zebrafish embryo models of inflammation, both of the two dosage forms of Forsythia Suspense significantly inhibited neutrophil aggregation, reduced the mRNA expressions of TNF-α, IL-6, P38, Jnk, Erk and P65, and increased the survival rate of zebrafish. They both showed obvious inhibitory effects against xenografts of different human cancer cells including colon cancer cells (HCT116), pancreas adenocarcinoma cells (PANC-1), lung cancer cells (A549), liver cancer cells (Hep3B) and cervical carcinoma cells (Hela) in zebrafish embryos, and exhibited strong anti-bacterial effects at the concentration of 15.63 mg/mL. The two dosage forms of Forsythia Suspense have similar anti-inflammatory, antitumor and antibacterial effects, but their effects for inhibiting IL-6, P65, and Jnk mRNA expressions and HCT116 cell proliferation differ significantly at low doses in zebrafish.

Synergistic effects of TiO2-ZnO/Poly(methyl methacrylate) nanocomposite films: Enhanced wettability, antimicrobial activity, and biocompatibility

The poly (methyl methacrylate) (PMMA) nanocomposite films were fabricated using a cost-effective solvent evaporation method. The dominance of the wurtzite Zinc oxide (ZnO) phase was observed, accompanied by a suppression in the phase intensity of anatase titanium dioxide (TiO2) in the PMMA matrix confirmed by the XRD. New peaks appeared at 1651 cm−1 for the Ti-OH of TiO2 and 590 cm−1 for the metal–oxygen vibration of ZnO in the nanocomposite films. SEM micrograph of the TZPMMA resulted in the formation of nanopores with the nanoparticles embedded in the PMMA matrix. The wettability was enhanced by 23 % with the incorporation of metallic nanoparticles. Zinc ions increased antimicrobial susceptibility against S. aureus by 20 % more than E. coli, demonstrating the potential for fighting bacterial infections. The porous surface created by the nanoparticles improved the cell viability of the PMMA. Zebrafish embryo mortality percentage (0.5 %) and cell viability percentage (80 %) against human gingival fibroblasts (hGFs) showed that the incorporation of metal oxides made the PMMA, a potential candidate for biomedical applications.

Structure-function relationship of phase-separated liposomes containing diacylglycerol analogues.

The composition and morphology of lipid-based nanoparticles can influence their overall in vivo behavior. Previously, we demonstrated that phase separation in liposomes composed of DSPC and a diacylglycerol lipid analogue (DOaG) drives the in vivo biodistribution towards a specific subset of endothelial cells in zebrafish embryos. In the absence of traditional targeting functionalities (e.g., antibodies, ligands), this selectivity is mediated solely by the unique liposome morphology and composition, characterized by a DOaG-rich lipid droplet within the DSPC-rich phospholipid bilayer. The phase separation is induced due to the geometry of DOaG lipid and its ability to create non-bilayer phases in lipid membranes. To investigate the underlying principles of phase separation and to optimize the liposome colloidal stability, we performed a structure-function relationship study by synthesizing a library of DOaG analogues with varying molecular properties, such as the number, length and sn-position of the acyl chains, as well as the degree of saturation or carbonyl substituents. We assessed the ability of these lipid analogues to assemble into phase-separated liposomes and studied their morphology, colloidal stability, and in vivo biodistribution in zebrafish embryos. We found that analogues containing unsaturated, medium length (C16-C18) fatty acids were required to obtain colloidally stable, phase-separated liposomes with cell-specific biodistribution patterns. Moreover, we observed that using the pure DOaG isomer, with acyl chains at the sn-1,3 positions, leads to more colloidally stable liposomes than when a mixture of sn-1,2 and sn-1,3 isomers is used. Similarly, we observed that incorporating a DOaG analogue with fatty tails shorter than DSPC, as well as PEGylation, endows liposomes with long term stability while retaining cell-selective biodistribution. Diacylglycerols are known to promote fusion, lipid polymorphism, signaling and protein recruitment on lipid membranes. In this study, we showed that diacylglycerol derivatives can induce phase separation in liposomes, unlocking the potential for cell-specific targeting in vivo. We believe that these findings can be the foundation for future use of diacylglycerols in lipid-based nanomedicines and could lead to the development of novel targeted delivery strategies.

