https://orcid.org/0009-0002-9151-544X
Figures
Abstract
The zebrafish (Danio rerio) is a valuable model organism for studying human biology due to its easy genetic manipulation and small size. It is optically transparent and shares genetic similarities with humans, making it ideal for studying developmental processes, diseases, and drug screening via imaging-based approaches. Solid malignant tumors often contain hypoxic areas that stimulate the release of extracellular vesicles (EVs), lipid-bound structures released by cells into the extracellular space, that facilitate short- and long-range intercellular communication and metastatization. Here we investigate the effects of EVs derived from neuroblastoma (NB), a pediatric solid tumor, on metastatic niche formation using the zebrafish as an in vivo model. Intravascular injection in zebrafish embryos allows a non-invasive visualization of EVs dispersion, uptake, and interactions with host cells. To improve repeatability of our results and ease the injection steps, we used an agarose device replica molded from a custom designed micromilled aluminum mold. We first demonstrated that EVs released under hypoxic conditions promote angiogenesis and are more easily internalized by endothelial cells than those purified from normoxic cells. We also showed that injection of with hypoxic EVs increased macrophages mobilization. We then focused on the caudal hematopoietic tissue (CHT) region of the embryo as a potential metastatic site. After hypoxic EVs injection, we highlighted changes in the expression of mmp-9 and cxcl8b genes. Furthermore, we investigated the ability of NB-derived EVs to prime a metastatic niche by a two-step injection of EVs first, followed by NB cells. Interestingly, we found that embryos injected with hypoxic EVs had more proliferating NB cells than those injected with normoxic EVs. Our findings suggest that EVs released by hypoxic NB cells alter the behavior of recipient cells in the zebrafish embryo and promote metastatic outgrowth. In addition, we demonstrated the ability of the zebrafish embryo to be a suitable model for studying the interactions between EVs and recipient cells in the metastatic process.
Citation: Fietta A, Fusco P, Germano G, Micheli S, Sorgato M, Lucchetta G, et al. (2024) Neuroblastoma-derived hypoxic extracellular vesicles promote metastatic dissemination in a zebrafish model. PLoS ONE 19(12): e0316103. https://doi.org/10.1371/journal.pone.0316103
Editor: Hodaka Fujii, Hirosaki University Graduate School of Medicine, JAPAN
Received: January 23, 2024; Accepted: December 4, 2024; Published: December 23, 2024
Copyright: © 2024 Fietta et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All original data are available at: https://researchdata.cab.unipd.it/id/eprint/1087.
Funding: The work was supported by the European Research Council, ERC (759467, StG-MICRONEX, PI E Cimetta). The funders provided financial support, and had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Abbreviations: CHT, Caudal hematopoietic tissues; CTR, Control; DZHEVs, Extracellular vesicles derived from SK-N-DZ cells cultured in hypoxic conditions; DZNEVs, Extracellular vesicles derived from SK-N-DZ cells cultured in normoxic conditions; ECM, Extracellular matrix; EVs, Extracellular vesicles; hpf, Hours post fertilization; hpi, Hours post injection; IMRHEVs, Extracellular vesicles derived from IMR32 cells cultured in hypoxic conditions; IMRNEVs, Extracellular vesicles derived from IMR32 cells cultured in normoxic conditions; ISV, Intersegmental vessel; mmps, Matrix metalloproteinases; NB, Neuroblastoma; SIV, Sub-intestinal veins; TAMs, Tumor associated macrophages
Introduction
Metastasis is the major contributor to cancer mortality, accounting for 90% of all cancer-related deaths. This complex process involves the mobilization of tumor cells from the primary site, their intravasation into the bloodstream, subsequent arrest, and extravasation at the target site, while favoring angiogenesis and proliferation at distant locations within the body [1]. Among the several active players in this process, extracellular vesicles (EVs), cell-secreted membrane-enclosed particles, emerged over the past decade for their ability to facilitate short- and long-range communication between various cell types at different metastatic stages [2]. EVs population is extremely heterogenous and ranges in size from 30 to 10,000 nm in diameter [3]. EVs contain various biomolecules derived from the donor cells, including proteins, nucleic acids, and lipids, and can deliver this molecular information over long distances to recipient cells or tissues within the body. Tumor progression can be promoted by tumor cell derived EVs via induction of cell migration and invasion, stimulation of tumor angiogenesis, and impairment of tumor immunity [4, 5].
Neuroblastoma (NB) is a solid pediatric tumor of neural crest origin [6] characterized by the amplification of the MYC family member MYCN oncogene in ∼25% of cases, which correlates with high-risk disease and poor prognosis. Amplification of MYCN remains the best-characterized genetic marker of risk in NB. Adrenal medulla, paraspinal ganglia and sympathetic nervous system are common primary NB tumor sites whereas bone, bone marrow and lymph nodes are mainly metastatic sites [7, 8].
The zebrafish (Danio rerio) is a powerful in vivo model showing a high degree of physiological and genetic similarity to mammals. In its simplicity, it can be used to mimic the complex tumor microenvironment to study human cancer biology [9]. Zebrafish xenotransplantation enables to study cancer progression, neo-angiogenesis, drug response and resistance, as well as metastasis [10, 11], with fluorescent-stained tumor cells easily tracked in the optically transparent embryos allowing live monitoring of the process of metastatization. Zebrafish has been used in numerous studies as a model for NB metastasis [12, 13], but a study investigating the effect of NB derived EVs on metastatic dissemination is still lacking. We here investigated the role of EVs released by NB cells in metastatic progression as a function of the oxygenation level of the parent cells. In NB, evidence point towards a role of hypoxia in tumor initiation during development, tumor cell differentiation, survival, and metastatic spreading [14]. However, the heterogeneous nature of NB, its developmental origin, and the lack of suitable experimental models have hampered a clear understanding of the effect of hypoxia in its progression and the molecular mechanisms implicated [15]. Here, we injected fluorescently labeled NB-derived EVs into zebrafish larvae at 2 days post fertilization (dpf) and investigated their contribution to tumor progression and metastatic colonization. An agarose molded device eased the injection steps and increased repeatability of our results. We report that hypoxic NB-derived EVs enhance angiogenesis and macrophages mobilization and modify the expression of mmp-9 and cxcl8b, genes involved in metastatic progression. Moreover, a two-steps injection of EVs and NB cells allowed us to mimic the metastatic colonization process, suggesting that hypoxic EVs promote metastatic outgrowth compared to their normoxic counterpart. Taken together, our data demonstrate that zebrafish larvae xenotransplants are promising in vivo models to investigate the role of EVs-dependent tumorigenesis and metastatic dissemination and show a potential use of EVs as a therapeutic target for NB metastasis.
