Published: April 1st, 2021
Here, we present how to generate a non-alcoholic fatty liver disease (NAFLD)-associated Hepatocellular Carcinoma (HCC) zebrafish model to study the impact of cholesterol surplus on liver microenvironment and immune cell landscape.
Liver cancer is currently the third leading cause of cancer related death worldwide, and Hepatocellular Carcinoma (HCC) accounts for 75-90% of all liver cancer cases. With the introduction of effective treatments to prevent and treat hepatitis B/C, non-alcoholic fatty liver disease (NAFLD), and the more aggressive form know as non-alcoholic steatohepatitis (NASH), are quickly becoming the number one risk factors to develop HCC in modern societies. To better understand the role NASH has on the development of HCC we designed a NASH-associated HCC zebrafish. The optical clarity and genetic tractability of the zebrafish larvae make them an appealing and powerful model to study the liver microenvironment and immune cell composition using non-invasive fluorescent live imaging. This protocol describes how to use a NASH-associated HCC zebrafish model to investigate the effect of cholesterol surplus in the liver microenvironment and its impact on immune cell composition at early stages of the disease. First, we feed HCC larvae (s704Tg), which express hepatocyte-specific activated beta-catenin, with a 10% high cholesterol diet for 8 days to develop a NASH-associated HCC model. Here we describe how to make use of different transgenic lines to evaluate several early malignancy features in the liver by non-invasive confocal microscopy, such as liver area, cell, and nuclear morphology (hepatocytes area, nuclear area, nuclear:cytoplasmic ratio (N:C ratio), nuclear circularity, micronuclei/nuclear herniation scoring) and angiogenesis. Then, using transgenic lines with tagged immune cells (neutrophils, macrophages, and T cells) we show how to analyze liver immune cell composition in NASH-associated HCC larvae. The described techniques are useful to evaluate liver microenvironment and immune cell composition at early hepatocarcinogenesis stages, but they can also be modified to study such features in other liver disease models.
Hepatocellular Carcinoma (HCC) is an aggressive cancer with limited therapeutic options. It has been found that upwards of 30% of all patients with HCC are obese and have NASH, an aggressive form of NAFLD1,2,3,4. Consumption of calorie rich diets drastically increase fatty acid availability that causes local and systemic metabolic shifts and triggers steatosis, hepatocyte injury, inflammation, and fibrosis - all key features of NASH. NASH progression to HCC involves the accumulation of lipids in the liver, which triggers inflammation and altered immune cell composition5,6,7. It is of particular interest and importance to understand how the liver microenvironment and immune cell landscape are altered during liver disease progression, and how it changes due to certain etiological factors. To better identify the impact that cholesterol surplus has on liver microenvironment and immune cell landscape, we have developed a unique zebrafish model of NASH-associated HCC. The use of this model has given us a better understanding of the impact of diet and overnutrition on liver microenvironment and liver disease progression.
Mammalian models, such as mice and human tissue samples, have been essential in understanding the pathogenesis of steatohepatitis and steatosis8. Mice are the preferred model for liver disease and cancer, but they lack optical clarity at a cellular level, while human tissue samples often lack the 3D environment that animal models are able to imitate. These obstacles have made Zebrafish a powerful model in the research community. Zebrafish have remarkable similarities to humans, with at least 70% gene conservation. They maintain liver microenvironment, hepatic cellular composition, function, signaling, and response to injuries9,10. Using a high-cholesterol diet (HCD) in combination with an established transgenic zebrafish model of HCC, we have developed a zebrafish model of NASH-associated HCC.
Here we present a protocol explaining how to generate a NASH-associated HCC zebrafish model and how to study liver microenvironment and address early malignancy features in vivo. Using non-invasive confocal microscopy in combination with zebrafish transgenic lines with fluorescently tagged hepatocyte membrane and nucleus, we can address early malignancy features by analyzing liver morphology (area, volume, and surface area), cell and nuclear morphology (hepatocytes area, nuclear area, N:C ratio, nuclear circularity, micronuclei/nuclear herniation scoring) and angiogenesis (vessel density). Immune cell microenvironment is also an important feature on hepatocarcinogenesis11,12,13,14, therefore, we also show how to analyze liver immune cell composition in NASH-associated HCC larvae, using transgenic zebrafish lines with tagged immune cells (neutrophils, macrophages, and T cells). The described techniques are unique to the model and extremely useful to evaluate liver microenvironment and immune cell composition in liver disease progression.
Animal studies are carried out following procedures approved by the institutional animal care and use committee (IACUC) of Albert Einstein College of Medicine. For recipes for buffers and solutions used in the protocol please refer to Supplementary Table 1.