Modeling Gilteritinib Resistance By Enforced cxcl8a Expression in Zebrafish HSPCs

In Acute Myeloid Leukemia (AML), the FMS-like tyrosine kinase III Internal Tandem Duplication (FLT3-ITD) is common and increases the risk of relapse which is only partially mitigated by tyrosine kinase inhibitor (TKI) therapy. Gilteritinib is the current mainstay for treating relapsed FLT3-ITD AML, but therapy is non-curative, and the duration of remission is transient. Mutations in the RAS pathway are known to promote gilteritinib resistance, however non-mutational mechanisms of resistance remain under-explored and could provide new avenues for combination therapy. Our previous work has associated AML cell-autonomous expression of chemokines such as CXCL8 (IL-8) as a mechanism of resistance to gilteritinib therapy. Here we enforce expression of cxcl8a ( cxcl8, ENSDARG00000104795) by hematopoietic stem and progenitor cells (HSPCs) in transgenic zebrafish embryos using a defined Runx1 enhancer element. In the absence of gilteritinib, Runx1:cxcl8(+) animals had greater numbers of HPSCs compared to Runx1:cxcl8(-) animals (4.0 vs 2.1, p = 0.04). When treated with gilteritinib at 100 nM from 48-72 hours post-fertilization (hpf), HSPC numbers were decreased in both groups; however, Runx1:cxcl8(+) animals still had more HSPCs compared to Runx1:cxcl8(-) animals (1.6 vs 0.4, p = 0.02). This suggests that cxcl8 may act as a resistance factor for gilteritinib in vivo and that understanding regulators of cxcl8 expression may provide insight into gilteritinib resistance. Reanalysis of published single cell RNA-seq data in AML patients revealed an association between expression of CXCL8 and BTK and BMX but not other Tec-family kinases such as ITK and TEC itself. Zebrafish do not have an annotated BMX gene; BTK is expressed in HSPCs and neutrophils but not the erythroid lineage. CHMFL is a specific and potent inhibitor of human BMX with an unknown spectrum of activity in zebrafish. In Runx1:cxcl8(-) Runx1:GFP zebrafish, CHMFL treatment reduced HSPC numbers, but this was not rescued in Runx1:cxcl8(+) animals (untreated vs treated, cxcl8(-): 2.9 vs 1.5, p = 0.02, untreated vs treated, cxcl8(+): 4.0 vs 1.4, p = 0.0006). This contrasts with our prior work with the non-specific Tec family kinase inhibitor, ibrutinib, where HSPC numbers were rescued in Runx1:cxcl8(+) animals. Ongoing studies are focused on understanding the targets of ibrutinib and CHMFL in the zebrafish and how these pathways may affect gilteritinib resistance.

Apollo-NADP+ reveals in vivo adaptation of NADPH/NADP+ metabolism in electrically activated pancreatic β cells.

Several genetically encoded sensors have been developed to study live cell NADPH/NADP+ dynamics, but their use has been predominantly in vitro. Here, we developed an in vivo assay using the Apollo-NADP+ sensor and microfluidic devices to measure endogenous NADPH/NADP+ dynamics in the pancreatic β cells of live zebrafish embryos. Flux through the pentose phosphate pathway, the main source of NADPH in many cell types, has been reported to be low in β cells. Thus, it is unclear how these cells compensate to meet NADPH demands. Using our assay, we show that pyruvate cycling is the main source of NADP+ reduction in β cells, with contributions from folate cycling after acute electrical activation. INS1E β cells also showed a stress-induced increase in folate cycling and further suggested that this cycling requires both increased glycolytic intermediates and cytosolic NAD+. Overall, we show in vivo application of the Apollo-NADP+ sensor and reveal that β cells are capable of adapting NADPH/NADP+ redox during stress.