Materials and methods
Cell culture
SK-N-DZ (ATCC, CRL 2149) and IMR32 (kindly provided by Dr G.P. Tonini) cell lines were cultured in DMEM supplemented with 10% FBS (ATCC), 100 U/mL penicillin/streptomycin (Life Technologies) and 1% MEM NEAA (Gibco) at 37°C in 5% CO2. Mesenchymal Stem Cells (MSCs) (ATCC, PCS-500-030) were cultured in Mesenchymal Stem Cell Basal Medium (ATCC) supplemented with 125 pg/mL rh FGF-b, 15 ng/mL rh IGF-1, 7% FBS, 2.4 mM L-alanyl-L-glutamine and 0.5% penicillin/streptomycin at 37°C, 5% CO2 (studies involving MSCs are reported as Supplementary Information). For hypoxic studies, cells were maintained at 37°C with 5% CO2 and 1.5% O2 in Whitley H35 Hypoxystation (Don Whitley Scientific).
Zebrafish
Fish handling procedures and care maintenance were accomplished in accordance with EU Directive 2010/63/EU for animal experiments. All experiments were conducted in non-feeding zebrafish embryos and their protocols were approved by the local OPBA (Organismo Preposto al Benessere Animale) of Fondazione Istituto di Ricerca Pediatrica Città della Speranza (Authorization Number: N02ZFU5). Adult Casper (Danio rerio) [16] and the transgenic zebrafish lines Tg(fli1:EGFP) [17], Tg(mpeg1:mCherry) [18] and Tg(gata1:dsRed) [19], were maintained using established temperature and light cycle conditions as previously described [20]. Fertilized eggs were obtained from natural spawning. Embryos were kept at 28.5°C in methylene blue-containing water (E3 medium) until injection. For EVs transplantation experiments, 2 dpf embryos were dechorionated and anaesthetized in 0.3× Danieau’s solution containing Phenythiourea (PTU, Sigma-Aldrich), excluded for the Casper embryos, and 0.04 mg/ml Tricaine (Sigma-Aldrich) before injection.
EVs isolation and labeling
EVs were isolated from conditioned media (CM) by ultrafiltration using 100-kDa MWCO Spin-X 20 mL Concentrator (Corning) following the manufacturer’s instructions. To generate CM, 4.5x106 cells were cultured in complete growth medium for 48 hours (80% confluency) and maintained in normoxic (20% O2) or hypoxic (1.5% O2) condition. Media was then replaced with DMEM supplemented with 100 U/mL penicillin/streptomycin (Life Technologies) and 1% MEM NEAA (Gibco) without FBS for 48 hours. CM was harvested, centrifuged to remove cellular debris (2000g for 5 min), and filtered by 0.22 μm syringe filters (Millipore). 20 mL of CM were then loaded into the concentrator tube and centrifuged at 3000g for 30 minutes at room temperature. This last step was repeated adding 15 mL of PBS 1x and centrifuged at 3000g for 15 minutes at room temperature. EVs derived from SK-N-DZ and IMR32 cells cultured under normoxic (DZNEVs and IMRNEVs respectively) or hypoxic (DZHEVs and IMRHEVs respectively) conditions, were collected and either used fresh or stored at -80°C. The total amount of proteins in EVs was quantified using Pierce BCA Protein Assays (ThermoFisher). EVs were labeled with the fluorescent Vybrant DiO Cell-Labeling Solution (ThermoFisher) or Vybrant DiI Cell-Labeling Solution (ThermoFisher) as recommended by the manufacturer for in vivo imaging of Tg(gata1:dsRed) and Tg(fli1:EGFP) embryos and the evaluation of EVs internalization. Briefly, 100 μg/mL of isolated EVs were resuspended in 1 mL of PBS 1X mixed with 5 μL of Cell-Labeling Solution for 30 minutes at 37°C. Labeled EVs were washed with 13 mL of PBS 1X, loaded into the concentrator tube, centrifuged at 3000g for 15 minutes at room temperature to remove free dye, and immediately injected. For the control group we used 100 nm fluorescent polystyrene beads (Phosphorex Inc.). For angiogenesis assessment using Tg(fli1:EGFP) embryos, macrophages mobilization in Tg(mpeg1:mCherry) embryos, and RNA extraction using Casper strain embryos, 100 μg/mL of EVs were resuspended in a solution of Fluorescein isothiocyanate–dextran (Sigma-Aldrich) in PBS 1X prior to injection. Control group was injected with dextran solution without EVs.
EVs characterization
For transmission electron microscopy (TEM) analysis, one drop of purified EVs (~ 25 μL) was deposited on 400 mesh holey film grid. Filter paper (Whatman) was used to remove the excess liquid. EVs were then stained with 1% uranyl acetate for 2 minutes. Samples were observed with a Tecnai G2 (FEI) transmission electron microscope operating at 100 kV equipped with a Veleta CCD camera (Olympus Soft Imaging System) at the BioImaging Facility (Department of Biology, University of Padova). The concentration of EVs was analyzed using Nanosight NS300 (Malvern) according to the manufacturer’s instructions. Briefly, samples were diluted 1:100 in PBS and analyzed under constant flow conditions at 25°C. For each measurement, three consecutive 60s videos were captured with a camera level of 11. Data were analyzed using NTA software with a detection threshold of 5. For western blot analysis, 30 μg of EVs or fresh cell culture medium (considered as CTR), were diluted 1:4 in Cell Extraction Buffer (ThermoFisher), separated by SDS-PAGE using Bolt 4–12% Bis-Tris Plus gels (Invitrogen). Gels were transferred onto nitrocellulose membranes using Power Blotter Select Transfer Stacks and the Power Blotter System (Invitrogen). Membranes were blocked with I-Block reagent (ThermoFisher) and incubated overnight at 4°C with the following primary antibodies: mouse monoclonal anti-calnexin (Santa Cruz); mouse monoclonal anti-CD63 (abcam); mouse monoclonal anti-CD81 (abcam). Membranes were then incubated with peroxidase-conjugated secondary antibody Goat anti-Mouse (G-21040). Images were captured using Westar Hypernova ECL substrate (Cyanagen) and iBright Western Blot Imaging Systems (Invitrogen).
EmbryoAligner device: Design, production, and characterization
The injection of exogenous substances such as EVs or cells can be invasive for zebrafish embryos. To minimize the manipulation while easing and speeding up embryo transfer processes before and after the injections of EVs or cells, we created a device we named “EmbryoAligner”. The master was fabricated by micro-milling an aluminum block (Kugler MICROMASTER® 5X) based on the chosen design digitalized using 3D-CAD software. The device could then be produced by replica molding from the aluminum master using either Polydimethylsiloxane (PDMS, Sylgard 184 Dow Corning, USA) or agarose using standard techniques [21]. Briefly, PDMS was prepared by mixing the base and the curing agent in a 10:1 w/w ratio, degassed, and cast on the aluminum master. After an additional degassing step, the mold was baked in an oven for 60 minutes at 80°C. Similarly, hot 1.5% agarose was poured over the master and allowed to solidify (approximately 5 minutes). The PDMS and agarose replicas could then easily be peeled from the mold. Optical metrological tests were performed using a 3D optical profiler (Sensofar® S Neox) on both the aluminum master and the PDMS replicas to evaluate the precision of the features. This is important since variations in the heights of the structural elements may compromise the proper alignment and manipulation of the zebrafish embryos. The key measurements are the height of the tail and the main body of the embryo impressed in the mold. PDMS was used for the metrological characterization thanks to its optical properties, while agarose proved ideal for all other experiments.
