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

투명 제브라피시 배아에서 종양 이종 이식 분석을 위한 간단하고 빠르며 효과적인 방법

종양 이종 이식을 평가하기 위한 유세포 분석을 위한 단세포 배아 현탁액의 준비

시작하려면 CM-Dil로 염색된 백혈병 세포가 주입된 제브라피시 배아를 얻습니다. 마취된 배아를 1.5밀리리터 원심분리 튜브에 옮기고 P200 피펫을 사용하여 난황을 100마이크로리터의 무칼슘 링거 용액에 5분 동안 녹입니다. 노른을 제거한 배아의 각 샘플을 섭씨 29도에서 30-35분 동안 트립신 콜라겐분해효소 용액 1mL로 배양합니다.P1000 팁을 사용하여 백본 구조가 더 이상 보이지 않을 때까지 5분마다 이 용액에서 배아를 위아래로 피펫팅합니다. 반응을 중지하려면 튜브에 200마이크로리터의 FBS를 첨가하십시오. 잘 섞고 5분 더 배양합니다.세포 현탁액을 섭씨 4도에서 5분 동안 300G로 펠릿화합니다. 상층액을 버리면 세포 펠릿을 냉각된 PBS에서 두 번 세척하고 70마이크로미터 세포 여과기를 통해 세포를 변형시킵니다. 세포를 세척한 후, 이식된 세포와 반응하는 항체를 포함하는 염색 매체에 세포 펠릿을 재현탁시킵니다.섭씨 4도에서 20분 동안 세포를 배양합니다. 세포를 펠릿화하고 1개의 마이크로몰 나선과 P Blue를 포함하는 200마이크로리터의 염색 매체에 재현탁시킵니다. 세포 혼합물을 5밀리리터의 둥근 바닥 폴리카보네이트 튜브로 옮기고 유세포 분석을 수행합니다.이중 표지를 통해 양호한 볼루스 접종과 열등한 볼루스 접종 간의 종양 부담 차이를 측정할 수 있었습니다.

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 ...

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Injecting Labeled Leukemia Cells into Zebrafish Embryos for Assessing Tumor Behavior

종양 거동을 평가하기 위해 표지된 백혈병 세포를 제브라피시 배아에 주입