We thank the Zebrafish Facility from the Centre for Comparative Medicine Research (CCMR) at The University of Hong Kong. We thank Ms Jo Yiu Ling Wong for animal assistance. The works were supported by the Theme-based Research Scheme (T12-702/20-N), Health and Medical Research Fund Projects No.08192066 and No. 08193106, National Natural Science Foundation of China (NSFC)/Research Grants Council (RGC) Joint Research Scheme 2021/22 N_HKU745/21, the National Key R &#38; D program of China (2023YFA1800100) and the Centre for Oncology and Immunology under the Health@InnoHK Initiative funded by the Innovation and Technology Commission, the Government of Hong Kong SAR, China (A.Y.H.L.).

All animal procedures were approved by the Committee of the Use of Laboratory and Research Animals (CULATR) at the University of Hong Kong (HKU).
1. Preparation of injection material
<ol>
	<li>Prepare cell suspension medium: 0.9x PBS + 5% fetal bovine serum (FBS) + 1% penicillin/streptomycin (Pen/Strep).</li>
	<li>Sterilize the Hamilton syringe and attached needle (34 G, 10 mm length, 10 &#181;L capacity, see Table of Materials) under UV light with a 270-280 nm wavelength and expose the items for 2 h.</li>
	<li>Prepare sterilized cotton buds, foam pads, and tissue paper.</li>
	<li>Prepare the anesthesia mixture by diluting tricaine with sterilized fish water to a final concentration of 0.2 mg/mL.</li>
	<li>For the transplantation of WKM cells in Casper fish, 2 days before the transplantation, administer a sublethal irradiation dose of 25 Gy, divided into two sessions of 12.5 Gy each, given on the same day with a 7-8 h interval between doses. For the irradiation process, place 5-10 adult fish in a 100 mm Petri dish containing fish water and expose them in a gamma irradiator. 
	NOTE: The dose was determined to be sublethal based on survival analysis after exposing Casper fish to various irradiation levels up to 35 Gy, monitored over 30 days (Figure 1A). The exact duration of exposure should be calibrated according to the settings of the irradiator. No irradiation is needed for procedures involving only drug delivery.</li>
	<li>Use a laboratory autoclave steam sterilizer to sterilize the fish water intended for housing treated fish (see Table of Materials) to prevent infections and ensure a safe recovery environment for the fish post-injection. 
	NOTE: The sterilization process is tailored to the specific autoclave unit used, and we adhere to the manufacturer&#39;s guidelines for autoclaving liquid solutions to prepare the fish water.</li>
	<li>Prepare needle washing buffer: several tubes of 75% ethanol and ultrapure water in 1.5 mL microcentrifuge tubes.</li>
	<li>Prepare transplantation buffer by dissolving FITC-Dextran (Molecular Weight: 10,000) in the previously prepared cell suspension medium (0.9x PBS with 5% FBS and 1% Pen/Strep). Adjust the concentration to 100 &#181;g/mL. 
	NOTE: This buffer will be used for the injections, with a planned volume of 2 &#181;L per fish.</li>
</ol>
2. Preparation of whole kidney marrow for transplantation
<ol>
	<li>Euthanize the necessary number of Tg(mpo: EGFP) fish by submerging them in a 0.4 mg/mL tricaine solution, ensuring they remain in the solution for at least 10 min following the cessation of opercular movement.</li>
	<li>Dissect the Tg(mpo: EGFP) fish to isolate and transfer the kidney marrow (KM) into the cell suspension medium within a 1.5 mL microcentrifuge tube as described previously22.</li>
	<li>Disaggregate the KM cells by pipetting and filtering the suspension through a 40 &#181;m nylon cell strainer.</li>
	<li>Centrifuge the suspension at 500 &#215; g for 8 min.</li>
	<li>Discard the supernatant and resuspend the pellet in 500 &#181;L of cell suspension medium.</li>
	<li>Count the cells using an automated counter with a 10 &#181;L aliquot of the cell suspension.</li>
	<li>Centrifuge again at 500 &#215; g for 8 min.</li>
	<li>Adjust the cell concentration to 150,000 cells/&#181;L using the transplantation buffer.</li>
</ol>
3. Injection procedure
<ol>
	<li>Thoroughly wash the Hamilton syringe 3-4x with 75% ethanol before the injection. Rinse the syringe 3-4x with ultrapure water to remove any ethanol residue.</li>
	<li>Administer anesthesia to the zebrafish by immersing them in the anesthesia mixture until they are fully anesthetized, evidenced by diminished movement and loss of reflex responses. Once the fish are sedated, position them with their dorsal side facing upward and their head oriented to the left (Figure 1B). Carefully lay the fish on clean, damp tissue paper, which will help stabilize them for the injection. Make sure the tissue is sufficiently moist to prevent the fish from drying out, yet not so wet as to cause slipping.</li>
	<li>Grasp the Hamilton syringe firmly and angle the needle at ~45&#176; to the primary vessel (the caudal vein) to target it effectively while minimizing damage to surrounding tissues. For Capser zebrafish, aim for a section of the vessel that is visible through its translucent body for accurate needle placement (Figure 1B). For wild-type zebrafish (Tubingen, TU) with pigmented skin, aim for primary vessels which are along the body axis and posterior to the anus in the region of the primary vessels (Figure 1B). Gently and smoothly insert the needle into the designated vessel, administer the injection (2 &#181;L) slowly to minimize stress, ensure optimal uptake of the substance, and apply gentle pressure to the site with a cotton bud for 10 s to control any bleeding after the injection.</li>
	<li>Observe under a microscope to confirm the presence of GFP signals, ensuring the effectiveness of the injection. Confirm that the cells or dyes appear as green signals within the vessel (Figure 2A).</li>
	<li>Allow the fish to recover in freshly sterilized fish water post injection for 10 min till transfer to a static tank.</li>
	<li>Clean the needle as previously described between injections of different reagents.</li>
	<li>To prevent cell clumping, flick the cell suspension every 10 min to resuspend the cells.</li>
	<li>Maintain a maximum of 15 transplanted fish in a 4 L tank under static water conditions for the treatment duration. Alternatively, house the transplanted fish individually in 1 L static tanks. Equip the tank with an oxygen pump and perform daily water changes to minimize the risk of infection.</li>
	<li>Monitor and assess GFP expression by flow cytometry in the fish approximately 14 days post-transplantation to evaluate the engraftment rate. Record the daily mortality of fish to accurately calculate survival rates. Perform one-way ANOVA for engraftment analysis and the Log-rank test for the survival analysis.</li>
</ol>