Zebrafish EVs transplantation
For EVs transplantation experiments, 2 dpf embryos were dechorionated and anaesthetized in 0.3× Danieau’s solution containing PTU, excluding for the Casper embryos, and 0.04 mg/ml Tricaine. Embryos were then positioned in the EmbryoAligner. A total of 23 nL of labeled EVs or dextran resuspended EVs, were back-loaded into pulled borosilicate glass capillary needles (Narishige PC-10, Drummond Scientific) and injected into the Duct of Cuvier of each embryo using the Drummond Nanoject II microinjector (Drummond Scientific) under a fluorescent stereomicroscope (SMZ1500 Nikon). Upon bloodstream assessment, incorrectly injected embryos were discarded, while other were transferred to E3 medium and either immediately imaged under a confocal microscope or incubated at 34°C until the desired time point. At 5 dpf, zebrafish were sacrificed with 2.4 mM Tricaine. The control group was injected with dextran solution without EVs.
Confocal imaging and Image-J analysis
Confocal imaging was performed using a Zeiss LSM800 Airyscan. To visualize EVs in the blood circulation, Tg(gata1:dsRed) and Tg(fli1:EGFP) embryos were imaged immediately after injection. To determine the proportion of EVs internalized by endothelial cells, 6 to 20 Z-stacks were acquired in the caudal plexus of each EVs-injected Tg(fli1:EGFP) embryo 54 hpf at 25X magnification. We merged stacks into a single image using ImageJ software (National Institutes of Health, MD, United States), measured the colocalization of the green and red signals using the JACoP BIOP plugin, and obtained the Pearson’s correlation coefficient (values ranging from 0 to 1) for each image. To determine the effect of EVs on angiogenesis, we analyzed the increase of sprout area in the sub-intestinal veins (SIVs) of Tg(fli1:EGFP) embryos [22] 24 hours after EVs injection. 2 to 10 Z-stacks were captured in each zebrafish SIVs at 10X magnification. Using ImageJ software, stacks were merged into a single image and the area was measured after setting a threshold and selecting the SIVs perimeter. To determine the effect of EVs on the immune compartment, we used Tg(mpeg1:mCherry) embryos, which express fluorescent macrophages. Six hours after EVs injection, embryos were imaged at 25X magnification (10 to 18 Z-stacks). Using ImageJ, stacks were merged into a single image and the area covered by macrophages was measured after setting the proper threshold.
Tube formation assay
Human Umbilical Vein Endothelial Cells (HUVEC, Lonza) were seeded into a Matrigel Basement Membrane Matrix (354234, Corning) precoated 96-well plate at 2×104 cells/well and cultured in Endothelial Basal Medium, in the presence of EVs (20 μg/mL) or PBS (control). Tube formation was examined using the EVOS FL Cell Imaging System. The angiogenic property was assessed by measuring the total branching point and tube length from five random microscopic fields using Python Angiogenesis analyzer. Results are provided as Supplementary information (Please see S1 Fig).
RNA extraction and qPCR of CHT tissues
To assess the effects of EVs in the caudal hematopoietic tissue (CHT) 24 hours after EVs injection, 30 Casper strain embryos per condition were placed in a petri dish and anesthetized with 200 mg/L Tricaine to cut CHT sections with a sterile scalpel (Swann-Morton). Sections were collected in 1.5 mL tubes and washed with PBS 1X. Total RNA was extracted using Trizol reagent (Invitrogen), and RNA was quantified using a Nanodrop spectrophotometer (Thermo Fisher Scientific). Isolated RNA was used for cDNA synthesis using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystem) according to the manufacturer’s instructions. Real-time PCR was performed using Power Up SYBR Green Master Mix (Applied Biosystems) according to the manufacturer’s instructions. Relative mRNA expression for each gene was analyzed by the ddCt method using β-Actin as a housekeeping gene. A list of all primers is provided in the S1 Table.
Two-steps injection, xenotransplantation, imaging, and analysis
Tg(fli1:EGFP) zebrafish embryos were dechorionated at 48 hpf, anesthetized with 200 mg/L Tricaine and placed on the EmbryoAligner containing a solution of 1.5% agarose in E3 medium. 100 μg/mL of EVs were resuspended in a solution of Fluorescein isothiocyanate–dextran in PBS 1X, loaded into borosilicate glass capillary needles, and 23 nL of suspension was injected into the Duct of Cuvier of each embryo using the Drummond Nanoject II microinjector under the fluorescent stereomicroscope. Control group was injected with dextran solution without EVs. After stereomicroscope evaluation of proper EVs injection, embryos were transferred to fresh E3 medium for recovery for 6 h at 28.5°C. 54 hpf, prior to cells injection, 1x106 SK-N-DZ, IMR32 cells and MSCs were labeled with Vybrant DiI Cell-Labeling Solution according to the manufacturer’s instructions. Cells were resuspended in PBS 1X and kept on ice until transplantation. Embryos were anesthetized with 200 mg/L Tricaine and placed on the EmbryoAligner containing a solution of 1.5% agarose in E3 medium. Cells suspension was loaded into borosilicate glass capillary needles (1.0 mm in diameter, World Precision Instruments, Sarasota, FL, USA) and injected into the Duct of Cuvier of the embryos using a pneumatic microinjector (World Precision Instruments) under the fluorescent stereomicroscope. By setting pressure and duration, we injected approximately 100 cells per embryo, as determined by standard cell counting within droplets. Injected embryos were transferred to fresh E3 medium for recovery for 1 h at 28.5°C. The presence of fluorescent tumor cells at the injection site and in the bloodstream was evaluated using the fluorescent stereomicroscope. Images after cells injection were captured by confocal microscope at 10X magnification. Embryos were then incubated at 34°C for the rest of the experiment. Caudal plexus images were captured by confocal microscopy at 72 hpf (10X magnification, Z-stack) and tumor cells area in the caudal plexus was analyzed using ImageJ software.
Statistical analysis
Graphs and statistical analyses were performed using GraphPad Prism software. All data in graphs are from at least three independent experiments±SEM. Statistical significance was determined by unpaired Student’s t-test or two-way ANOVA. Asterisks indicate a significant difference between the treated and the control groups, unless otherwise specified. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.
