Zebrafish handling
The care, use and treatment of zebrafish were performed in agreement with the Animal Care and Use Committee of the University of Santiago de Compostela and the standard protocols of Spain (Directive 2012-63-UE). The protocol was approved by the Animal Care and Use Committee of the University of Santiago de Compostela. One-year-old adult zebrafish (Danio rerio, wild-type) were maintained at 28.5 °C in 30 L aquaria at a rate of 1 fish per liter of water, with a light-dark cycle of 14:10. Zebrafish embryos were obtained from mating adults according to previously described procedures [24]. When needed, embryos were euthanized by tricaine overdose.
Reagents and cell culture
The human colorectal cancer cell line HCT116 was obtained from American Type Culture Collection (ATCC, Catalog No. CCL-247) and cultured using McCoy’s 5A Medium containing 10% FBS (GIBCO, Invitrogen) and 1% Pen/Strep (GIBCO, Invitrogen) at 37 °C with 5% CO2 in a humidified atmosphere. The HCT116 cell line was transfected to express GFP constitutively. The HCT116 line was tested monthly for contamination.
Fluorescent GFP cell labeling
HCT116 cells were transduced using a lentiviral-driven GFP construct (Sigma, Mission TurboGFP, SHC003 V). Cells were placed 72 h post infection under selective pressure using 10 μg/ml puromycin. The rate of GFP positive cells was tested using flow cytometry (BD FACS Aria I, software FACSDiva 6.0.3).
Cell proliferation assays
Cell proliferation was determined using xCELLigence Real-Time Cell Analyzer; Acea Biosciences (Roche) following the manufacturer instructions. In brief, cells were seeded on E-plates containing electric nodes in their surface that allow the measurement of changes in impedance attributed to cell proliferation. Measurements were performed in quadruplicate, normalizing the initial cell index once the cells were completely adhered.
Cell injection
Two days post fertilization (dpf), zebrafish embryos were dechorionated (if needed) and anesthetized with 0.003% tricaine (Sigma). Cells were suspended at 10,000-20,000 cells/μl in complete McCoy and maintained at room temperature for no longer than 2 h before they were injected. The cell suspension was loaded into borosilicate glass capillary needles (1 mm O.D. × 0.78 mm I.D.; Harvard Apparatus), and injections were performed using IM-31 Electric Microinjector (Narishige) with an output pressure of 34 kPa and 30 ms injection time. The injections were performed manually right into the yolk of the embryo. Incorrectly injected embryos without cells inside of the yolk, or showing them in the circulation after xenotransplantation were discarded.
Incubation, imaging and cell quantification
After injection, 2dpf embryos were incubated at two different conditions (34 °C or 36 °C) in 24-well plates with salt dechlorinate tap water (SDTW, chlorine free water obtained with a reverse osmosis filter system) for 72 h to check the proliferation of the cell line by ZFtool. Each plate contained at least 2 negative controls (injected with complete McCoy medium) and 2 blanks (not injected). Apart from those plates, another plate with 12 negative controls and 12 blanks were included in some experiments to test the viability of the embryos. No development abnormalities were observed during incubation at this temperature.
In order to reach a 36 °C incubation temperature without a large amount of embryo mortality, plates were covered with a transparent sealing tape (PCR Plastics) to prevent evaporation and reduction of dissolved oxygen. After that, plates were placed inside an incubator with minimal contact between the plate and the incubator structure to prevent water overheating.
Each embryo was photographed with AZ-100 Nikon fluorescence stereomicroscope at 0 hpi and 72 hpi to be analyzed by ZFtool software. The objective of this software is to automatize and improve the task of measuring the number and mean value of GFP pixels in order to compare them for these two conditions and compute the proliferation index. Finally, this analysis yields the number of GFP pixels in the image (nGFP), which represents the area of the cells inside the yolk sac at two different times and the GFP intensity Medium Value (GMV), which represents the medium intensity of the fluorescence inside the yolk. By multiplying the nGFP number by the GMV of each image, we determined the proliferation ratio between 0 hpi and 72 hpi to estimate the cell growth. The result obtained at 72 hpi was divided by that obtained at 0 hpi, yielding a proliferation index value (PI):
$$ \frac{nGFP_{72 hpi}\bullet {GMV}_{72 hpi}}{nGFP_{0 hpi}\bullet {GMV}_{0 hpi}} $$
A PI value =1 means that cells remain stable during incubation, a PI higher than 1 indicates tumor cell proliferation and a PI lower than 1 indicates tumor cell death.
