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

Bruk av larvesebrafisk for effektive in vivo tumorstudier

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).

Forfatter Spotlight: Fremme kreftterapi ved bruk av tumorxenotransplantasjon hos sebrafisk

En enkel, rask og effektiv metode for tumorxenotransplantasjonsanalyse i gjennomsiktige sebrafiskembryoer

We seek to gain molecular insight into the control of normal and malignant hematopoiesis and then to exploit those insights to develop new therapies for the treatment of blood cancers. Tumor xenografts are an indispensable tool for evaluating the impact of a genetic alteration or treatment on tumor behavior. However, mice xenografts are expensive, time consuming, and limited in scope.Using zebrafish for tumor xenotransplantation overcomes all these drawbacks. In this protocol, we demonstrate an easy and rapid mounting process for xenotransplantation, which increases throughput. We further employ cell staining to know the quality of the inoculum and then flow cytometry to assess the tumor progression by evaluating or quantifying the disease burden.We intend to use this approach to identify novel ways of blocking the function of the Ras/MAP kinase cascade, which drives the vast majority of human cancers. And we'll do that not by trying to block the catalytic activity of the Ras/MAP kinase cascade but rather by preventing its ability to phosphorylate those cancer-driving substrates it modifies.

Utarbeidelse av encellet embryosuspensjon for flowcytometrianalyse for å vurdere tumorxenotransplantasjon

For å begynne, få sebrafiskembryoer injisert med leukemiceller forhåndsfarget med CM-Dil. Overfør de bedøvede embryoene til et 1,5 milliliters sentrifugerør og bruk en P200-pipette, oppløs eggeplommen i 100 mikroliter kalsiumfri ringeløsning i fem minutter. Inkuber hver prøve av aveggede embryoer med en milliliter trypsinkollagenaseoppløsning ved 29 grader Celsius i 30 til 35 minutter.Bruk en P1000-spiss til å pipettere embryoene opp og ned i denne løsningen hvert femte minutt til ryggradsstrukturen ikke lenger er synlig. For å stoppe reaksjonen, tilsett 200 mikroliter FBS i røret. Bland godt og rug i ytterligere fem minutter.Pellet cellesuspensjonen ved 300 G i fem minutter ved fire grader celsius. Når supernatanten er kastet, vask cellepelleten to ganger i avkjølt PBS og sil cellene gjennom en 70 mikrometer cellesil. Etter å ha vasket cellene, resuspender cellepelleten i fargemediet som inneholder et antistoff som er reaktivt med de transplanterte cellene.Inkuber cellene ved fire grader Celsius i 20 minutter. Pellet cellene og resuspender dem i 200 mikroliter fargemedium, som inneholder en mikromolar helix og P Blue. Overfør celleblandingen til et fem milliliter polykarbonatrør med rund bunn og utfør flowcytometri.Den doble merkingen tillot måling av forskjeller i tumorbyrden mellom god versus dårligere bolusinokulering.

preparation-single-cell-embryo-suspension-for-flow-cytometry-analysis

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 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.

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.

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 ...