Results
Characterization of EVs isolated from SK-N-DZ and IMR32 cells cultured at different oxygen conditions
All experiments were performed using two MYCN-amplified NB cell lines: SK-N-DZ derived from a metastatic site and IMR-32 derived from a primary tumor. NB cells were cultured at 20% O2 (normoxia) or 1.5% O2 (hypoxia) before conditioned media (CM) collection and EVs isolation. The morphology of EVs isolated from normoxic (DZNEVs IMRNEVs) and hypoxic (DZHEVs, IMRHEVs) conditions, was analyzed by transmission electron microscopy (TEM). Isolated EVs had a homogeneous size, consistent for both cell lines and unaffected by the oxygen tension at which the parent NB cells were cultured (Fig 1A). The expression of the characteristic EVs markers CD63, CD81 and the absence of negative marker Calnexin was verified by western blotting (Fig 1B). Furthermore, Nanoparticle Tracking Analysis (NTA) resulted in a conserved distribution profile of EVs with an average size of ~100 nm for all conditions (Fig 1C). These data indicate that EVs were successfully isolated from the CM, regardless of the oxygenation condition of the parent cells.
A. Representative images of EVs morphology taken by TEM. Scale bar: 100 nm. B. Validation of EVs markers CD63, CD81, and negative marker Calnexin expression by western blot analysis. Panels represent different blots. Fresh cell culture medium was considered as CTR. C. Size distribution profile of EVs analyzed by NTA.
Zebrafish EmbryoAligner mold design and application
The microinjection of zebrafish embryos is a critical technique employed in numerous models. To simplify and streamline key pre- and during-injection steps such as embryo manipulation for alignment and securing in place, we designed and produced a customized tool (Fig 2). Panels A-B show the final design, the aluminum mold, and an agarose replica of the EmbryoAligner. Each replica housed up to 6 fish, aligned and kept in place in the respective slots to perform injections at different points. Given our experimental needs, the mold was designed replicating the size of embryos at 48 hpf. Panels C-E report the results of the profilometer analysis. For the aluminum master, the mean height of the tail was 0.113 ± 0.003 mm, slightly below the nominal CAD value set at 0.12 mm. The body measured 0.237 ± 0.003 mm, with a CAD value of 0.24 mm. These small deviations can be correlated to the default machine error and are within the expected tolerances. Given the poor optical properties of agarose, measures on replicas were performed using PDMS. Here, the height for the tail and body were 0.107 ± 0.0017 and 0.229 ± 0.003 mm, respectively. Thanks to the EmbryoAligner device, embryos were quickly aligned by gently pushing them with a pipette tip. After the injection, embryos were removed from the agarose device with a Pasteur pipette and transferred to a petri dish with E3 medium. The mold that we created is an easy and quick method that greatly reduces embryo manipulation while improving injection accuracy. The device is reusable, and several replicas can be created for each experiment.
A. 3D-CAD design of the master; dimensions in mm. B. Finished EmbryoAligner aluminum mold and PDMS replica. C. Selected areas of interest for the metrological analyses. D. Metrological characterization of the tail height. Comparisons between master mold and PDMS replicas measured in the highlighted areas of interest A-C. CAD reference value was 0.12 mm. E. Metrological characterization of the body height. Comparisons between master mold and PDMS replicas measured in the highlighted areas of interest 1–4. CAD reference values were 0.12 mm for the region 1–2 and 0.24 mm for 1–4. D. and E. are box mean bars with square error.
EVs visualization in the blood flow
Circulating EVs play crucial roles as mediators of cell communication between different tissues and are involved in a wide range of cellular processes [23]. In vivo distribution and kinetics of EVs shedding and internalization can be accurately assessed using non-invasive imaging techniques, for a more comprehensive understanding of their therapeutic effects in vivo [24]. The transgenic line Tg(gata1:dsRed) has dsRed-positive primitive erythroid cells, enabling visualization of labeled EVs in the blood flow. Briefly, we labeled normoxic EVs using the Vybrant DiO Cell-Labeling Solution, injected them into the zebrafish embryo at 48 hpf and immediately evaluated their presence in vivo. EVs could be seen as clusters in the blood circulation, with the majority homing within 30 minutes from injection (S1 Video). EVs stopped mainly in the CHT, a highly vascularized region of the zebrafish embryo [25]. These observations suggest that in vivo interactions between cells and EVs are a rapid process, supporting the choice of relatively short timepoints through our investigation.
EVs internalization by endothelial cells
The aggressiveness and propensity to metastasize of a solid tumor like NB also depend on changes in the tumor niche, including angiogenesis. To address whether EVs affected angiogenesis in vitro we analyzed changes in the ability of endothelial cells to form capillary-like structures using a tube formation assay on Matrigel (S1 Fig). Our findings indicate that EVs from hypoxic conditions significantly increased the number of nodes and total tube length in network structures compared to untreated controls. To assess the ability of EVs to be internalized by recipient cells and study their effect on endothelial cells in vivo, we used the Tg(fli1:EGFP) transgenic line expressing enhanced green fluorescent protein (EGFP) in endothelial cells (Fig 3). To evaluate whether different oxygen tensions could affect the internalization of EVs by endothelial cells, we injected 23 nL of labeled NB-derived normoxic and hypoxic EVs into Tg(fli1:EGFP) embryos at 48 hpf (Fig 3A and 3B). EVs were rapidly internalized by endothelial cells 30 minutes post injection (S2 Video). The CHT (Fig 3C and 3D) were then imaged at 6 hpi, proving that EVs derived from NB cells cultured in hypoxic conditions were internalized more when compared with their normoxic counterparts. Quantifications of the correlation coefficient gave values of 0.316±0.02 and 0.175±0.02 for DZHEVs and DZNEVs, respectively, and 0.323±0.02 and 0.150±0.02 for IMRHEVs and IMRNEVs (Fig 3E). This suggests that a greater internalization of EVs leads to an increased transportation of their cargo into recipient cells, thereby impacting them.
A-B. Injection site, imaging site, and timeline for the transgenic line Tg(fli1:EGFP) injected with labeled NB-derived EVs (red) at 48 hpf. The CHT was imaged after 6 hours to evaluate internalization by endothelial cells (green). C. Representative 6 hpi CHT images of internalized EVs derived from each cell line and oxygenation condition as indicated. Scale bars 50 μm. D. Higher magnification images in selected areas. White arrows indicate overlapping areas (yellow) of labeled EVs (red) internalized by endothelial cells (green). Scale bars 10 μm. E. Evaluation of internalization of labeled EVs by endothelial cells via ImageJ software. n = 88 from at least 3 independent experiments. *** p < 0.001.