<ol>
	<li>Choi, T. Y., Choi, T. I., Lee, Y. R., Choe, S. K., Kim, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+as+an+animal+model+for+biomedical+research.">Zebrafish as an animal model for biomedical research.</a> Exp Mol Med. 53 (3), 310-317 (2021).</li><li>Lieschke, G. J., Currie, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+human+disease:+zebrafish+swim+into+view.">Animal models of human disease: zebrafish swim into view.</a> Nat Rev Genet. 8 (5), 353-367 (2007).</li><li>Wu, J. Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patient-derived+xenograft+in+zebrafish+embryos:+a+new+platform+for+translational+research+in+gastric+cancer.">Patient-derived xenograft in zebrafish embryos: a new platform for translational research in gastric cancer.</a> J Exp Clin Cancer Res. 36 (1), 160 (2017).</li><li>Welker, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correction:+Standardized+orthotopic+xenografts+in+zebrafish+reveal+glioma+cell-line-specific+characteristics+and+tumor+cell+heterogeneity.">Correction: Standardized orthotopic xenografts in zebrafish reveal glioma cell-line-specific characteristics and tumor cell heterogeneity.</a> Dis Model Mech. 9 (9), 1063-1065 (2016).</li><li>Stoletov, K., Montel, V., Lester, R. D., Gonias, S. L., Klemke, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+imaging+of+the+dynamic+tumor+cell+vascular+interface+in+transparent+zebrafish.">High-resolution imaging of the dynamic tumor cell vascular interface in transparent zebrafish.</a> Proc Natl Acad Sci U S A. 104 (44), 17406-17411 (2007).</li><li>Etchin, J. P. K. J., Look, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+as+a+model+for+the+study+of+human+cancer.">Zebrafish as a model for the study of human cancer.</a> Methods Cell Biol. 105, 309-337 (2011).</li><li>Khan, N., Mahajan, N. K., Sinha, P., Jayandharan, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+efficient+method+to+generate+xenograft+tumor+models+of+acute+myeloid+leukemia+and+hepatocellular+carcinoma+in+adult+zebrafish.">An efficient method to generate xenograft tumor models of acute myeloid leukemia and hepatocellular carcinoma in adult zebrafish.</a> Blood Cells Mol Dis. 75, 48-55 (2019).</li><li>Corkery, D. P., Dellaire, G., Berman, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukaemia+xenotransplantation+in+zebrafish--chemotherapy+response+assay+in+vivo.">Leukaemia xenotransplantation in zebrafish--chemotherapy response assay in vivo.</a> Br J Haematol. 153 (6), 786-789 (2011).</li><li>He, B. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functions+of+flt3+in+zebrafish+hematopoiesis+and+its+relevance+to+human+acute+myeloid+leukemia.">Functions of flt3 in zebrafish hematopoiesis and its relevance to human acute myeloid leukemia.</a> Blood. 123 (16), 2518-2529 (2014).</li><li>Wang, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+IDH2(R172K)+and+IDH2(R140Q)+zebrafish+models+recapitulated+features+of+human+acute+myeloid+leukemia.">Transgenic IDH2(R172K) and IDH2(R140Q) zebrafish models recapitulated features of human acute myeloid leukemia.</a> Oncogene. 42 (16), 1331 (2023).</li><li>Fan, R. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+xenograft+model+for+studying+mechanism+and+treatment+of+non-small+cell+lung+cancer+brain+metastasis.">Zebrafish xenograft model for studying mechanism and treatment of non-small cell lung cancer brain metastasis.