Abstract A009: Distinct activity of androgen receptor splice variants in promoting prostate cancer metastasis

Abstract Introduction: Constitutively active androgen receptor splice variant 7 (AR-V7) correlates with the adaptation to androgen receptor signaling inhibition treatment in prostate cancer (PCa) patients. Despite intensive studies of AR-V7, it is still unclear how the distinct genomic and epigenomic characteristics of AR-V7-dependent cistrome and transcriptome are distinct from full-length AR (AR-FL) and whether AR-V7 may play a role in promoting the metastatic progression of castration-resistant PCa (CRPC). Experiments: To accurately compare the activity of AR-V7 with AR-FL, we generated lentiviral stable cell lines with inducible overexpression of AR-V7 or AR-FL using AR-V7 negative LNCaP and C4-2 cell lines. We assessed the in vivo impact of AR-V7 or AR-FL expression on PCa metastasis by injecting cells in zebrafish embryos and the tibias of castrated male mice. ChIP-seq analyses of AR-V7, AR-FL, FOXA1, and H3K27ac, ATAC-seq analysis, and RNA-seq analysis were conducted in to characterize the global cistrome and transcriptome activity of AR-V7 in comparison with AR-FL. In addition, we also examined how a posttranslational modification, Ser81 phosphorylation, affects AR-V7 activity by generating an S81A mutant of AR-V7 and using S81-phosphorylation inhibitors. Results: Our in vivo study revealed that overexpressed-AR-V7, but not AR-FL, strongly promotes tumor cell invasion into blood vessels and induces osteoblastic bone lesions. Through integrated ChIP-, ATAC- and RNA-seq analysis, we determined that AR-V7 has a “pioneer factor-like” ability to bind to androgen-responsive elements (AREs) located at compact chromatin regions and activates the enhancers by increasing the chromatin accessibility. This mechanism leads to a unique AR-V7-regulated transcriptional program that is completely independent of AR-FL activity and is highly enriched for genes mediating epithelial-mesenchymal transition and metastatic functions. We then identified a subset of AR-V7-specific targets with significant overexpression in CRPC tumor samples and associated with poor clinical outcomes. In particular, we found that SOX9, a critical metastasis driver gene, was a direct target and downstream effector of AR-V7, and its protein expression was dramatically upregulated at AR-V7-induced bone lesions. Furthermore, we found that the pro-metastasis function of AR-V7 can be enhanced by Ser81 phosphorylation through chromatin-binding-independent mechanisms, and that targeting this phosphorylation by CDK9 inhibitors can block AR-V7-induced SOX9 expression and impair AR-V7-mediated metastasis function. Conclusion: Our study provides novel molecular insights into the role of AR splice variants in driving the metastatic progression of CRPC. In specific, these data have demonstrated the distinctive transcriptional program of AR-V7 from AR-FL, identified critical AR-V7 targets, and determined the function of Ser81 phosphorylation on AR-V7. Our data also suggest a new therapeutic strategy to target AR-V7-induced metastasis cascade in CRPC by using CDK9 inhibitor treatment. Citation Format: Maryam Labaf, Dong Han, Yawei Zhao, Jude Owiredu, Songqi Zhang, Kavita Venkataramani, Jocelyn S. Steinfeld, Wanting Han, Muqing Li, Mingyu Liu, Zifeng Wang, Anna Besschetnova, Susan C. Patalano-Salsman, Jill A. Macoska, Xin Yuan, Steven P. Balk, Stephen R. Plymate, Shuai Gao, Kellee R. Siegfried-Harris, Ruihua Liu, Gabrielle Foxa, Mary M. Stangis, Piotr J. Czernik, Bart Williams, Kourosh Zarringhalam, Xiaohong Li, Changmeng Cai. Distinct activity of androgen receptor splice variants in promoting prostate cancer metastasis [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A009.

Induction of Apoptosis by Matrine Derivative ZS17 in Human Hepatocellular Carcinoma BEL-7402 and HepG2 Cells through ROS-JNK-P53 Signalling Pathway Activation.