Hypoxic NB-derived EVs promote angiogenesis
Angiogenesis plays an important role in tumor growth, invasion, and metastasis, and is known to be induced, among other factors, by hypoxia [26]. SIVs sprout from the Duct of Cuvier and connect to the posterior cardinal vein and are used as a model to study venous angiogenesis [9, 27, 28]. To evaluate whether hypoxic EVs can stimulate angiogenesis, we measured the SIVs area of Tg(fli1:EGFP) embryos at 24 hpi (Fig 4A–4C). We found that the injection of DZHEVs induced 60% more sprouting of vessels in the SIVs compared to the embryos injected with DZNEVs. Similarly, the injection of IMRHEVs induced 50% more sprouting compared to the embryos injected with IMRNEVs (Fig 4D). These results demonstrate that hypoxic NB EVs are indeed strong effectors of angiogenesis in vivo. Furthermore, as shown in S3A and S3B Fig, the injection of EVs isolated from mesenchymal stem cells (MSCs), cultured under normoxic and hypoxic conditions, revealed that these EVs had no significant effect on angiogenesis compared to the control group. This indicates that non-tumor EVs did not stimulate angiogenesis in zebrafish embryos.
A-B. Injection site, imaging site, and timeline for the transgenic line Tg(fli1:EGFP) injected with NB-derived EVs at 48 hpf. C. SIVs were imaged 24 hpi to evaluate the sprouting of vessels. SIVs areas analyzed are highlighted by white dotted lines. Scale bars 100 μm. D. Evaluation of SIVs area via ImageJ software. n = 137 from at least 3 independent experiments. * p < 0.05.
EVs are internalized by macrophages and can affect their proliferation
EVs are also responsible for the modulation of inflammation in disease progression [29]. EVs derived from cancer cells have the potential to significantly alter the phenotypes and functions of macrophages, thereby promoting tumor development [30]. To investigate the effects of EVs on macrophages, we injected the transgenic line Tg(mpeg1:mCherry) with 23 nL of NB-derived EVs at 48 hpf (Fig 5A and 5B). This transgenic line specifically labels macrophages, enabling the visualization of their behavior in vivo [18]. Patrolling macrophages successfully internalized tumor EVs (Fig 5C, S3 Video). To evaluate the effects of EVs on macrophages proliferation/mobilization, we analyzed the area covered by macrophages in the zebrafish embryo at 6 hpi (Fig 5D). Compared to normoxic DZNEVs and IMRNEVs (areas of 1.287±0.1, and 0.936±0.1 respectively), we demonstrated that hypoxic DZHEVs and IMRHEVs (1.701±0.14 1.496±0.2, respectively) significantly promoted macrophages proliferation (Fig 5E). Although the association between the number of macrophages and their mobilization in tumor progression is still uncertain, these data indicate that hypoxic EVs isolated from NB SK-N-DZ and IMR32 cell lines target macrophages by inducing their mobilization.
A-B. Injection site, imaging site, and timeline for the transgenic line Tg(mpeg1:mCherry) injected with NB-derived EVs at 48 hpf. C. Representative image of labeled EVs (green, white arrows) internalized by macrophages (red). Scale bar 20 μm. D. Macrophages mobilization in the CHT 6 hpi. Scale bars 100 μm. E. Evaluation of macrophages area via ImageJ software. n = 125 from at least 2 independent experiments. * p < 0.05.
Hypoxic EVs influence the expression of mmp9 and cxcl8b in the CHT
Like the phenotype of the mammalian bone marrow, the CHT is the first homing and expansion site for hematopoietic stem cells in the zebrafish embryo [31]. Zebrafish and mammals also share similarities in key cytokines and chemokines such as CXCL12, necessary for the hematopoietic stem and progenitor cells guidance through the CXCL2–CXCR4 signaling axis [32, 33]. To mimic metastatic colonization in the CHT and investigate the effects of NB-derived EVs on the CHT, we injected the EVs in the blood circulation of Casper zebrafish embryos at 48 hpf, isolated CHT tissues at 24 hpi (Fig 6A), and analyzed the expression of the inflammatory chemokine cxcl8b [34], and the matrix metalloproteinase-9 (mmp-9) and mmp-2, which correlate with neuroblastoma metastasis and progression (Fig 6B) [35–38]. We found that the expression of mmp-9 was significantly increased in the embryos injected with DZNEVs and DZHEV (2.4- and 2.8-fold, respectively) compared to the control group. The expression of mmp-9 increased 2.3-fold in the group injected with IMRHEVs and decreased 1.6 fold in the IMRNEVs-injected group compared to the control group. No significant changes were measured in mmp-2 expression between normoxic and hypoxic-EVs from both cell lines. Finally, we demonstrated that the expression of cxcl8b significantly increased in the embryos injected with DZNEVs and DZHEVs (1.6 and 2.4-fold, respectively) compared to the control group. The expression of cxcl8b did not differ in the embryos injected with IMR32-derived EVs. These findings indicate that EVs indeed influence the expression of genes involved in metastatic progression, with different effects depending on the parental cells of origin. EVs derived from the metastatic SK-N-DZ cells, are more potent than those from IMR32, a primary tumor cell line.
A. Injection site, analysis site, and timeline for Casper embryos injected with NB-derived EVs. B. Relative mRNA expression for mmp9, mmp2 and cxcl8b measured by qPCR in CHT tissues 24 hours after EVs injection. Data are represented as 2-ΔΔCt, β-Actin was used as housekeeping gene. n = 450 from at least 3 independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.
Hypoxic EVs promote NB cells proliferation in the CHT
Although the key role of EVs in the preparation of the pre-metastatic niche and tumor establishment has been extensively studied [39–41], it has never been investigated in NB using an in vivo zebrafish model. We mimicked metastatic colonization by a two-step injection in Tg(fli1:EGFP) embryos 48 hpf (Fig 7A): first with NB-derived EVs, and 6 hours later with NB cells (S2 Fig). NB cells proliferation in the CHT was evaluated at 24 hpi (Fig 7B). DZHEVs-injected embryos had a higher NB cell area compared to their normoxic counterpart (2.01±0.2 and 1.3±0.2, respectively). A similar trend was measured for IMREVs-injected embryos (1.72±0.2 and 1.11±0.13 for hypoxic and normoxic EVs, respectively) (Fig 7C). These data suggest that hypoxia induces NB cells to shed EVs that are more potent in preconditioning and targeting the potential metastatic site and modifying the CHT microenvironment by promoting NB cells proliferation. Furthermore, we proved that this effect is specific to NB cells and does not occur in non-tumor cells like MSCs (please see S3C–S3E Fig). Similarly, MSCs-derived EVs had no effect on tumor cell proliferation (S3G and S3H Fig). These findings, along with the results reported above, demonstrate that the homing and proliferation of tumor cells in the CHT is mediated by tumor-derived EVs, rather than by simple accumulation caused by blood circulation.
A. Injection site, analysis site, and timeline for Tg(fli1:EGFP) embryos injected with NB-derived EVs and NB cells. Embryos were injected with EVs isolated from SK-N-DZ or IMR32 cells cultured in normoxic or hypoxic conditions, and subsequently injected with SK-N-DZ and IMR32 cells, respectively. B. Representative CHT images 24 hpi of EVs and 18 hpi of SK-N-DZ cells (orange). Scale bar 100 μm. C. Representative CHT images 24 hpi of EVs and 18 hpi of IMR32 cells (orange). Scale bar 100 μm. D. Evaluation of proliferated SK-N-DZ cells area in the CHT at 72 hpf. n = 53 from at least 3 independent experiments. * p < 0.05. E. Evaluation of proliferated IMR32 cells area in the CHT at 72 hpf. n = 59 from at least 3 independent experiments. * p < 0.05.