</a> J Exp Clin Cancer Res. 40 (1), 371 (2021).</li><li>LeBlanc, J., Bowman, T. V., Zon, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+whole+kidney+marrow+in+adult+zebrafish.">Transplantation of whole kidney marrow in adult zebrafish.</a> J Vis Exp. (2), e159 (2007).</li><li>Pugach, E. K., Li, P., White, R., Zon, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retro-orbital+injection+in+adult+zebrafish.">Retro-orbital injection in adult zebrafish.</a> J Vis Exp. (34), e1645 (2009).</li><li>Benjamin, D. C., Hynes, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+imaging+of+metastasis+in+adult+zebrafish.">Intravital imaging of metastasis in adult zebrafish.</a> BMC Cancer. 17 (1), 660 (2017).</li><li>Hilligan, K. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravenous+administration+of+BCG+protects+mice+against+lethal+SARS-CoV-2+challenge.">Intravenous administration of BCG protects mice against lethal SARS-CoV-2 challenge.</a> J Exp Med. 219 (2), e20211862 (2022).</li><li>Moreo, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravenous+administration+of+BCG+in+mice+promotes+natural+killer+and+T+cell-mediated+antitumor+immunity+in+the+lung.">Intravenous administration of BCG in mice promotes natural killer and T cell-mediated antitumor immunity in the lung.</a> Nat Commun. 14 (1), 6090 (2023).</li><li>Wang, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravenous+administration+of+IL-12+encoding+self-replicating+RNA-lipid+nanoparticle+complex+leads+to+safe+and+effective+antitumor+responses.">Intravenous administration of IL-12 encoding self-replicating RNA-lipid nanoparticle complex leads to safe and effective antitumor responses.</a> Sci Rep. 14 (1), 7366 (2024).</li><li>Saleem, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+best+practice+for+validating+tail+vein+injections+in+rat+with+near-infrared-labeled+agents.">A new best practice for validating tail vein injections in rat with near-infrared-labeled agents.</a> J Vis Exp. (146), e59295 (2019).</li><li>Rojas, A. M., Shiau, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain-localized+and+Intravenous+Microinjections+in+the+Larval+Zebrafish+to+Assess+Innate+Immune+Response.">Brain-localized and Intravenous Microinjections in the Larval Zebrafish to Assess Innate Immune Response.</a> Bio Protoc. 11 (7), e3978 (2021).</li><li>van Soest, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+static+immersion+and+intravenous+injection+systems+for+exposure+of+zebrafish+embryos+to+the+natural+pathogen+Edwardsiella+tarda.">Comparison of static immersion and intravenous injection systems for exposure of zebrafish embryos to the natural pathogen Edwardsiella tarda.</a> BMC Immunol. 12, 58 (2011).</li><li>Cianciolo Cosentino, C., Roman, B. L., Drummond, I. A., Hukriede, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravenous+microinjections+of+zebrafish+larvae+to+study+acute+kidney+injury.">Intravenous microinjections of zebrafish larvae to study acute kidney injury.</a> J Vis Exp. (42), e2079 (2010).</li><li>Gerlach, G. F., Schrader, L. N., Wingert, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+of+the+adult+zebrafish+kidney.">Dissection of the adult zebrafish kidney.</a> J Vis Exp. (54), e2839 (2011).</li></ol>

