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

Utilizzo delle larve di pesce zebra per studi efficienti sui tumori in vivo

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).

Autore in primo piano: Avanzamento delle terapie antitumorali utilizzando lo xenotrapianto tumorale nel pesce zebra

Un metodo semplice, rapido ed efficace per l'analisi dello xenotrapianto tumorale in embrioni trasparenti di zebrafish

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.

Preparazione di sospensioni embrionali unicellulari per l'analisi di citometria a flusso per valutare lo xenotrapianto tumorale

Per iniziare, ottenere embrioni di pesce zebra iniettati con cellule leucemiche prestate con CM-Dil. Trasferire gli embrioni anestetizzati in una provetta da centrifuga da 1,5 millilitri e, utilizzando una pipetta P200, sciogliere il tuorlo in 100 microlitri di soluzione ringer priva di calcio per cinque minuti. Incubare ogni campione di embrioni deyolked con un millilitro della soluzione di tripsina collagenasi a 29 gradi Celsius per 30-35 minuti.Utilizzando un puntale P1000, pipettare gli embrioni su e giù in questa soluzione ogni cinque minuti fino a quando la struttura della spina dorsale non è più visibile. Per fermare la reazione, aggiungere 200 microlitri di FBS al tubo. Mescolare bene e incubare per altri cinque minuti.Pellettare la sospensione cellulare a 300 G per cinque minuti a quattro gradi Celsius. Una volta scartato il surnatante, lavare il pellet di cella due volte in PBS refrigerato e filtrare le cellule attraverso un colino di 70 micrometri. Dopo aver lavato le cellule, risospendere il pellet cellulare nel mezzo di colorazione contenente un anticorpo reattivo con le cellule trapiantate.Incubare le cellule a quattro gradi Celsius per 20 minuti. Pellettare le cellule e risospenderle in 200 microlitri di terreno di colorazione, contenente un'elica micromolare e P Blu. Trasferire la miscela cellulare in una provetta in policarbonato a fondo tondo da cinque millilitri ed eseguire la citometria a flusso.La doppia marcatura ha permesso di misurare le differenze nel carico tumorale tra l'inoculazione in bolo buona e quella inferiore.

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

225 Views.  Fox Chase Cancer Center. Per iniziare, ottenere embrioni di pesce zebra iniettati con cellule leucemiche prestate con CM-Dil. Trasferire gli embrioni anestetizzati in una provetta da centrifuga da 1,5 millilitri e, utilizzando una pipetta P200, sciogliere il tuorlo in 100 microlitri di soluzione ringer priva di calcio per cinque minuti. Incubare ogni campione di embrioni deyolked con un millilitro della soluzione di tripsina collagenasi a 29 gradi Celsius per 30-35 minuti.Utilizzando un puntale P1000, pipettar...

Video: Preparazione di sospensioni embrionali unicellulari per l'analisi di citometria a flusso per valutare lo xenotrapianto tumorale

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 ...