Discussion and conclusions
Cancer cell derived EVs emerged as potential mediators of metastatic spread and of the pre-metastatic niche formation [42], while hypoxia, a decline of tissue oxygen level, is a known hallmark of cancer favoring tumor growth, angiogenesis, and metastasis [43]. In our recent study, we demonstrated the ability of hypoxic EVs to increase the aggressiveness of NB cells in vitro [44]. Here, we studied the role of hypoxic and normoxic EVs in NB metastatic dissemination in an in vivo zebrafish (Danio Rerio) model using specific transgenic lines [45, 46]. We first characterized purified EVs released by two MYCN amplified NB cell lines cultured in normoxic and hypoxic conditions, proving their similarities in morphology, size, and surface markers independently on the culture conditions of the parent cells. Before moving to the in vivo experiments, we designed and fabricated a customized device to minimize manipulation of the zebrafish embryos and increase repeatability of our experiments. Each device could accommodate and correctly position 6 embryos, greatly facilitating and reducing errors during injections into the Duct of Cuvier. This site was chosen as it ensures a rapid and complete dissemination of exogenous components throughout the blood circulation [47].
Injected fluorescently labeled-EVs preferentially home in the CHT of Tg(gata1:dsRed) embryos. The CHT is a highly vascularized region in the zebrafish tail [25] sharing similarities with the cytoarchitecture of the mammalian bone marrow [31], the main metastatic site in NB. Furthermore, Tg(fli1:EGFP) embryos expressing endothelial cells in green, allowed visualization of EVs in the blood circulation proving the higher internalization by endothelial cells of EVs derived from hypoxic NB cells compared to their normoxic counterparts. These findings confirm the EVs-mediated preferential targeting of the bone marrow by NB, and a potent role for hypoxia in facilitating EVs uptake and cargo delivery by target cells. We then proved that hypoxic EVs induced an increase of sprout areas in the SIVs of transgenic zebrafish embryos [27], further supporting their role in promoting tumor spread by favoring neoangiogenesis [48, 49]. This finding is consistent with several reports on the higher angiogenic capacity of hypoxic EVs in various cancers, supporting the increased oxygen and nutrients demand in growing tumors [50, 51]. The complex chain of events dictating tumor metastatic spread also involves the immune compartment, with macrophages promoting angiogenesis and the formation of a pre-metastatic niche through the secretion of chemokines [52] and transforming into tumor-associated macrophages (TAMs) [53, 54]. Using the transgenic line Tg(mpeg1:mCherry) we here showed that injection of hypoxic EVs effectively led to increased mobilization of macrophages compared to their normoxic counterparts, further proving the role of hypoxia and the involvement of macrophages in favoring tumor progression and angiogenesis. However, given that macrophages are remarkably heterogenous, whether it is the pro- or anti-inflammatory phenotype that increases after EVs injection remains to be investigated. Considering the preferential homing of EVs [31, 55], we collected and analyzed CHT tissues 24 hpi to measure key matrix metalloproteinases and chemokines mainly released by TAMs and known to promote angiogenesis, tumor invasion, and metastasis [56, 57]. The expression of mmp-9 increased in embryos injected with hypoxic EVs, further evidence of the role of hypoxia on angiogenesis and macrophage function. No significant differences emerged for mmp-2, in line with reports where its increased expression in NB patients was observed at advanced stages and in those who died from progressive disease [37]. The slight variation we observed at our 24 hpi timepoint leads to the hypothesis that analyses at longer timepoints, with both normoxic and hypoxic EVs, might result in higher differences for both mmp-9 and mmp-2 expression. Cxcl8b expression increased only after injection of EVs derived from the metastatic SK-N-DZ cells, supporting the hypothesis that EVs released by a metastatic line have a greater effect compared to the EVs released by a primary tumor cell line.
Last, we mimicked the steps of NB metastatic niche establishment with a double injection of EVs at 48 hpf, followed by NB cells 6 hours later. Timings were determined based on the effects of EVs on angiogenesis and macrophages mobilization which we analyzed at 6 hpi and the fact that the duct of Cuvier physiologically disappears at later timepoints. Hypoxic EVs were again more potent drivers of NB aggressiveness by increasing NB cells proliferation in the CHT 24 hours post injections compared to their normoxic counterparts.
Our findings suggest that EVs derived from NB cells play a crucial part in NB spread and the formation of pre-metastatic niches with hypoxia emerging as a potent factor influencing the function of EVs by inducing angiogenesis, macrophages recruitment, and affecting the expression of mmp-9 and cxcl8b. Furthermore, mimicking the steps of NB metastatic niche establishment confirmed that the pre-conditioning of the CHT with hypoxic EVs promotes the proliferation of NB cells. Overall, we also highlight the ability of the zebrafish to serve as potent tool to study the in vivo interaction between EVs and cells, thanks to the combination of transgenic lines and high-resolution in vivo imaging. Although longer time points are necessary to fully follow the complex temporal sequence of events leading to metastatic spread, we believe that our study gives proof of the potency of this model. By enabling to better understand the key pathways at play and the underlying molecular mechanisms, we are confident that further studies will lead to better cures and targeted therapies for NB and beyond.
Supporting information
S1 Video. DiO-labeled NB-derived EVs in the blood stream.
Representative CHT video of labeled EVs (green) injected at 48 hpf in the Duct of Cuvier of Tg(gata1:dsRed) zebrafish embryos expressing red blood cells in red. Magnification 25X. Scale bar 10 μm.
https://doi.org/10.1371/journal.pone.0316103.s002
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S2 Video. DiI-labeled NB-derived EVs are internalized by endothelial cells.
ISV (intersegmental vessel) representative video of labeled EVs (red) injected in the Duct of Cuvier of Tg(fli1:EGFP) zebrafish embryos at 48 hpf internalized by endothelial cells (in green). Magnification 25X. Scale bar 5 μm.
https://doi.org/10.1371/journal.pone.0316103.s003
(AVI)
S3 Video. DiO-labeled NB-derived EVs are internalized by macrophages.
Representative video of labeled EVs (green) injected at 48 hpf in the Duct of Cuvier of Tg(mpeg1:mCherry) zebrafish embryos internalized by macrophages (in red). Magnification 25X. Scale bar 20 μm.
https://doi.org/10.1371/journal.pone.0316103.s004
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S1 Fig. Tube forming assay.
The angiogenic property was assessed by measuring the total branching point and tube length from five random microscopic fields using Python Angiogenesis analyzer.
https://doi.org/10.1371/journal.pone.0316103.s005
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S2 Fig. NB cells in the CHT after injection.