The authors have no conflicts of interest to declare.

In this study, we developed an IV injection protocol tailored for adult zebrafish to enhance the precision and consistency of delivering cells or drugs. A critical element of this method is the ability to localize and visualize the primary vein, which is crucial for the accurate administration of substances. While the translucent Casper zebrafish strain is recommended for optimal vessel visibility, our protocol is adaptable for use with various adult zebrafish strains, provided suitable adjustments are made for ...

Zebrafish (Danio rerio) has emerged as a valuable model organism in biomedical research, primarily due to its genetic similarity to humans, with more than 70% of human genes having zebrafish counterparts1,2. This genetic resemblance, combined with zebrafish&#39;s compact size, rapid developmental cycle, and capacity for extensive genetic manipulation, makes it a powerful tool for scientific exploration. These attributes are particularly advantageous for experiments involving cell transplantation, drug delivery, and cell tracing. Furthermore, the optical clarity of transparent larvae and specific pigmentation mutants, such as the Casper zebrafish, allows for precise visualization of transplanted cells or substances, offering a more efficient and cost-effective alternative to traditional animal models.
Zebrafish are widely used to develop cancer transplantation models and to conduct drug screenings for severe diseases like glioma, melanoma, pancreatic tumors, and leukemia3,4,5,6,7,8,9,10. Typically, these models are initiated at the larval stage to take advantage of the larvae&#39;s immature immune system and inherent transparency, which simplifies the injection process and enhances the feasibility of short-term studies. However, using larvae limits the duration for assessing engraftment and therapeutic interventions7,11. Transitioning these experimental protocols to adult zebrafish introduces challenges such as more complex injection procedures, reduced engraftment efficiency, higher mortality rates, and increased variability in individual responses. These challenges highlight the critical need for improved injection and substance delivery techniques, particularly for studies involving adult zebrafish that require extended observation periods.
Historically, intracardiac12 and retro-orbital (RO) injections13 have been the primary methods for drug delivery and cell transplantation in adult zebrafish. Intracardiac injection, which involves injecting substances directly into the heart, ensures immediate systemic circulation of drugs but is associated with significant risks, including potential cardiac injury and high mortality. On the other hand, RO injection, which delivers materials to the venous sinus behind the eye, also promotes rapid systemic distribution but can be stressful for the fish and demands high precision in execution14. IV injections, well-established for systemic substance delivery in mice and rats, are crucial for pharmacokinetics studies, drug efficacy assessments, and therapeutic interventions in these models15,16,17,18. Recently, IV injections have gained prominence in zebrafish research, particularly in studies of innate immune responses19,20 and acute kidney injury21 in larval zebrafish. However, their application in adult zebrafish remains limited.
In this study, we developed and optimized an IV injection procedure tailored specifically for adult zebrafish, which significantly improves survival rates, precision, and delivery efficiency. We visually demonstrate this method and provide a detailed protocol using this refined technique. We successfully administered IV injections of whole kidney marrow (WKM) cells from Tg(mpo: EGFP) fish and FITC-dextran dye into adult Casper fish, with confirmation of delivery and engraftment assessed by fluorescent microscopy. Our findings show that the refined IV injection technique is more efficient and consistent compared to the traditional intra-cardiac and RO methods. As the use of zebrafish in scientific research continues to expand, the adoption of this improved IV injection technique is likely to enhance our understanding of disease pathogenesis and accelerate the development of new therapeutic approaches.

<table><tbody><tr><td>Autoclaves Steam Sterilizer</td><td>HIRAYAMA&amp;nbsp;</td><td>HRG-140</td><td></td></tr><tr><td>Centrifuge 5424</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>Countess 3 Automated Cell Counter</td><td>Thermofisher</td><td></td><td></td></tr><tr><td>Countess II FL</td><td>Invitrogen</td><td></td><td></td></tr><tr><td>Ethyl 3-aminobenzoate methanesulfonate salt (Tricaine)</td><td>Sigma-aldrich</td><td>MKCL9483</td><td></td></tr><tr><td>Falcon 40 &amp;micro;m Cell Strainer</td><td>CORNING</td><td></td><td>Blue, Sterile, Individually Packaged, 50/Case</td></tr><tr><td>FBS, Qualified</td><td>Gibco</td><td>26140079</td><td></td></tr><tr><td>FITC-Dextran (MW 10000)&amp;nbsp;</td><td>MedChemExpress</td><td>60842-46-8</td><td></td></tr><tr><td>Injection needle</td><td>Hamilton&amp;nbsp;</td><td>HAMI207434</td><td>34 G, 10 mm length</td></tr><tr><td>Micro syringe</td><td>Hamilton&amp;nbsp;</td><td>7635-01&amp;nbsp;</td><td>10 &amp;micro;L capacity, Model 701 RN</td></tr><tr><td>Nikon SMZ18</td><td></td><td></td><td></td></tr><tr><td>PBS pH 7.4 (1x)</td><td>Gibco</td><td>10010023</td><td></td></tr><tr><td>Penicillin-Streptomycin (5,000 U/mL)</td><td>Gibco&amp;trade;</td><td>15070063</td><td>100x</td></tr><tr><td>Pipette Tips</td><td>Eppendorf&amp;nbsp;</td><td>epTIPS</td><td></td></tr><tr><td>Single Channel Pipette</td><td>Eppendorf&amp;nbsp;</td><td>05-403-151</td><td></td></tr><tr><td>UltraPure Distilled Water</td><td>Invitrogen</td><td>10977015</td><td></td></tr></tbody></table>

