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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

We describe a protocol for xenotransplantation into the yolk of transparent zebrafish embryos that is optimized by a simple, rapid staging method. Post-injection analyses include survival and assessing the disease burden of xenotransplanted cells by flow cytometry.

Abstract

In vivo studies of tumor behavior are a staple of cancer research; however, the use of mice presents significant challenges in cost and time. Here, we present larval zebrafish as a transplant model that has numerous advantages over murine models, including ease of handling, low expense, and short experimental duration. Moreover, the absence of an adaptive immune system during larval stages obviates the need to generate and use immunodeficient strains. While established protocols for xenotransplantation in zebrafish embryos exist, we present here an improved method involving embryo staging for faster transfer, survival analysis, and the use of flow cytometry to assess disease burden. Embryos are staged to facilitate rapid cell injection into the yolk of the larvae and cell marking to monitor the consistency of the injected cell bolus. After injection, embryo survival analysis is assessed up to 7 days post injection (dpi). Finally, disease burden is also assessed by marking transferred cells with a fluorescent protein and analysis by flow cytometry. Flow cytometry is enabled by a standardized method of preparing cell suspensions from zebrafish embryos, which could also be used in establishing the primary culture of zebrafish cells. In summary, the procedure described here allows a more rapid assessment of the behavior of tumor cells in vivo with larger numbers of animals per study arm and in a more cost-effective manner.

Introduction

Analysis of the behavior of tumors in response to genetic alteration or drug treatment in vivo is an essential element of cancer research1,2,3,4. Such studies most often involve the use of immunocompromised mouse (Mus musculus) models5; however, xenotransplantation studies in mice are limited in many respects, including limited capacity, extended duration, significant expense, and the requirement for sophisticated imaging equipment to monitor the progression of internal tumors6

Protocol

Zebrafish maintenance, feeding, and husbandry occurred under standard aquaculture conditions at 28.5 °C, as described31. All zebrafish-related experiments were done at this temperature; however, following xenotransplantation, the animals were cultured at 34 °C for the duration of the experiment, in accordance with procedures approved by the Institutional Animal Care and Use Committee (IACUC).

1. Breeding (3 days before injection)

  1. P.......

Representative Results

Xenotransplantation
A comprehensive view of the entire experiment and analysis is depicted in Figure 1, spanning from embryo production to the assessment of disease progression by both survival and disease burden analysis by flow cytometry. This approach brings several improvements that enhance the reproducibility and scalability of xenotransplantation, as well as adding a new way to assess disease burden. The success of these experiments is highly dependent upon the h.......

Discussion

Zebrafish xenotransplantation has emerged as a rapid, robust, and cost-effective alternative to mouse studies12. Though several approaches to zebrafish xenotransplantation have been reported, our adaptation has resulted in significant improvements. In addition to standardizing parameters around the procedure, these improvements specifically focus on accelerating the rate at which tumor injections can be performed, thus enabling an increase in the number of animals per study arm and using tumor lab.......

Acknowledgements

This work was supported by NIH grants R37AI110985 and P30CA006927, an appropriation from the Commonwealth of Pennsylvania, the Leukemia and Lymphoma Society, and the Bishop Fund. This study was also supported by the core facilities at Fox Chase, including Cell Culture, Flow Cytometry, and Laboratory Animal facility. We thank Dr. Jennifer Rhodes for maintaining the zebrafish and microinjection facility at FCCC.

....

Materials

NameCompanyCatalog NumberComments
1-phenyl 2-thiourea (PTU)SigmaP7629
70 micron cell strainerCorning CLS431751-50EA
90 mm Petri dishThermo Fisher ScientificS43565
AgaroseApex bioresearch20-102GP
APC APC anti-mouse CD45.2 AntibodyBiolegend109814
BD FACSymphony A5 Cell AnalyzerBD BiosciencesBD FACSymphony A5
calibration capillariesSigma P1424-1PAK
Cell tracker CM-dil dyeInvitrogenC7001
Collageanse IVGibco17104019
Dumont forceps number 55Fine science tools11255-20
FBSCorning 35-015-CV
Fluorescence microscopeNikonmodel SMZ1500
Glass capillaries (Borosilicate)World precision instruments1B100-4
HBSSCorning 21-023-CV
Helix NP BlueBiolegend425305
Instant Ocean Sea SaltInstant oceanSS15-10
Light microscopeNikonmodel SMZ1000
Methylene blueSigmaM9140-100G
Microloader (long tips for laoding cells)eppendorf930001007
P1000 micropipette pullerSutter instrumentsmodel P-97
PM 1000 cell microinjectorMicroData Instruments, Inc. (MDI)PM1000
Tricaine methanesulphate (Ethyl 3- aminobenzoate methanesulphate)SigmaE10521-10G
Trypsin-EDTA (0.5%), no phenol redGibco15400054
Zebrafish adult irradiated diet (dry feed)Zeigler388763

References

  1. Sharma, G., Goyal, Y., Bhatia, S. Handbook of Animal Models and its Uses in Cancer Research. Preclinical Animal Models of Cancer: Applications and Limitations. , (2022).
  2. Singhal, S. S., et al. Recent ....

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Cancer ResearchIn Vivo StudiesCancer ResearchMurine ModelsCell TransplantationFlow CytometryCell SuspensionPrimary Culture

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