Acquisition at 1 hpi of Tg(fli1:EGFP) embryos injected at 54 hpf with NB cells. Magnification 10X. Scale bar 100 μm.
https://doi.org/10.1371/journal.pone.0316103.s006
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S3 Fig. Effects of MSCs-derived EVs on angiogenesis and on NB cells proliferation.
A. Tg(fli1:EGFP) embryos were injected with MSCs-derived EVs at 48 hpf. SIVs were imaged 24 hpi to evaluate the sprouting of vessels. SIVs areas analyzed are highlighted by white dotted lines. Scale bars 100 μm. B. Evaluation of SIVs area via ImageJ software. n = 32. C. Timeline for Tg(fli1:EGFP) embryos injected with NB-derived EVs and MSCs. Embryos were injected with EVs isolated from SK-N-DZ cells, and subsequently injected with MSCs. D. Representative CHT images 24 hpi of EVs and 18 hpi of MSCs cells (orange). Scale bar 100 μm. E. Evaluation of proliferated MSCs area in the CHT at 72 hpf. n = 37. F. Timeline for Tg(fli1:EGFP) embryos injected with MSCs-derived EVs and NB cells. Embryos were injected with EVs isolated from MSCs, and subsequently injected with SK-N-DZ cells. G. Representative CHT images 24 hpi of EVs and 18 hpi of SK-N-DZ cells (orange). Scale bar 100 μm. H. Evaluation of proliferated SK-N-DZ area in the CHT at 72 hpf. n = 35. All variations were not statistically significant.
https://doi.org/10.1371/journal.pone.0316103.s007
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Acknowledgments
We thank Dr. Georges Lutfalla for providing us the Tg(mpeg1:mCherry) zebrafish line. We acknowledge Fondazione Umberto Veronesi for supporting Dr. Pina Fusco.
References
- 1. Kogure A., Yoshioka Y. & Ochiya T. Extracellular Vesicles in Cancer Metastasis: Potential as Therapeutic Targets and Materials. Int J Mol Sci 21, 1–16 (2020). pmid:32585976
- 2. Wang S. E. Extracellular Vesicles and Metastasis. Cold Spring Harb Perspect Med 10, 1–18 (2020).
- 3. Willms E., Cabañas C., Mäger I., Wood M. J. A. & Vader P. Extracellular vesicle heterogeneity: Subpopulations, isolation techniques, and diverse functions in cancer progression. Front Immunol 9, 355368 (2018). pmid:29760691
- 4. Kanada M., Bachmann M. H. & Contag C. H. Signaling by Extracellular Vesicles Advances Cancer Hallmarks. Trends Cancer 2, 84–94 (2016). pmid:28741553
- 5. Sheehan C. & D’Souza-Schorey C. Tumor-derived extracellular vesicles: molecular parcels that enable regulation of the immune response in cancer. J Cell Sci 132, (2019). pmid:31615844
- 6. Luksch R. et al. Neuroblastoma (Peripheral neuroblastic tumours). Crit Rev Oncol Hematol 107, 163–181 (2016). pmid:27823645
- 7. Liu S. et al. Metastasis pattern and prognosis in children with neuroblastoma. World J Surg Oncol 21, 130 (2023). pmid:37046344
- 8. Huang M. & Weiss W. A. Neuroblastoma and MYCN. Cold Spring Harb Perspect Med 3, (2013).
- 9. Nicoli S. & Presta M. The zebrafish/tumor xenograft angiogenesis assay. Nat Protoc 2, 2918–2923 (2007). pmid:18007628
- 10. Gamble J. T., Elson D. J., Greenwood J. A., Tanguay R. L. & Kolluri S. K. The zebrafish xenograft models for investigating cancer and cancer therapeutics. Biology (Basel) 10, (2021). pmid:33804830
- 11. Wang X. et al. Zebrafish Xenograft Model for Studying Pancreatic Cancer-Instructed Innate Immune Microenvironment. Int J Mol Sci 23, (2022). pmid:35742884
- 12. Hurtado P. et al. Modelling metastasis in zebrafish unveils regulatory interactions of cancer-associated fibroblasts with circulating tumour cells. Front Cell Dev Biol 11, (2023). pmid:36949770
- 13. Paj Her Z., Siong Yeo K., Howe C., Levee T. & Zhu S. Zebrafish Model of Neuroblastoma Metastasis.
- 14. Påhlman S. & Mohlin S. Hypoxia and hypoxia-inducible factors in neuroblastoma. Cell Tissue Res 372, 269 (2018). pmid:29032465
- 15. Vega F. M., Huertas-Castaño C., Gómez-Muñoz M. A. & Pardal R. Hypoxia in the initiation and progression of neuroblastoma tumours. International Journal of Molecular Sciences vol. 21 Preprint at (2020). pmid:31861671
- 16. White R. M. et al. Transparent adult zebrafish as a tool for in vivo transplantation analysis. Cell Stem Cell 2, 183 (2008). pmid:18371439
- 17. Lawson N. D. & Weinstein B. M. In vivo imaging of embryonic vascular development using transgenic zebrafish. Dev Biol 248, 307–318 (2002). pmid:12167406
- 18. Ellett F., Pase L., Hayman J. W., Andrianopoulos A. & Lieschke G. J. mpeg1 promoter transgenes direct macrophage-lineage expression in zebrafish. Blood 117, e49 (2011). pmid:21084707
- 19. Traver D. et al. Transplantation and in vivo imaging of multilineage engraftment in zebrafish bloodless mutants. Nature Immunology 2003 4:12 4, 1238–1246 (2003). pmid:14608381
- 20. Fan R. Y. et al. Zebrafish xenograft model for studying mechanism and treatment of non-small cell lung cancer brain metastasis. Journal of Experimental and Clinical Cancer Research 40, 1–15 (2021).