optimized intravenous injection in adult zebrafish

Intravenous (IV) injection is widely recognized as the most effective and commonly utilized method for achieving systemic delivery of substances in mammalian research models. However, its application in adult zebrafish for drug delivery, stem cell transplantation, and regenerative and cancer studies has been limited due to the challenges posed by their small body size and intricate blood vessels. To overcome these limitations, alternative injection techniques such as intracardiac and retro-orbital (RO) injection have been explored in the past for stem cell transplantation in adult zebrafish. However, these techniques have their drawbacks, including the need for meticulous injection techniques or increased risk of mortality.
In this study, we have developed a refined and optimized IV injection procedure specifically tailored to adult zebrafish, addressing the challenges associated with their unique anatomy. To demonstrate the effectiveness of this technique, we performed successful IV injections of whole kidney marrow cells from Tg(mpo: EGFP) fish and FITC-dextran dye into adult Casper fish. The subsequent visualization of injected cells and dyes using a fluorescence microscope confirmed their successful delivery and engraftment within the zebrafish. Furthermore, we demonstrated that compared with the intracardiac and RO injections, the IV injection resulted in improved survival rates and engraftment efficiency in treated zebrafish. This approach enables precise delivery and localization of substances and holds great potential for large-scale drug and chemical screening using adult zebrafish. Additionally, the ability to visually track the injected cells and dyes provides invaluable insights into their engraftment, migration, and interactions with host tissues, enabling a more comprehensive evaluation of therapeutic effects and biological processes in zebrafish models.

To evaluate the effectiveness and precision of different injection methods in adult Casper fish, a comparative analysis was conducted using intracardiac, RO, and IV techniques (Figure 2A). Varying quantities of WKM cells from Tg(mpo: EGFP) fish were injected, each mixed with FITC-dextran dye at concentrations of 1 &#215; 105 and 3 &#215; 105 cells. The success of each method was determined through immediate microscopic assessment of GFP signal presence...

Here, we present an optimized and effective protocol for the intravenous injection of cells or pharmacological agents into adult zebrafish, resulting in enhanced cell engraftment and increased survival rates of the treated zebrafish.

Optimized Intravenous Injection in Adult Zebrafish

Intravenous (IV) injection is widely recognized as the most effective and commonly utilized method for achieving systemic delivery of substances in ...

optimized-intravenous-injection-in-adult-zebrafish

Universidad de Cartagena UDEC

Research

JoVE Journal

Cancer Research

382 Views.  The University of Hong Kong. Here, we present an optimized and effective protocol for the intravenous injection of cells or pharmacological agents into adult zebrafish, resulting in enhanced cell engraftment and increased survival rates of the treated zebrafish.

Video: Optimized Intravenous Injection in Adult Zebrafish

Intra-iliac Artery Injection for Efficient and Selective Modeling of Microscopic Bone Metastasis

Intra-iliac artery (IIA) injection is an efficient approach to introduce metastatic lesions of various cancer cells in animals. Compared to the widely used intra-cardiac and intra-tibial injections, IIA injection brings several advantages. First, it can deliver a large quantity of cancer cells specifically to hind limb bones, thereby providing spatiotemporally synchronized early-stage colonization events and allowing robust quantification and swift detection of disseminated tumor cells. Second, it injects cancer cells into the circulation without damaging the local tissues, thereby avoiding inflammatory and wound-healing processes that confound the bone colonization process. Third, IIA injection causes very little metastatic growth in non-bone organs, thereby preventing animals from succumbing to other vital metastases, and allowing continuous monitoring of indolent bone lesions. These advantages are especially useful for the inspection of progression from single cancer cells to multi-cell micrometastases, which has largely been elusive in the past. When combined with cutting-edge approaches of biological imaging and bone histology, IIA injection can be applied to various research purposes related to bone metastases.

This manuscript provides the detailed procedure of intra-iliac artery (IIA) injection, a technique to deliver cancer cells specifically to hind limb tissues including bones to establish experimental bone metastases. Although initially established with breast tumor models, this protocol can be easily extended to other cancer types.

Intra-iliac artery (IIA) injection is an efficient approach to introduce metastatic lesions of various cancer cells in animals. Compared to the widely ...

Testing the Vascular Invasive Ability of Cancer Cells in Zebrafish (Danio Rerio)

Cancer cell vascular invasion and extravasation is a hallmark of metastatic progression. Traditional in vitro models of cancer cell invasion of endothelia typically lack the fluid dynamics that invading cells are otherwise exposed to in vivo. However, in vivo systems such as mouse models, though more physiologically relevant, require longer experimental timescales and present unique challenges associated with monitoring and data analysis. Here we describe a zebrafish assay that seeks to bridge this technical gap by allowing for the rapid assessment of cancer cell vascular invasion and extravasation. The approach involves injecting fluorescent cancer cells into the precardiac sinus of transparent 2-day old zebrafish embryos whose vasculature is marked by a contrasting fluorescent reporter. Following injection, the cancer cells must survive in circulation and subsequently extravasate from vessels into tissues in the caudal region of the embryo. Extravasated cancer cells are efficiently identified and scored in live embryos via fluorescence imaging at a fixed timepoint. This technique can be modified to study intravasation and/or competition amongst a heterogeneous mixture of cancer cells by changing the injection site to the yolk sac. Together, these methods can evaluate a hallmark behavior of cancer cells and help uncover mechanisms indicative of malignant progression to the metastatic phenotype.