1. Preparation of 10% Cholesterol-enriched diet for acute cholesterol surplus.
2. NASH induction with short-term larvae feeding with cholesterol-enriched diet - static conditions.
3. Collection of 13 days post fertilization larvae from feeding boxes.
4. Control assay for diet induced hepatic steatosis - Oil Red O (ORO) staining, imaging, and scoring.
5. Non-invasive confocal imaging using the zebrafish Wounding and Entrapment Device for Growth and Imaging(zWEDGI).
6. Analysis of liver morphological variations.
NOTE: The steps listed below are performed for liver surface area and volume quantification:
NOTE: Perform the following steps for liver area quantification.
7. Hepatocyte and Nuclear morphological analysis.
8. Angiogenesis analysis.
9. Immune cell recruitment analysis.
By introducing a short-term high cholesterol diet into a Hepatocellular Carcinoma (HCC) zebrafish model, which overexpresses a hepatocyte specific constitutively active form of Beta-catenin (s704Tg; Tg(fabp10a:pt-B-cat, cryaa:Venus)17, we are able to create a non-mammalian vertebrate model of NASH-associated HCC. Liver disease progression can be monitored early by measuring hepatic steatosis, liver size, hepatocyte, nuclear morphology, angiogenesis, and immune cell infiltration (Figure 1).
HCC larvae fed with a normal diet show none to mild hepatic steatosis, measured by Oil Red O staining. However, HCC larvae fed with a high cholesterol diet show a significant increase in hepatic steatosis (Figure 2).
A well-known marker of liver disease is hepatomegaly17. To evaluate liver size, HCC larvae can be outcrossed with a transgenic line specifically expressing a fluorescent marker on hepatocytes, such as the Tg(fabp10a:H2BmCherry). To assess hepatomegaly, evaluation of the liver area (2D), liver surface area, and liver volume (3D) are performed. After 8 days of exposure to a cholesterol surplus, liver enlargement was observed in in HCC larvae (Figure 3).
Using non-invasive live imaging, transgenic fish lines expressing fluorescent proteins in hepatocyte membranes (such as Tg(fabp10a:Life-actin-EGFPP) and in hepatocytes nuclei (such as Tg(Fabp10a:H2B-mCherry) can be used to assess cellular and nuclear morphology alterations associated with malignancy in hepatocytes. Hepatocyte area was increased in NASH-associated HCC (Figure 4A,B,D), as well as nuclear area (Figure 4C,E) and nuclear:cytoplasmic ratio (Figure 4F). A significant decrease in nuclear circularity was also observed in the HCD+HCC group (Figure 4G). Lipotoxicity triggers DNA damage, a feature of carcinogenesis in the presence of micronuclei. Using the H2B-mCherry marker we observed a greater incidence of micronuclei in the HCC larvae fed with a high cholesterol diet (Figure 4H).
Hepatic vasculature can easily be evaluated in zebrafish models using a transgenic tagged line, such as Tg (kdrl:mCherry or Tg(fli:EGFP), which label vasculature. A significant increase of vessel density was observed in HCC+HCD larvae (Figure 5).
To observe the inflammatory response triggered early in NASH-associated HCC, a transgenic fish line expressing fluorescent proteins in macrophages and neutrophils, such as Tg(mfap4:tdTomato-CAAX; lyz:BFP), was outcrossed with the HCC transgenic line. Infiltration of neutrophils and macrophages occur in both HCC and HCC fed with HCD, which was assessed by quantification of number and density of neutrophils/macrophages in the liver and vicinity (surrounding area up to 75µm) (Figure 6A-H). Nevertheless, the HCC+HCD displayed a significant increase in neutrophil number and density (Figures 6F-H). At 13 days post fertilization, the adaptive immune system is already functional. Using a transgenic fish line expressing fluorescent protein in T-Cells, such as the Tg(lck:EGFP), and in combination with the HCC transgenic line; we evaluated the impact of cholesterol surplus on recruitment of T-cells to the liver. A significant decrease in T-Cell density and overall number was observed in HCC larvae fed with HCD (Figure 6I-K).
|Transgenic Zebrafish lines
|Tg(fabp10a:pt-β-catenin_cryaa:Venus / fabp10a:H2B-mCherry )
|s704Tg / uwm41Tg
|Tg(fabp10a:pt-β-catenin_cryaa:Venus; fabp10a:H2B-mCherry; fabp10a:LIFEACT-EGFP)
|s704Tg / uwm41Tg / uwm42Tg
|Hepatocytes and nuclear morphology
|Tg(fabp10a:pt-β-catenin_ cryaa:Venus; fli:EGFP)
|s704Tg / y1Tg
|Tg(fabp10a:pt-β-catenin_cryaa:Venus; mfap4:Tomato-CAAX; lyzC:BFP)
|s704Tg / xt6Tg / zf2171Tg
|Macrophage and Neutrophil recruitment
|s704Tg / cz1Tg
Table 1: Transgenic zebrafish lines to use in different assays.