- 21. Beebe D. J., Mensing G. A. & Walker G. M. Physics and applications of microfluidics in biology. Annual Review of Biomedical Engineering vol. 4 261–286 Preprint at (2002). pmid:12117759
- 22. Serbedzija G. N., Flynn E. & Willett C. E. Zebrafish angiogenesis: A new model for drug screening. Angiogenesis 3, 353–359 (1999). pmid:14517415
- 23. Alberro A., Iparraguirre L., Fernandes A. & Otaegui D. Extracellular Vesicles in Blood: Sources, Effects, and Applications. Int J Mol Sci 22, 8163 (2021). pmid:34360924
- 24. Gangadaran P., Hong C. M. & Ahn B. C. An update on in vivo imaging of extracellular vesicles as drug delivery vehicles. Front Pharmacol 9, 334362 (2018). pmid:29541030
- 25. Karthik S. et al. Synergistic interaction of sprouting and intussusceptive angiogenesis during zebrafish caudal vein plexus development. Scientific Reports 2018 8:1 8, 1–15 (2018). pmid:29959335
- 26. Jiang X. et al. The role of microenvironment in tumor angiogenesis. Journal of Experimental & Clinical Cancer Research 2020 39:1 39, 1–19 (2020). pmid:32993787
- 27. Isogai S., Horiguchi M. & Weinstein B. M. The vascular anatomy of the developing zebrafish: an atlas of embryonic and early larval development. Dev Biol 230, 278–301 (2001). pmid:11161578
- 28. Goi M. & Childs S. J. Patterning mechanisms of the sub-intestinal venous plexus in zebrafish. Dev Biol 409, 114 (2016). pmid:26477558
- 29. Wang T. et al. Functions of Exosomes in the Triangular Relationship between the Tumor, Inflammation, and Immunity in the Tumor Microenvironment. J Immunol Res 2019, 4197829 (2019). pmid:31467934
- 30. Tian J. W. et al. Tumor Cell-derived Extracellular Vesicles in Modulating Phenotypes and Immune Functions of Macrophages: Mechanisms and Therapeutic Applications. Journal of Cancer vol. 14 1321–1334 Preprint at (2023). pmid:37283792
- 31. Murayama E. et al. Tracing hematopoietic precursor migration to successive hematopoietic organs during zebrafish development. Immunity 25, 963–975 (2006). pmid:17157041
- 32. Glass T. J. et al. Stromal cell-derived factor-1 and hematopoietic cell homing in an adult zebrafish model of hematopoietic cell transplantation. Blood 118, 766–774 (2011). pmid:21622651
- 33. Wattrus S. J. & Zon L. I. Stem cell safe harbor: the hematopoietic stem cell niche in zebrafish. Blood Adv 2, 3063–3069 (2018). pmid:30425071
- 34. Sommer F., Torraca V. & Meijer A. H. Chemokine Receptors and Phagocyte Biology in Zebrafish. Front Immunol 11, 325 (2020). pmid:32161595
- 35. Egeblad M. & Werb Z. New functions for the matrix metalloproteinases in cancer progression. Nature Reviews Cancer 2002 2:3 2, 161–174 (2002). pmid:11990853
- 36. Sugiura Y, Shimada H, Seeger RC, Laug WE & DeClerck YA. Matrix metalloproteinases-2 and -9 are expressed in human neuroblastoma: contribution of stromal cells to their production and correlation with metastasis. Cancer Res 58, 2209–2216 (1998). pmid:9605768
- 37. Tasnim Ara B. et al. Determination of Imbalance Between MMP-2 and TIMP-2 in Human Neuroblastoma by Reverse-Transcription Polymerase Chain Reaction and Its Correlation With Tumor Progression. (2000).
- 38. Chantrain C. F. et al. Stromal Matrix Metalloproteinase-9 Regulates the Vascular Architecture in Neuroblastoma by Promoting Pericyte Recruitment. CANCER RESEARCH vol. 64 http://aacrjournals.org/cancerres/article-pdf/64/5/1675/2523355/zch00504001675.pdf (2004). pmid:14996727
- 39. dos Anjos Pultz B. et al. The multifaceted role of extracellular vesicles in metastasis: Priming the soil for seeding. Int J Cancer 140, 2397–2407 (2017). pmid:28090647
- 40. Wang X. et al. Investigation of the roles of exosomes in colorectal cancer liver metastasis. Oncol Rep 33, 2445–2453 (2015). pmid:25760247
- 41. Costa-Silva B. et al. Pancreatic cancer exosomes initiate pre-metastatic niche formation in the liver. Nat Cell Biol 17, 816–826 (2015). pmid:25985394
- 42. van der Pol E., Böing A. N., Harrison P., Sturk A. & Nieuwland R. Classification, functions, and clinical relevance of extracellular vesicles. Pharmacol Rev 64, 676–705 (2012). pmid:22722893
- 43. Venturella M., Criscuoli M., Carraro F., Naldini A. & Zocco D. Interplay between Hypoxia and Extracellular Vesicles in Cancer and Inflammation. Biology (Basel) 10, (2021). pmid:34209290
- 44. Fusco P. et al. miR-210-3p enriched extracellular vesicles from hypoxic neuroblastoma cells stimulate migration and invasion of target cells. Cell Biosci 13, 89 (2023). pmid:37202777
- 45. Astell K. R. & Sieger D. Zebrafish In Vivo Models of Cancer and Metastasis. Cold Spring Harb Perspect Med 10, 1–17 (2020). pmid:31615862
- 46. Hyenne V. et al. Studying the Fate of Tumor Extracellular Vesicles at High Spatiotemporal Resolution Using the Zebrafish Embryo. Dev Cell 48, 554–572.e7 (2019). pmid:30745140
- 47. Pontes K. C. de S. et al. Evaluation of (fli:GFP) Casper Zebrafish Embryos as a Model for Human Conjunctival Melanoma. Invest Ophthalmol Vis Sci 58, 6065–6071 (2017). pmid:29204645
- 48. Nathan J. & Kannan R. R. Antiangiogenic molecules from marine actinomycetes and the importance of using zebrafish model in cancer research. Heliyon 6, (2020). pmid:33319107
- 49. Chávez M. N., Aedo G., Fierro F. A., Allende M. L. & Egaña J. T. Zebrafish as an Emerging Model Organism to Study Angiogenesis in Development and Regeneration. Front Physiol 7, 56 (2016). pmid:27014075
- 50. Muz B., Puente P. de la Azab, F. & Azab A. K. The role of hypoxia in cancer progression, angiogenesis, metastasis, and resistance to therapy. Hypoxia 3, 83 (2015). pmid:27774485
- 51. Jung K. O., Youn H., Lee C. H., Kang K. W. & Chung J. K. Visualization of exosome-mediated miR-210 transfer from hypoxic tumor cells. Oncotarget 8, 9899 (2017). pmid:28038441
- 52. Waugh D. J. J. & Wilson C. The Interleukin-8 Pathway in Cancer. Clinical Cancer Research 14, 6735–6741 (2008). pmid:18980965
- 53. Chen X. W. et al. CYP4A in tumor-associated macrophages promotes pre-metastatic niche formation and metastasis. Oncogene 36, 5045 (2017). pmid:28481877
- 54. Guo W., Li Y., Pang W. & Shen H. Exosomes: A Potential Therapeutic Tool Targeting Communications between Tumor Cells and Macrophages. Molecular Therapy 28, 1953–1964 (2020). pmid:32563274
- 55. Matthay K. K. et al. Neuroblastoma. Nature Reviews Disease Primers 2016 2:1 2, 1–21 (2016).
- 56. Dandekar R. C., Kingaonkar A. V. & Dhabekar G. S. Role of macrophages in malignancy. Ann Maxillofac Surg 1, 150 (2011). pmid:23482819
- 57. Dunn G. P., Old L. J. & Schreiber R. D. The immunobiology of cancer immunosurveillance and immunoediting. Immunity 21, 137–148 (2004). pmid:15308095