Testing the Vascular Invasive Ability of Cancer Cells in Zebrafish Danio Rerio

This method utilizes zebrafish embryos to efficiently test the vascular invasive ability of cancer cells. Fluorescent cancer cells are injected into the precardiac sinus or yolk sac of developing embryos. Cancer cell vascular invasion and extravasation is assessed via fluorescence microscopy of the tail region 24 to 96 hr later.

Cancer cell vascular invasion and extravasation is a hallmark of metastatic progression.  Traditional in vitro models of cancer cell invasion of ...

Invasive Behavior of Human Breast Cancer Cells in Embryonic Zebrafish

In many cases, cancer patients do not die of a primary tumor, but rather because of metastasis. Although numerous rodent models are available for studying cancer metastasis in vivo, other efficient, reliable, low-cost models are needed to quickly access the potential effects of (epi)genetic changes or pharmacological compounds. As such, we illustrate and explain the feasibility of xenograft models using human breast cancer cells injected into zebrafish embryos to support this goal. Under the microscope, fluorescent proteins or chemically labeled human breast cancer cells are transplanted into transgenic zebrafish embryos, Tg (fli:EGFP), at the perivitelline space or duct of Cuvier (Doc) 48 h after fertilization. Shortly afterwards, the temporal-spatial process of cancer cell invasion, dissemination, and metastasis in the living fish body is visualized under a fluorescent microscope. The models using different injection sites, i.e., perivitelline space or Doc are complementary to one another, reflecting the early stage (intravasation step) and late stage (extravasation step) of the multistep metastatic cascade of events. Moreover, peritumoral and intratumoral angiogenesis can be observed with the injection into the perivitelline space. The entire experimental period is no more than 8 days. These two models combine cell labeling, micro-transplantation, and fluorescence imaging techniques, enabling the rapid evaluation of cancer metastasis in response to genetic and pharmacological manipulations.

Here, we describe xenograft zebrafish models using two different injection sites, i.e., perivitelline space and duct of Cuvier, to investigate the invasive behavior and to assess the intravasation and extravasation potential of human breast cancer cells, respectively.

In many cases, cancer patients do not die of a primary tumor, but rather because of metastasis. Although numerous rodent models are available for studying ...

Unraveling Key Players of Humoral Immunity: Advanced and Optimized Lymphocyte Isolation Protocol from Murine Peyer's Patches

In the gut mucosa, immune cells constitute a unique immunological entity, which promotes immune tolerance while concurrently conferring immune defense against pathogens. It is well established that Peyer&#39;s patches (PPs) have an essential role in the mucosal immune network by hosting several effector T and B cell subsets. A certain fraction of these effector cells, follicular T helper (TFH) and germinal center (GC) B cells are professionalized in the regulation of humoral immunity. Hence, the characterization of these cell subsets within PPs in terms of their differentiation program and functional properties can provide important information about mucosal immunity. To this end, an easily applicable, efficient and reproducible method of lymphocyte isolation from PPs would be valuable to researchers. In this study, we aimed to generate an effective method to isolate lymphocytes from mouse PPs with high cell yield. Our approach revealed that initial tissue processing such as the use of digestive reagents and tissue agitation, as well as cell staining conditions and selection of antibody panels, have great influence on the quality and identity of the isolated lymphocytes and on experimental outcomes.
Here, we describe a protocol enabling researchers to efficiently isolate lymphocyte populations from PPs allowing reproducible flow cytometry-based assessment of T and B cell subsets primarily focusing on&#160;TFH and GC B cell subsets.

In this study, we present a novel and effective protocol for the isolation of lymphocytes from Peyer&#39;s Patches (PPs), which can be subsequently used for in vivo and in vitro functional assays as well as flow cytometric studies of follicular T helper and germinal center B cells.

In the gut mucosa, immune cells constitute a unique immunological entity, which promotes immune tolerance while concurrently conferring immune defense ...

In Vivo Imaging and Quantitation of the Host Angiogenic Response in Zebrafish Tumor Xenografts

Tumor angiogenesis is a key target of anti-cancer therapy and this method has been developed to provide a new model to study this process in vivo. A zebrafish xenograft is created by implanting mammalian tumor cells into the perivitelline space of two days-post-fertilization zebrafish embryos, followed by measuring the extent of the angiogenic response observed at an experimental endpoint up to two days post-implantation. The key advantage to this method is the ability to accurately quantitate the zebrafish host angiogenic response to the graft. This enables detailed examination of the molecular mechanisms as well as the host vs tumor contribution to the angiogenic response. The xenografted embryos can be subjected to a variety of treatments, such as incubation with potential anti-angiogenesis drugs, in order to investigate strategies to inhibit tumor angiogenesis. The angiogenic response can also be live-imaged in order to examine more dynamic cellular processes. The relatively undemanding experimental technique, cheap maintenance costs of zebrafish and short experimental timeline make this model especially useful for the development of strategies to manipulate tumor angiogenesis.