Hepatocellular carcinoma (HCC) is one of the most common malignancies and ranks third among cancer-related deaths worldwide. Using matrine as a lead compound, 12 matrine derivatives were designed and synthesised, and their antiproliferative activities were evaluated in four cancer cell lines. Eight of the twelve compounds showed strong antiproliferative activity, with an IC50 of <10 μM. The compound ZS17 exhibited strong antiproliferative activity in hepatocellular carcinoma cell lines with IC50 values in the range of 3.014-3.388 μM, which was much lower than that of matrine. Furthermore, we explored the role of ZS17 in inducing apoptosis in HCC cells in vitro and in vivo, as well as possible mechanisms involved. ZS17 inhibited the proliferation of BEL-7402 and HepG2 cells in time- and dose-dependent manners. In addition, we found that ZS17 significantly induced apoptosis and ROS (reactive oxygen species) production, promoted JNK phosphorylation, activated p53, and activated the caspase signalling pathway. Furthermore, the antioxidant NAC, JNK inhibitor SP600125, and Si-JNK increased cell viability, re-established cell metastasis, and inhibited ZS17-induced apoptosis. An in vivo antitumour assay demonstrated that ZS17 significantly reduced the number of migrating HepG2 cells in zebrafish embryos and suppressed the growth of HepG2 xenografts in nude mice without any obvious side effects. Our study demonstrated that the ROS-JNK-P53 pathway plays an important role in the destruction of liver tumour cells by ZS17. Thus, ZS17 may represent a promising chemotherapeutic agent for the treatment of HCC patients.

Establishment of Patient-Derived Xenograft (PDX) Zebrafish Model of Multiple Myeloma and Its Application in Drug Screening

Objective: To confirm the in vivo anti-myeloma activity of I3MO (Indirubin-3' -monoxime) in primary patient samples, a patient-derived xenograft (PDX) zebrafish model of MM was utilized. Methods: zebrafish embryos were obtained by the natural spawning of Tubingen strain and incubated at 28°C in embryo medium. The purified CD138+ MM cells fluorescently labeled with Calcein-AM or Dil were injected into the perivitelline space of each embryo of zebrafish at 48 hpf. Engrafted embryos were transferred into 24-well plates and incubated at 28°C in freshly prepared E3 medium containing DMSO, BTZ, I3MO. At 24 hours post injection (hpi), engrafted embryos were detected by fluorescent microscopy to qualify the positive areas with calcein (green) or Dil (red). Results:1. The zebrafish PDX model of MM was successfully applied in the drug effects evaluation. 2. The results in vivo demonstrated that BTZ or I3MO eliminates the engraftment and growth of MM cells in zebrafish embryos (p<0.05). Conclusion: The zebrafish is an ideal model to investigate the cytotoxicity in vivo of compounds on primary MM patient samples.

Zebrafish Embryos Display Characteristic Bioelectric Signals during Early Development.

Bioelectricity is defined as endogenous electrical signaling mediated by the dynamic distribution of charged molecules. Recently, increasing evidence has revealed that cellular bioelectric signaling is critical for regulating embryonic development, regeneration, and congenital diseases. However, systematic real-time in vivo dynamic electrical activity monitoring of whole organisms has been limited, mainly due to the lack of a suitable model system and voltage measurement tools for in vivo biology. Here, we addressed this gap by utilizing a genetically stable zebrafish line, Tg (ubiquitin: ASAP1), and ASAP1 (Accelerated sensor of action potentials 1), a genetically encoded voltage indicator (GEVI). With light-sheet microscopy, we systematically investigated cell membrane potential (Vm) signals during different embryonic stages. We found cells of zebrafish embryos showed local membrane hyperpolarization at the cleavage furrows during the cleavage period of embryogenesis. This signal appeared before cytokinesis and fluctuated as it progressed. In contrast, whole-cell transient hyperpolarization was observed during the blastula and gastrula stages. These signals were generally limited to the superficial blastomere, but they could be detected within the deeper cells during the gastrulation period. Moreover, the zebrafish embryos exhibit tissue-level cell Vm signals during the segmentation period. Middle-aged somites had strong and dynamic Vm fluctuations starting at about the 12-somite stage. These embryonic stage-specific characteristic cellular bioelectric signals suggest that they might play a diverse role in zebrafish embryogenesis that could underlie human congenital diseases.

Fenpropathrin exposure induces neurotoxicity in zebrafish embryos.

