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

Een eenvoudige, snelle en effectieve methode voor tumor xenotransplantatie analyse in transparante zebravis embryo's

Bereiding van eencellige embryosuspensie voor flowcytometrieanalyse om tumorxenotransplantatie te beoordelen

Verkrijg om te beginnen zebravisembryo's die zijn geïnjecteerd met leukemiecellen die vooraf zijn gekleurd met CM-Dil. Breng de verdoofde embryo's over in een centrifugebuis van 1,5 milliliter en los de dooier met behulp van een P200-pipet gedurende vijf minuten op in 100 microliter calciumvrije ringeroplossing. Incubeer elk monster van gededooierde embryo's met één milliliter trypsinecollagenase-oplossing bij 29 graden Celsius gedurende 30 tot 35 minuten.Gebruik een P1000-tip om de vijf minuten de embryo's op en neer in deze oplossing totdat de ruggengraatstructuur niet meer zichtbaar is. Om de reactie te stoppen, voegt u 200 microliter FBS toe aan de buis. Meng goed en incubeer nog vijf minuten.Pellet de celsuspensie bij 300 G gedurende vijf minuten bij vier graden Celsius. Zodra het supernatans is weggegooid, wast u de celpellet twee keer in gekoelde PBS en zeeft u de cellen door een celzeef van 70 micrometer. Na het wassen van de cellen, resuspendeert u de celkorrel in het kleuringsmedium dat een antilichaam bevat dat reactief is met de getransplanteerde cellen.Incubeer de cellen bij vier graden Celsius gedurende 20 minuten. Pellet de cellen en resuspendeer ze in 200 microliter kleuringsmedium, bestaande uit één micromolaire helix en P Blue. Breng het celmengsel over in een polycarbonaatbuis van vijf milliliter met ronde bodem en voer flowcytometrie uit.De dubbele etikettering maakte het mogelijk om verschillen in de tumorlast tussen goede versus inferieure bolusinoculatie te meten.

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 ...

Injecting Labeled Leukemia Cells into Zebrafish Embryos for Assessing Tumor Behavior

Injecteren van gelabelde leukemiecellen in zebravisembryo's voor het beoordelen van tumorgedrag