The aim of this method is to generate an in vivo model of tumor angiogenesis by xenografting mammalian tumor cells into a zebrafish embryo that has fluorescently-labelled blood vessels. By imaging the xenograft and associated vessels, a quantitative measurement of the angiogenic response can be obtained.

Tumor angiogenesis is a key target of anti-cancer therapy and this method has been developed to provide a new model to study this process in vivo. A ...

Drug Screening of Primary Patient Derived Tumor Xenografts in Zebrafish

Patient derived xenograft models are critical in defining how different cancers respond to drug treatment in an in vivo system. Mouse models are the standard in the field, but zebrafish have emerged as an alternative model with several advantages, including the ability for high-throughput and low-cost drug screening. Zebrafish also allow for in vivo drug screening with large replicate numbers that were previously only obtainable with in vitro systems. The ability to rapidly perform large scale drug screens may open up the possibility for personalized medicine with rapid translation of results back to clinic. Zebrafish xenograft models could also be used to rapidly screen for actionable mutations based on tumor response to targeted therapies or to identify new anti-cancer compounds from large libraries. The current major limitation in the field has been quantifying and automating the process so that drug screens can be done on a larger scale and be less labor-intensive. We have developed a workflow for xenografting primary patient samples into zebrafish larvae and performing large scale drug screens using a fluorescence microscope equipped imaging unit and automated sampler unit. This method allows for standardization and quantification of engrafted tumor area and response to drug treatment across large numbers of zebrafish larvae. Overall, this method is advantageous over traditional cell culture drug screening as it allows for growth of tumor cells in an in vivo environment throughout drug treatment, and is more practical and cost-effective than mice for large scale in vivo drug screens.

Zebrafish xenograft models allow for high-throughput drug screening and fluorescent imaging of human cancer cells in an in vivo microenvironment. We developed a workflow for large scale, automated drug screening on patient-derived leukemia samples in zebrafish using an automated fluorescence microscope equipped imaging unit.

Patient derived xenograft models are critical in defining how different cancers respond to drug treatment in an in vivo system. Mouse models are the ...

In Vivo Targeting of Xenografted Human Cancer Cells with Functionalized Fluorescent Silica Nanoparticles in Zebrafish

Developing nanoparticles capable of detecting, targeting, and destroying cancer cells is of great interest in the field of nanomedicine. In vivo animal models are required for bridging the nanotechnology to its biomedical application. The mouse represents the traditional animal model for preclinical testing; however, mice are relatively expensive to keep and have long experimental cycles due to the limited progeny from each mother. The zebrafish has emerged as a powerful model system for developmental and biomedical research, including cancer research. In particular, due to its optical transparency and rapid development, zebrafish embryos are well suited for real-time in vivo monitoring of the behavior of cancer cells and their interactions with their microenvironment. This method was developed to sequentially introduce human cancer cells and functionalized nanoparticles in transparent Casper zebrafish embryos and monitor in vivo recognition and targeting of the cancer cells by nanoparticles in real time. This optimized protocol shows that fluorescently labeled nanoparticles, which are functionalized with folate groups, can specifically recognize and target metastatic human cervical epithelial cancer cells labeled with a different fluorochrome. The recognition and targeting process can occur as early as 30 min postinjection of the nanoparticles tested. The whole experiment only requires the breeding of a few pairs of adult fish and takes less than 4 days to complete. Moreover, zebrafish embryos lack a functional adaptive immune system, allowing the engraftment of a wide range of human cancer cells. Hence, the utility of the protocol described here enables the testing of nanoparticles on various types of human cancer cells, facilitating the selection of optimal nanoparticles in each specific cancer context for future testing in mammals and the clinic.

Described here is a method for utilizing zebrafish embryos to study the ability of functionalized nanoparticles to target human cancer cells in vivo. This method allows for the evaluation and selection of optimal nanoparticles for future testing in large animals and in clinical trials.

Developing nanoparticles capable of detecting, targeting, and destroying cancer cells is of great interest in the field of nanomedicine. In vivo animal ...