Zebrafish embryos have variable autofluorescence, especially in the yolk area. To accurately quantify the injected cells fluorescence a pre-processing is needed to only count the GFP pixels belonging to injected cells filtering autofluorescence. To achieve this, the software counts the number of GFP pixels with different intensity thresholds, from 0 (no threshold) to 50 (Fig. 2) and the ZFtool algorithm provides a homogeneous measurement of the GFP area for all fish analyzed comparing nGFP for each threshold analyzed with nGFP for threshold = 0, where fish auto fluorescence is mostly present. When the relation between measured nGFP compared to nGFP at threshold = 0 surpass a fixed value, we consider the GFP area to be stable and the threshold is fixed at this point. In case there is no autofluorescence in the embryo, the threshold is established based on a tolerance parameter and a correction is included to assure the accuracy of the measurement in this cases. The ZFtool algorithm automatic thresholding for each analyzed embryo is one of the main automation components of the software, making it efficient in producing reliable fish to fish measurements.
Cell counting software
The ZFTool extension for cell counting was developed. A drop of cells was placed on a microscope slide and photographed to obtain a fluorescence image. The algorithm detects circular objects of the fluorescence input image with a fixed diameter. The output yields a fluorescence image with nearly every cell or group of cells delimited by a contour and an estimation of the number of cells inside the input image. This algorithm is based on the circular Hough transform and has several parameters fixing the strength of the edge, and a minimum and maximum radius of the circles to detect. As we know the approximate size of the cells, we can fix these parameters in order to obtain an estimation of the number of cells. The method will be more accurate as the cells are more isolated, but as the number of cells injected increases over 400, we do not need the exact number of cells, but only an estimation, so this method still fits our purposes.
Anticancer drugs toxicity and treatment
In order to test the toxicity of an anticancer drug (5-Fluorouracil), experiments were performed according to the OECD (Organisation for Economic Co-operation and Development) guideline for the testing of chemicals [25]. This procedure consists of exposing 0 h post fecundation (hpf) eggs to dissolved chemicals in 24-well plates, for a period of 96 h. Various indicators (such as coagulation of embryos, lack of somite formation, non-detachment of the tail or lack of heartbeat) were checked every 24 h during the experiment, to test the mortality of the embryos and calculate the LC50 (lethal concentration 50%) at the end of the test. The drug was tested to determine a concentration range that included 0–100% mortality. Experiments were considered valid when egg fertilization was ≥ 70%. At the beginning, the oxygen concentration should have ≥ 80% saturation, and the water temperature should be 26 ± 1 °C. During the test, the negative control embryos mortality could not be ≥ 10% at any time of the experiment. Exposure to the positive control resulted in a minimum mortality of 30% at the end, and the hatching rate of the negative control embryos was higher than 80 % at 96 h. The concentrations tested were 250 μM, 500 μM, 1000 μM, 1500 μM, 2000 μM, with 1% DMSO. Another analog experiment was conducted changing the treatment starting point from 0 hpf to 48hpf in order to evaluate how the toxicity changed with a dechorionated embryo at 36 °C.
Statistical analysis
Homoscedasticity and statistical analyses were performed using the SPSS software (IBM). An excel outlier analysis was performed using interquartile range (IQR), while the outliers were discarded. One factor ANOVA for non-parametrical data was applied to non-homoscedastic data with confidence intervals of 95% or 99%, and a Student’s t-test was applied to homoscedastic data with confidence intervals of 95% or 99%. Number of embryos analyzed is represented by nrep and ntotal, being nrep the number of embryos in each replica, and ntotal the total number of embryos statistically analyzed for the experiment.