Analysis of Liver Microenvironment During Early Progression of Non-Alcoholic Fatty Liver Disease-Associated Hepatocellular Carcinoma in Zebrafish

Our zebrafish liver cancer models and protocol provide a unique opportunity to visualize in vivo, non-invasively, the liver microenvironment and immune landscape using intravital microscopy. The transparency of the zebrafish larvae is undoubtedly the main advantage of this system, which allows us to perform non-invasive intravital microscopy and gain insight into the microenvironment and immune cell infiltration of a deep tissue and organ, such as liver. Our diet-induced nephrotic model and the imaging techniques described in this protocol can easily be applied to the convoluted cell-cell interactions in other liver diseases or research fields, including metabolic syndrome and cardiovascular diseases.

Demonstrating the procedure will be Cassia Michael, our lab technician, and Dr.Francisco Juan Martinez-Navarro, a postdoctoral fellow in my laboratory. To begin, weigh four grams of commercial dry food diet for zebrafish larvae into two 25-milliliter glass beakers, one for normal diet and the other for the 10%high cholesterol diet, or HCD. Weigh 0.4 grams of cholesterol in a 10-milliliter glass beaker and cover the beaker with foil.

In a fume hood, measure five milliliters of diethyl ether using a 10-milliliter syringe with a 20-gauge needle. Then, add the diethyl ether to the normal diet beaker and immediately mix it with the dry food using a spatula. Measure another five milliliters of diethyl ether using the same syringe and needle and add it to the HCD-containing beaker.

Mix immediately by aspirating up and down with the syringe. Quickly add the cholesterol solution to the HCD beaker and immediately mix it with a spatula until the solution is uniform. Leave beakers in the hood for up to 24 hours to completely evaporate the diethyl ether.

On the next day, grind the diets into fine particles using a pestle and mortar. Transfer each diet to a small, labeled plastic bag or a 50-milliliter centrifuge tube and store them at minus 20 degrees Celsius. Set up transgenic fish lines on day minus one.

On day zero, collect eggs in a mesh strainer after the fish have spawned. Using a wash bottle with E3, rinse the eggs thoroughly and carefully transfer them to 10-centimeter Petri dishes with E3 medium. Clean the plates of all debris that might have accumulated in the breeding boxes, including any dead or unfertilized eggs to avoid the uncontrolled growth of microorganisms and consequent defects in larvae development.

Then divide the eggs at a density of 70 or 80 eggs per dish containing 25 milliliters of E3.On day one, check and clean dead embryos or embryos with developmental defects under a dissection scope equipped with a transillumination base. On day five, combine larvae from all the dishes in a 15-centimeter Petri dish and add E3 without methylene blue. Divide larvae in the feeding boxes and feed them as described in the text manuscript.

Keep the larvae in the zebrafish room or an incubator at 28 degrees Celsius in a dark-light cycle and feed them twice a day from day 5 to day 12. Remove food debris daily as well as 90 to 95%of the medium using a vacuum system attached to a one-milliliter pipette tip. Then, carefully pour the new E3 without methylene blue into one corner of the feeding box to avoid damaging the larvae.

Alternatively, the larvae can be divided into three-liter tanks with system water and placed as a standalone system unit with a low water flow. This alternative saves the time required for the daily cleaning process, and the filtration system helps keep the larvae healthy until endpoint. On day 13, prepare collection dishes according to the number of experimental conditions that were set up.

Pour enough E3 without methylene blue to cover the bottom of each dish. Then, carefully, using the vacuum system, aspirate the water from the feeding boxes. When the water level starts to get low, slowly lift the feeding box to make larvae swim to one of the corners, then aspirate in the opposite direction.

Once only about 20 to 30 milliliters of the liquid remain, carefully decant larvae into the prepared collection Petri dish and place the labeled tape from the feeding box on the lid of the dish. On a fluorescence stereomicroscope, screen the anesthetized larvae for desired fluorescent markers. Then add tricaine E3 into the chambers of the wounding and entrapment device.

Remove air bubbles from the chambers and the restraining channel using a P-200 micropipette. Remove all excess tricaine E3, leaving only enough volume to fill the chambers. Next, transfer an anesthetized larvae into the loading chamber of the wounding and entrapment device and position it for imaging of the left lobe using an eyelash tool.

Image the morphology of all the required cells in the liver under a confocal microscope as described in the text manuscript. Open the Fiji software. Then open the image file and tick the Split channels option from the Bio-Format Import Options tab.

After creating maximum intensity projections for each channel, create an ROI surrounding the larval liver and measure the liver area. Then, add a scale bar as a reference and create a second ROI including the liver area and 75 micrometers of the surrounding area. In the Plugins menu, select the Analyze option followed by Cell Counter and count the immune cells inside the recruitment area.

Record the number of recruited immune cells on a spreadsheet. Calculate the neutrophil, macrophage, and T-cell densities by normalizing the number of immune cells per liver area. HCC zebrafish larvae fed with a normal diet show no hepatic steatosis, as measured by Oil Red O staining.

However, HCC larvae fed with HCD show a significant increase in hepatic steatosis. After eight days of exposure to a cholesterol surplus, liver enlargement was observed in HCC larvae. To assess hepatomegaly, the liver area, liver surface area, and liver volume were evaluated.

In non-alcoholic steatohepatitis-associated HCC, the hepatocyte area, along with the nuclear area, and nuclear-to-cytoplasmic ratio were increased. A significant decrease in nuclear circularity was also observed in the high fat diet-fed HCC group. Using the H2B-mCherry marker, a greater incidence of micronuclei was detected in the HCC larvae fed with HCD.

Hepatic vasculature evaluation showed a significant increase in vessel density in HCC larvae fed with HCD. Infiltration of macrophages and neutrophils occurred in both HCC and HCC fed with HCD larvae. Quantification of neutrophils in macrophages in the liver and its vicinity showed a significant increase in the number and density of the cells in HCC larvae fed with HCD.

In contrast, a significant decrease in T-cell density in overall number was observed in HCC larvae fed with HCD. The cleaning of the tanks and the feeding are the most important steps to assure larvae viability and avoiding development of steatosis or liver and systemic chronic inflammation in the controls due to poor quality of the water or improper feeding. Other methods such as time-lapse microscopy and analysis of the interaction between the different cells populating the liver can be performed.

Additionally, single-cell RNA-seek of dissected livers at different liver cancer stages can be performed to understand in full how the liver immune landscape evolves with disease progression.