preparation of single-cell embryo suspension for flow cytometry analysis to assess tumor xenotransplantation

Utilizing Larval Zebrafish for Efficient In Vivo Tumor Studies

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 tumors6,7. By contrast, the zebrafish model (Danio rerio) enables greater capacity, shorter duration, lower expense, and, due to their transparency, simple monitoring of disease progression8,9.
Zebrafish is a well-developed vertebrate model system with ex-utero development and high fecundity, with individual females producing more than 100 embryos10. Moreover, zebrafish embryos are transparent, enabling easy visualization of developmental processes using fluorescence-related techniques such as reporters. Finally, the conservation of critical developmental processes makes them an ideal model for many types of studies, including the behavior of transplanted malignant cells11,12. Wild-type zebrafish embryos develop melanocytes, which render them optically opaque by 2 weeks of age, but this has been overcome by the generation of casper embryos (roya9; mitfaw2), which remain transparent throughout life13. Because of their optical properties, casper zebrafish are ideal recipients of transplanted tumor cells14,15,16. Xenotransplantation of tumor cells into zebrafish has gained importance in the past 2 decades17,18,19,20,21. Zebrafish embryos have innate immunity; however, they lack adaptive immunity during their larval stage, rendering them functionally immunocompromised, which enables them to serve as effective hosts for transplanted tumor xenografts22.
Protocols have been developed for tumor engraftment in zebrafish embryos as well as adults that have considered a number of different variables23,24,25,26,27. These have explored numerous sites of tumor deposition in zebrafish, including injections in yolk, peri-vitelline space, and heart and at different developmental stages16,28. The ambient temperature of aquaculture for zebrafish xenografts is also important as zebrafish rearing typically occurs at 28 &#176;C, while mammalian cells grow at 37 &#176;C. Consequently, a compromise temperature must be employed that is tolerated by the fish yet supports tumor growth, and 34 &#176;C appears to achieve both goals29. Analysis of the behavior and progression of tumors following xenotransplantation is another major area of focus, and this involves the use of a variety of imaging modalities as well as survival analysis30. One of the major advantages of the zebrafish model is the availability of large numbers of study animals to provide immense statistical power to in vivo studies of tumor behavior; however, previous approaches have severely limited this potential because of the requirement of tedious mounting procedures for injections.
Here, we address this limitation through the development of a simple, rapid method with which to stage embryos that enables high throughput and monitoring of injection quality using the transparent casper zebrafish line. This entails the injection of xenografts into the yolk sac of the casper zebrafish embryos at 2 days post fertilization (dpf). We observe the survival of embryos following xenotransplantation as part of tumor behavior analysis. We further show the assessment of disease burden after xenotransplantation by making single cell suspensions and analyzing by flow cytometry (Figure 1).

Author Spotlight: Advancing Cancer Therapeutics Using Tumor Xenotransplantation in Zebrafish

Une méthode simple, rapide et efficace pour l’analyse de la xénotransplantation tumorale dans les embryons de poisson-zèbre transparent

Préparation d’une suspension d’embryon unicellulaire pour l’analyse par cytométrie en flux afin d’évaluer la xénotransplantation tumorale

Pour commencer, obtenez des embryons de poisson-zèbre injectés avec des cellules leucémiques pré-colorées avec CM-Dil. Transférez les embryons anesthésiés dans un tube à centrifuger de 1,5 millilitre et, à l’aide d’une pipette P200, dissolvez le jaune dans 100 microlitres de solution sans anneau de calcium pendant cinq minutes. Incuber chaque échantillon d’embryons déjaunes avec un millilitre de solution de trypsine collagénase à 29 degrés Celsius pendant 30 à 35 minutes.À l’aide d’une pointe P1000, pipetez les embryons de haut en bas dans cette solution toutes les cinq minutes jusqu’à ce que la structure de la colonne vertébrale ne soit plus visible. Pour arrêter la réaction, ajoutez 200 microlitres de FBS dans le tube. Bien mélanger et incuber pendant cinq minutes supplémentaires.Granulez la suspension cellulaire à 300 G pendant cinq minutes à quatre degrés Celsius. Une fois le surnageant jeté, lavez la pastille cellulaire deux fois dans du PBS réfrigéré et filtrez les cellules à travers une passoire à cellules de 70 micromètres. Après avoir lavé les cellules, mettez à nouveau en suspension la pastille cellulaire dans le milieu de coloration contenant un anticorps réactif avec les cellules transplantées.Incuber les cellules à quatre degrés Celsius pendant 20 minutes. Granulez les cellules et mettez-les en suspension dans 200 microlitres de milieu de coloration, contenant une hélice micromolaire et P Blue. Transférez le mélange de cellules dans un tube en polycarbonate à fond rond de cinq millilitres et effectuez une cytométrie en flux.Le double marquage a permis de mesurer les différences de charge tumorale entre une bonne inoculation de bolus et une inoculation en bolus inférieure.

Research

JoVE Journal

Cancer Research

Preparation of Single-Cell Embryo Suspension for Flow Cytometry Analysis to Assess Tumor Xenotransplantation

A Simple, Rapid, and Effective Method for Tumor Xenotransplantation Analysis in Transparent Zebrafish Embryos

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 ...

Recherche

Recherche en cancérologie

Injecting Labeled Leukemia Cells into Zebrafish Embryos for Assessing Tumor Behavior

Injection de cellules leucémiques marquées dans des embryons de poisson-zèbre pour évaluer le comportement tumoral