Fenpropathrin has been a commonly used insecticide to control agricultural and household insects over a few decades. Up to now, fenpropathrin residue in soil and water has been often determined due to its widespread use, which poses serious threat to environment and aquatic organisms. The potential of fenpropathrin to affect aquatic lives is still poorly understood. In this study, we used zebrafish (Danio rerio) embryo as an experimental model system to evaluate the toxicity of fenpropathrin to the development of zebrafish nervous system. Zebrafish embryos were separately exposed to fenpropathrin at the dose of 0.016mg/L, 0.032mg/L, 0.064mg/L, starting at 6h post-fertilizationhpf (hpf) up to 96 hpf. The results showed that fenpropathrin exposure gives rise to physiological, behavioral, and neurodevelopmental impairments in zebrafish embryos, including enhanced acetylcholinesterase (AChE) activity, abnormal swimming behavior, karyopyknosis in brain cells, increased intercellular space, and uneven migration of neuron in brain area. In addition, the expressions of genes concerning neurodevelopment and neurotransmitter system were inhibited following fenpropathrin exposure. We also found that fenpropathrin exposure distinctly induced oxidative stress by increasing reactive oxygen species (ROS) generation and inhibiting the production of antioxidant enzymes catalase (CAT) and superoxide dismutase (SOD). Expectedly, some apoptosis-associated genes were induced and the apoptosis appeared in the brain and heart cells of zebrafish embryos. Moreover, fenpropathrin exposure also inhibited the expressions of genes in Nrf2 signaling pathway, such as heme oxygenase-1 (HO-1) and SOD. In summary, the results of this study indicate that oxidative stress-triggered apoptosis may be an underlying fundamental of fenpropathrin-induced neurotoxicity in zebrafish embryos.

Gene expression and functional analysis of Aha1a and Aha1b in stress response in zebrafish.

Activator of heat shock protein 90 (hsp90) ATPase (Aha1) is a Hsp90 co-chaperone required for Hsp90 ATPase activation. Aha1 is essential for yeast survival and muscle development in C. elegans under elevated temperature and hsp90-deficeiency induced stress conditions. The roles of Aha1 in vertebrates are poorly understood. Here, we characterized the expression and function of Aha1 in zebrafish. We showed that zebrafish genome contains two aha1 genes, aha1a and aha1b, that show distinct patterns of expression during development. Under the normal physiological conditions, aha1a is primarily expressed in skeletal muscle cells of zebrafish embryos, while aha1b is strongly expressed in the head region. aha1a and aha1b expression increased dramatically in response to heat shock induced stress. In addition, Aha1a-GFP fusion protein exhibited a dynamic translocation in muscle cells in response to heat shock. Moreover, upregulation of aha1 expression was also observed in hsp90a1 knockdown embryos that showed a muscle defect. Genetic studies demonstrated that knockout of aha1a, aha1b or both had no detectable effect on embryonic development, survival, and growth in zebrafish. The aha1a and aha1b mutant embryos showed normal muscle development and stress response in response to heat shock. Single or double aha1a and aha1b mutants could grow into normal reproductive adults with normal skeletal muscle structure and morphology compared with wild type control. Together, data from these studies indicate that Aha1a and Aha1b are involved in stress response. However, they are dispensable in zebrafish embryonic development, growth, and survival.

CDH17 nanobodies facilitate rapid imaging of gastric cancer and efficient delivery of immunotoxin