Zebrafish Model of Neuroblastoma Metastasis

Zebrafish has emerged as an important animal model to study human diseases, especially cancer. Along with the robust transgenic and genome editing technologies applied in zebrafish modeling, the ease of maintenance, high-yield productivity, and powerful live imaging altogether make the zebrafish a valuable model system to study metastasis and cellular and molecular bases underlying this process in vivo. The first zebrafish neuroblastoma (NB) model of metastasis was developed by overexpressing two oncogenes, MYCN and LMO1, under control of the dopamine-beta-hydroxylase (d&#946;h) promoter. Co-overexpressed MYCN and LMO1 led to the reduced latency and increased penetrance of neuroblastomagenesis, as well as accelerated distant metastasis of tumor cells. This new model reliably reiterates many key features of human metastatic NB, including involvement of clinically relevant and metastasis-associated genetic alterations; natural and spontaneous development of metastasis in vivo; and conserved sites of metastases. Therefore, the zebrafish model possesses unique advantages to dissect the complex process of tumor metastasis in vivo.

This paper introduces the method of developing, characterizing, and tracking in real-time the tumor metastasis in zebrafish model of neuroblastoma, specifically in the transgenic zebrafish line with overexpression of MYCN and LMO1, which develops metastasis spontaneously.

Zebrafish has emerged as an important animal model to study human diseases, especially cancer. Along with the robust transgenic and genome editing ...

Digital Spatial Profiling for Characterization of the Microenvironment in Adult-Type Diffusely Infiltrating Glioma

Diffusely infiltrating gliomas are associated with high morbidity and mortality due to the infiltrative nature of tumor spread. They are morphologically complex tumors, with a high degree of proteomic variability across both the tumor itself and its heterogenous microenvironment. The malignant potential of these tumors is enhanced by the dysregulation of proteins involved in several key pathways, including processes that maintain cellular stability and preserve the structural integrity of the microenvironment. Although there have been numerous bulk and single-cell glioma analyses, there is a relative paucity of spatial stratification of these proteomic data. Understanding differences in spatial distribution of tumorigenic factors and immune cell populations between the intrinsic tumor, invasive edge, and microenvironment offers valuable insight into the mechanisms underlying tumor proliferation and propagation. Digital spatial profiling (DSP) represents a powerful technology that can form the foundation for these important multilayer analyses.
DSP is a method that efficiently quantifies protein expression within user-specified spatial regions in a tissue specimen. DSP is ideal for studying the differential expression of multiple proteins within and across regions of distinction, enabling multiple levels of quantitative and qualitative analysis. The DSP protocol is systematic and user-friendly, allowing for customized spatial analysis of proteomic data. In this experiment, tissue microarrays are constructed from archived glioblastoma core biopsies. Next, a panel of antibodies is selected, targeting proteins of interest within the sample. The antibodies, which are preconjugated to UV-photocleavable DNA oligonucleotides, are then incubated with the tissue sample overnight. Under fluorescence microscopy visualization of the antibodies, regions of interest (ROIs) within which to quantify protein expression are defined with the samples. UV light is then directed at each ROI, cleaving the DNA oligonucleotides. The oligonucleotides are microaspirated and counted within each ROI, quantifying the corresponding protein on a spatial basis.

Proteomic dysregulation plays an important role in the spread of diffusely infiltrating gliomas, but several relevant proteins remain unidentified. Digital spatial processing (DSP) offers an efficient, high-throughput approach for characterizing the differential expression of candidate proteins that may contribute to the invasion and migration of infiltrative gliomas.

Diffusely infiltrating gliomas are associated with high morbidity and mortality due to the infiltrative nature of tumor spread. They are morphologically ...

Modeling Brain Metastasis by Internal Carotid Artery Injection of Cancer Cells

Brain metastasis is a cause of severe morbidity and mortality in cancer patients. Critical aspects of metastatic diseases, such as the complex neural microenvironment and stromal cell interaction, cannot be entirely replicated with in vitro assays; thus, animal models are critical for investigating and understanding the effects of therapeutic intervention. However, most brain tumor xenografting methods do not produce brain metastases consistently in terms of the time frame and tumor burden. Brain metastasis models generated by intracardiac injection of cancer cells can result in unintended extracranial tumor burden and lead to non-brain metastatic morbidity and mortality. Although intracranial injection of cancer cells can limit extracranial tumor formation, it has several caveats, such as the injected cells frequently form a singular tumor mass at the injection site, high leptomeningeal involvement, and damage to brain vasculature during needle penetration. This protocol describes a mouse model of brain metastasis generated by internal carotid artery injection. This method produces intracranial tumors consistently without the involvement of other organs, enabling the evaluation of therapeutic agents for brain metastasis.

Brain metastasis is a cause of severe morbidity and mortality in cancer patients. Most brain metastasis mouse models are complicated by systemic metastases confounding analysis of mortality and therapeutic intervention outcomes. Presented here is a protocol for internal carotid injection of cancer cells that produces consistent intracranial tumors with minimal systemic tumors.

Brain metastasis is a cause of severe morbidity and mortality in cancer patients. Critical aspects of metastatic diseases, such as the complex neural ...