The online version of this article (https://doi.org/10.1186/s12885-017-3919-8) contains supplementary material, which is available to authorized users.
Zebrafish (Danio rerio) is a model organism that has emerged as a tool for cancer research, cancer being the second most common cause of death after cardiovascular disease for humans in the developed world. Zebrafish is a useful model for xenotransplantation of human cancer cells and toxicity studies of different chemotherapeutic compounds in vivo. Compared to the murine model, the zebrafish model is faster, can be screened using high-throughput methods and has a lower maintenance cost, making it possible and affordable to create personalized therapies. While several methods for cell proliferation determination based on image acquisition and quantification have been developed, some drawbacks still remain. In the xenotransplantation technique, quantification of cellular proliferation in vivo is critical to standardize the process for future preclinical applications of the model.
This study improved the conditions of the xenotransplantation technique – quantification of cellular proliferation in vivo was performed through image processing with our ZFtool software and optimization of temperature in order to standardize the process for a future preclinical applications. ZFtool was developed to establish a base threshold that eliminates embryo auto-fluorescence and measures the area of marked cells (GFP) and the intensity of those cells to define a ‘proliferation index’.
The analysis of tumor cell proliferation at different temperatures (34 °C and 36 °C) in comparison to in vitro cell proliferation provides of a better proliferation rate, achieved as expected at 36°, a maintenance temperature not demonstrated up to now. The mortality of the embryos remained between 5% and 15%. 5- Fluorouracil was tested for 2 days, dissolved in the incubation medium, in order to quantify the reduction of the tumor mass injected. In almost all of the embryos incubated at 36 °C and incubated with 5-Fluorouracil, there was a significant tumor cell reduction compared with the control group. This was not the case at 34 °C.
Our results demonstrate that the proliferation of the injected cells is better at 36 °C and that this temperature is the most suitable for testing chemotherapeutic drugs like the 5-Fluorouracil.
Additional file 1: Figure S6. Revision of parameters regarding xenotransplantation conditions. (PDF 296 kb)12885_2017_3919_MOESM1_ESM.pdf
Additional file 2: Figure S1. ZFtool automatically elimination of fish autofluorescence. ZFtool software detects all the green pixels in the image (red/pink line) but eliminates all those pixels corresponding to fish autofluorescence and keeps pixels above an established threshold (blue line). (TIFF 7994 kb)12885_2017_3919_MOESM2_ESM.tif
Additional file 3: Figure S2. Automated counting of cells. This image shows the process of the software to count the cells on the microscope slide performed before the injection of the zebrafish embryos. (A) Fluorescent image of low cell number. (B) Cells of the A image counted (179). (C) Fluorescent image of high cell number. D: Cells of the C counted (404). Scale bar = 100 μm. (TIFF 1115 kb)12885_2017_3919_MOESM3_ESM.tif
Additional file 4: Figure S3. OECD protocol toxicity results. (DOCX 18 kb)12885_2017_3919_MOESM4_ESM.docx
Additional file 5: Figure S4. Cell proliferation inside the zebrafish embryos at 34 °C and 34 °C with 5-FU (A) Zebrafish embryo incubation at 34 °C analyzed with ZFtool yielding a proliferation index of 0.4748. (B) Zebrafish embryo incubation at 34 °C, with 5-FU analyzed with the ZFtool yielding a proliferation index of 0.5415. All images are a superposition of a fluorescence field image over a bright field image. In all panels, the left image is a 48 hpf or 0 hpi zebrafish embryo, and the right image is the same zebrafish embryo with 120 hpf or 72 hpi. Scale bar = 100 μm. (TIFF 8180 kb)12885_2017_3919_MOESM5_ESM.tif
Additional file 6: Figure S5. Cell proliferation inside the zebrafish embryos at 36 °C and 36 °C with 5-FU (A) Zebrafish embryo incubation at 36 °C analyzed with ZFtool yielding a proliferation index of 2.6653. (B) Zebrafish embryo incubation at 36 °C, with 5-FU analyzed with the ZFtool yielding a proliferation index of 1.9592. All images are a superposition of a fluorescence field image over a bright field image. In all panels, the left image is a 48 hpf or 0 hpi zebrafish embryo, and the right image is the same zebrafish embryo with 120 hpf or 72 hpi. Scale bar = 100 μm. (TIFF 10158 kb)12885_2017_3919_MOESM6_ESM.tif
Jo DH, Son D, Na Y, Jang M, Choi JH, Kim JH, et al. Orthotopic transplantation of retinoblastoma cells into vitreous cavity of zebrafish for screening of anticancer drugs. Mol Cancer. 2013;12:71.
Annila T, Lihavainen E, Marques IJ, Williams DR, Yli-Harja O, Ribeiro A. ZebIAT, an image analysis tool for registering zebrafish embryos and quantifying cancer metastasis. BMC Bioinformatics. 2013;14(Suppl 10):S5.
Westerfield M. The zebrafish book. A guide for the laboratory use of zebrafish ( Danio rerio). 4th ed. Eugene: University of Oregon Press; 2000.
OECD. Test no. 236: fish embryo acute toxicity (FET) test, OECD guidelines for the testing of chemicals. In: Section 2. Paris: OECD Publishing; 2013.
Kovacs R, Bakos K, Urbanyi B, Kovesi J, Gazsi G, Csepeli A, et al. Acute and sub-chronic toxicity of four cytostatic drugs in zebrafish. Environ Sci Pollut Res Int. 2016;23(15):14718–29.
- Improving zebrafish embryo xenotransplantation conditions by increasing incubation temperature and establishing a proliferation index with ZFtool
María J. Carreira
Juan A. Rubiolo
Andrés A. Sciara
Luis M. Botana
- BioMed Central
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