|Number of larvae
|Feeding box size
|Amount of food per day (mg)
|E3 Volume (ml)
|Small breeding box
|Small/Big breeding box
|Big breeding box
Table 2: Set up conditions of feeding boxes.
|Normal Nuclei, No micronuclei or herniation
|Low number of micronuclei (less than 5 per field of view) and/or herniation
|Moderate to high number of micronuclei (more than 5 per field of view) and/or herniation
Table 3: Micronuclei and Nuclear Herniation Scoring.
Figure 1: Protocol Diagram summarizing main experimental steps and analysis approach. Please click here to view a larger version of this figure.
Figure 2: Control Assay for Liver Steatosis - ORO staining. HCC larvae were fed with normal or high cholesterol diet and Oil Red O (ORO) staining was performed to assess hepatic steatosis. (A) Diagram of the 12 well plate to perform sequential ORO staining using the mesh well inserts. (B) Image of eyelash tool used to manipulate larvae. (C) Representative images of livers stained with Oil Red; HCC and HCC+HCD larvae. (D) Chi-square graph showing percentage of larvae with different scoring of liver steatosis. Scale bar= 50 µm. Please click here to view a larger version of this figure.
Figure 3: Representative images from liver size. Transgenic HCC larvae expressing a liver marker (Tg(fabp10a:H2B-mCherry)) were imaged live and non-invasively, on an inverted spinning disk confocal microscope using a zWEDGI. (A) Representative 3D reconstructions of livers in HCC and HCC+HCD larvae. (B-D) Graph showing liver morphological alterations including liver area (B) liver surface area (C) and liver volume (D) in HCC and HCC+HCD larvae. Scale bar = 50 µm. Please click here to view a larger version of this figure.
Figure 4: Representative images from hepatocytes and nuclei morphology. Transgenic HCC lines expressing fluorescent proteins in hepatocyte membranes (Tg(fabp10a:Life-actin-EGFP) and in hepatocytes nuclei (Tg(fabp10a:H2B-mCherry) were imaged. (A-C) Representative 3D reconstructions of F-actin and hepatocyte nuclei of HCC and HCC+HCD larvae. Open arrowheads show enlarged nuclei; white arrowheads show nucleus with altered shape; and red arrows show micronuclei and nuclear herniation. (D-G) Graphs showing averages of cell and nuclear parameters in HCC and HCC+HCD 13-day old larvae. (D) Hepatocytes area. (E) Nuclear Area. (F) Nuclear:Cytoplasm ratio. (G) Nuclear circularity. Each dot represents averages per larva. Dot plots show mean ±SEM (H) Chi-square graphs showing percentage of larvae with different scoring of Micronuclei and Nuclear herniation. Scale bar = 10 µm. Please click here to view a larger version of this figure.
Figure 5: Representative images from hepatic vasculature. Transgenic HCC lines expressing fluorescent proteins in hepatocyte nuclei (Tg(Fabp10a:H2B-mCherry) and in endothelial cells (Tg)fli:EGFP)). (A) Representative 3D reconstructions of hepatic vasculature in HCC and HCC+HCD larvae. (B-C) Graph showing vessel density index by liver surface area (B) volume (C) in HCC and HCC+HFCD larvae. Dot plots show mean ±SEM. Scale bar = 50 µm. Please click here to view a larger version of this figure.
Figure 6: Representative images from liver immune cell landscape. Transgenic HCC line expressing fluorescent proteins in macrophages and neutrophils (Tg(mfap4:tdTomato-CAAX; lyz:BFP) or in T-cells (Tg(lck-EGFP). (A,C,F) Representative 3D reconstructions of livers and leukocyte recruitment to liver area in 13-day old HCC and HCC+HCD larvae. (B) Diagram of imaged liver area. (D-E) Graph showing macrophage density (D) and number (E) in liver area in HCC and HCC+HCD larvae. (G-H) Graph showing neutrophils density (G) and number (H) in liver area in HCC and HCC+HCD larvae. (G) Representative 3D reconstructions of T cell recruitment to liver area in HCC and HCC+HCD larvae. (H-I) Graph showing T cell density (H) and number (I) in liver area in HCC and HCC+HCD larvae. Dot plots show mean ±SEM. Scale bar = 50 µm. Please click here to view a larger version of this figure.