BackgroundIt is highly desirable to develop new therapeutic strategies for gastric cancer given the low survival rate despite improvement in the past decades. Cadherin 17 (CDH17) is a membrane protein highly expressed in cancers of digestive system. Nanobody represents a novel antibody format for cancer targeted imaging and drug delivery. Nanobody targeting CHD17 as an imaging probe and a delivery vehicle of toxin remains to be explored for its theragnostic potential in gastric cancer.MethodsNaïve nanobody phage library was screened against CDH17 Domain 1-3 and identified nanobodies were extensively characterized with various assays. Nanobodies labeled with imaging probe were tested in vitro and in vivo for gastric cancer detection. A CDH17 Nanobody fused with toxin PE38 was evaluated for gastric cancer inhibition in vitro and in vivo.ResultsTwo nanobodies (A1 and E8) against human CDH17 with high affinity and high specificity were successfully obtained. These nanobodies could specifically bind to CDH17 protein and CDH17-positive gastric cancer cells. E8 nanobody as a lead was extensively determined for tumor imaging and drug delivery. It could efficiently co-localize with CDH17-positive gastric cancer cells in zebrafish embryos and rapidly visualize the tumor mass in mice within 3 h when conjugated with imaging dyes. E8 nanobody fused with toxin PE38 showed excellent anti-tumor effect and remarkably improved the mice survival in cell-derived (CDX) and patient-derived xenograft (PDX) models. The immunotoxin also enhanced the anti-tumor effect of clinical drug 5-Fluorouracil.ConclusionsThe study presents a novel imaging and drug delivery strategy by targeting CDH17. CDH17 nanobody-based immunotoxin is potentially a promising therapeutic modality for clinical translation against gastric cancer.Graphical

Quality assurance of hematopoietic stem cells by macrophages determines stem cell clonality.

Tissue-specific stem cells persist for a lifetime and can differentiate to maintain homeostasis or transform to initiate cancer. Despite their importance, there are no described quality assurance mechanisms for newly formed stem cells. We observed intimate and specific interactions between macrophages and nascent blood stem cells in zebrafish embryos. Macrophage interactions frequently led to either removal of cytoplasmic material and stem cell division, or complete engulfment and stem cell death. Stressed stem cells were marked by surface Calreticulin, which stimulated macrophage interactions. Using cellular barcoding, we found that calreticulin knock-down or embryonic macrophage depletion reduced the number of stem cell clones that established adult hematopoiesis. Our work supports a model in which embryonic macrophages determine hematopoietic clonality by monitoring stem cell quality.

Preclinical Evaluation of Novel Tyrosine-Kinase Inhibitors in Medullary Thyroid Cancer.

Simple SummaryMedullary thyroid carcinoma (MTC) is a neuroendocrine tumor arising from parafollicular calcitonin-secreting C cells of the thyroid. Most of the patients affected by MTC, especially the familial form, harbor a mutation of the RET proto-oncogene. In patients with advanced disease, medical therapy is represented by two tyrosine-kinase inhibitors: cabozantinib and vandetanib. However, their usage is limited by several adverse events and drug-resistance onset. The aim of this preclinical study was to evaluate the antitumor activity of novel molecules for the therapy of MTC: SU5402, an inhibitor of the fibroblast growth factor receptor type 1 (FGFR-1) and vascular endothelial growth factor receptor (VEGFR)-2; sulfatinib, a multi-target kinase inhibitor selective for FGFR-1 and the VEGFR-1, -2, and -3; SPP86, a RET-specific inhibitor. Our results suggest a potential role in targeting the FGFR and VEGFR signaling pathways as an alternative strategy for resistant tumors and a significative antitumor activity of this new RET-specific inhibitor.Medullary thyroid carcinoma (MTC) is a neuroendocrine tumor arising from parafollicular C cells of the thyroid gland. In this preclinical study, we tested three tyrosine-kinase inhibitors (TKIs): SU5402, a selective inhibitor of fibroblast growth factor receptor (FGFR)-1 and vascular endothelial growth factor receptor (VEGFR)-2; sulfatinib, an inhibitor of FGFR-1 and VEGFR-1, -2, -3; and SPP86, a RET-specific inhibitor. The effects of these compounds were evaluated in vitro in two human MTC cell lines (TT and MZ-CRC-1), and in vivo using xenografts of MTC cells in zebrafish embryos. SU5402, sulfatinib and SPP86 decreased cell viability. Sulfatinib and SPP86 significantly induced apoptosis in both cell lines. Sulfatinib and SPP86 inhibited the migration of TT and MZCRC-1 cells, while SU5402 was able to inhibit migration only in TT cells. In vivo we observed a significant reduction in TT cell-induced angiogenesis in zebrafish embryos after incubation with sulfatinib and SPP86. In conclusion, sulfatinib and SPP86 displayed a relevant antitumor activity both in vitro and in vivo. Moreover, this work suggests the potential utility of targeting FGFR and VEGFR signaling pathways as an alternative therapy for MTC.