Supplementary Table 1: Table of Buffers and solutions Please click here to download this Table.
With an increased incidence of HCC, specifically NASH induced HCC, it is of great importance to have more efficient models to study the cellular and molecular mechanisms involved in NASH-associated HCC. Deconvolution of cell-cell interactions at the liver are crucial to better understand liver disease progression and hepatocarcinogenesis. The approach described in this protocol offers a unique way to analyze liver disease progression in vivo and non-invasively.
Preparation of diet is critical to the success of establishing a NASH-associated HCC model. It is important to let diethyl ether completely evaporate inside the fume hood to avoid harmful effects, while preparing diets for zebrafish. To use these diets with larvae (5-12 days post fertilization), it is extremely important to grind up the diets into fine particles to assure the food intake by the larvae. The use of fluorescently tagged fatty acid analogs can be used to evaluate food intake in larvae.
Before placing the larvae into the breeding boxes and starting the feeding procedure, it is crucial to ensure that experimental sampling is uniformized by mixing larvae from different plates. This step is important since different microenvironments, beginning at Day 0, are promoted in each of the plates and might affect inflammatory response.
Another important step is to count the larvae, to know how much food is needed for each feeding box. If the amount of food is not adequate for the number of larvae present in each box, one of the two scenarios will occur: 1) larvae will be underfed; 2) larvae will be overfed. Inaccurate feeding will lead to unhealthy conditions associated with malnutrition or overnutrition, such as inflammation, which will drastically affect the liver microenvironment. If inaccurate feeding occurs, larvae will show motion issues. At this development stage, larvae should be swimming intensively, therefore if motion issues are noticed larvae were raised under unhealthy conditions (malnutrition or exposure to toxic doses of cholesterol due to overfeeding). For this reason, feeding procedures need to be tightly controlled for both diets, normal and cholesterol enriched. Some assays can be performed to quickly address accuracy of the feeding and health of larvae including presence of hepatomegaly (liver size) and inflammation of tissue and organs, particularly liver and intestine (visible increased infiltration of neutrophils and macrophages).
Daily cleaning and replacement of 95% of E3 is pivotal to reduce the growth of microorganisms in the feeding boxes and improve larvae health and survival. Alternatively, larvae can be placed in a Stand-alone Rack System. For best results, place 60-80 larvae in a 3-liter tank. Keep water flow at the minimum level by adjusting the flow to a fast-dripping mode and feeding larvae 3-4 mg twice a day (AM and PM). Water flow should be checked regularly to assure correct flow in each tank. In our laboratory, this method gives us 95-100% survival with short-term feeding of 10% HCD. In addition, this method greatly reduced the workload inherent to the daily cleaning and water exchange necessary in the static feeding protocol described.
While we utilized a 10% cholesterol-enriched diet to induce NASH in a short-term exposure (5 days is enough to induce steatohepatitis), diet alterations can be performed and expanded to use fructose 18, fatty acids (such as Palmitic Acid)19, or feeding protocols that can be extended using a 4% cholesterol-enriched diet20. Currently, there are few successful therapeutic targets for HCC and none for NASH. The use of zebrafish models offers a unique opportunity to expand our knowledge on hepatocarcinogenesis but also an unparallel vertebrate system to perform large throughput drug screenings. The techniques described in this protocol will facilitate future findings and therapeutic targets for liver disease and hepatocarcinogenesis.
The authors have nothing to disclose.
The author would like to acknowledge the Albert Einstein College of Medicine Zebrafish Core Facility technicians Clinton DePaolo, and Spartak Kalinin for assistance and maintenance of our zebrafish lines. FJMN is supported by the Cancer Research Institute and Fibrolamellar Cancer Foundation.
|Easily degraded. Store -20°C.
|Corning Netwells carrier kit 15 mm
|Corning Netwells inserts
|Dumont forceps #55 dumostar
|Fisherbrand Pasteur Pipets 5.75in
|4% paraformaldehyde (PFA)
|Electron Microscopy Science
|Golden Pearl Diet 5–50 nm Active Spheres
|Brine Shrimp Direct
|Any commercial dry powder food for larvae can be used.
|Graduated Transfer Pipets
|PBS, pH7.4, 10X, 10 Pack
|Petri Dishes 100X20MM
|Oil Red O solution 0.5% isopropanol
|Vacuum system for larvae collection
|M205 FCA THUNDER Imager Model Organism Large
|Spinning Disk Confocal Microscope
|S9i with transilluminated base
|Open-source Java image processing program.
|Bitplane; Oxford Instruments.
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