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Protocol
An Orthotopic Implantation Mouse Model of
Hepatocellular Carcinoma with Underlying
Liver Steatosis

Hiroaki Kasashima,
Angeles Duran,
Tania Cid-Diaz,
Yotaro Kudo, Maria
T. Diaz-Meco, Jorge
Moscat
m1165063@med.
osaka-cu.ac.jp (H.K.)
mtd4001@med.cornell.
edu (M.T.D.-M.)
jom4010@med.cornell.
edu (J.M.)

HIGHLIGHTS
Liver orthotopic
implantation is a
useful tool, but
consistency is
challenging

Pre-conditioning liver
with a high-fat diet
improved tumor cell
engraftment

Three injection sites

increase the
This protocol provides the steps required for a mouse liver orthotopic implantation model. The probability of mice
reliable pre-clinical animal models that have similar characteristics to hepatocellular carcinoma harboring tumors
(HCC) are a powerful tool to unveil the mechanisms controlling tumor initiation and progression.
Here, we describe a syngeneic orthotopic HCC model that recapitulates the role of a host pro-
tumorigenic microenvironment by pre-conditioning mouse livers with a high-fat diet (HFD).

Kasashima et al., STAR

Protocols 1, 100185
December 18, 2020 ª 2020
The Authors.
https://doi.org/10.1016/
j.xpro.2020.100185
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Protocol
An Orthotopic Implantation Mouse Model of
Hepatocellular Carcinoma with Underlying Liver
Steatosis
Hiroaki Kasashima,1,2,4,5,6,* Angeles Duran,1,3 Tania Cid-Diaz,1,3 Yotaro Kudo,2
Maria T. Diaz-Meco,1,2,3,* and Jorge Moscat1,2,3,7,*
1Department of Pathology and Laboratory Medicine, Weill Cornell Medicine, New York, NY 10065, USA
2Sanford Burnham Prebys Medical Discovery Institute, 10901 N. Torrey Pines Road, La Jolla, CA 92037, USA
3Sandra and Edward Meyer Cancer Center, Weill Cornell Medicine, New York, NY 10065, USA
4Department of Gastroenterological Surgery, Osaka City University Graduate School of Medicine, Osaka city, 545-8585, Japan
5Department of Surgery, Kashiwara Municipal hospital, Kashiwara city, 582-0005, Japan
6Technical Contact
7Lead Contact
*Correspondence: m1165063@med.osaka-cu.ac.jp (H.K.), mtd4001@med.cornell.edu (M.T.D.-M.),
jom4010@med.cornell.edu (J.M.)
https://doi.org/10.1016/j.xpro.2020.100185

SUMMARY
This protocol provides the steps required for a mouse liver orthotopic implantation
model. The reliable pre-clinical animal models that have similar characteristics to he-
patocellular carcinoma (HCC) are a powerful tool to unveil the mechanisms control-
ling tumor initiation and progression. Here, we describe a syngeneic orthotopic HCC
model that recapitulates the role of a host pro-tumorigenic microenvironment by
pre-conditioning mouse livers with a high-fat diet (HFD).
For complete details on the use and execution of this protocol, please refer to
Kudo et al. (2020).
BEFORE YOU BEGIN

Note: All mouse experiments must be approved by an Animal Care Committee in your
research institution. Personal protective equipment, autoclaved sterile surgical instruments
and frequent disinfection of equipment with 70% ethanol are essential for handling mice.

Induction of Fatty Liver by a High-Fat Diet

Timing: 8 weeks

1. Acquire High Fat, Fat Calories (60%), Mouse Diet (HFD, Bio-Serv F3282).
2. Mice should be started on HFD at approximately 6–8 weeks-old.
3. Mice will be fed HFD ad libitum for 8 weeks.

CRITICAL: Measuring body weight routinely during the experiment is important. Allocate
mice to be equally distributed for each experimental group according to body weight.

CAUTION: Be aware of skin dermatitis. It is important to notice dermatitis as soon as possible and
stop HFD if it happens. Autoclaved pieces of absorbant paper can help to prevent skin dermatitis.

4. Eight weeks after starting HFD, proceed to the next step to orthotopically implant HCC cells in
the fatty liver. One day before implantation, shave the abdominal region of the mouse.

STAR Protocols 1, 100185, December 18, 2020 ª 2020 The Authors. 1

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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Protocol

Preparation for Mouse Surgery

Timing: 30 min

5. All the surgical instruments must be autoclaved and opened on a clean bench.
6. Weigh and prepare the mice for the following procedure.
7. For anesthesia of mice, an isoflurane chamber with nose cone attachment is needed.
8. Prepare a set of autoclaved surgical instruments per mouse as follows: Sharp and blunt scissors; 1
curved fine forceps; 1 Straight blunt forceps: 2 Hartman hemostats.
9. Prepare 1 mL aliquots of Matrigel basement membrane matrix and store at 20 C. Thaw them on
ice 2 h before use.

KEY RESOURCES TABLE

REAGENT or RESOURCE SOURCE IDENTIFIER

Chemicals, Peptides, and Recombinant Proteins
DPBS Corning Cat# 21-031-CV
Penicillin-Streptomycin Corning Cat# 30-002-CI
DMEM Corning Cat# 10-017CV
Fetal Bovine Serum Omega Scientific, inc. Cat# FB-01
L-Glutamine Corning Cat# 25-005-CI
Trypsin-EDTA (0.25%), phenol red GIBCO Cat# 25200056
Matrigel basement membrane matrix Corning Cat# 354234
Trypan Blue Solution Gibco Cat# 15250061
Experimental Models: Cell Lines
DihXD3 cells Moscat lab N/A
Experimental Models: Organisms/Strains
Mouse: WT (C57BL/6), (*) Moscat lab N/A
Other
Insulin syringe, 0.3 mL with 31-gauge needle Becton Dickinson Cat# 328438
15- and 50-mL centrifuge tubes Genesee Scientific Cat# 28-103 and 28-108
Bright-field microscope EVOS M5000 Cat# AMF5000
Caliper Mitutoyo Cat# 500-171-30
Scale (capable of measuring mouse weight) Ohaus Cat# SP202
Sterile gauze N/A N/A
Heating pad N/A N/A
Isoflurane VETONE Vet-Rx-MW 502017
Curved fine forceps FST Cat# 11159-10
Straight brunt forceps FST Cat# 11002-12

Straight scissors FST Cat# 14040-10

Hartman Hemostats FST Cat# 13002-10
Autoclip applier plus 9-mm autoclips Kent Scientific Cat# INS750345; Cat# INS750546
PERMA-HAND SILK 5-0 VWR Cat# 95057-064
Germinator 500 Braintree Scientific, inc. Cat# GER 5287-120V
Surgical microscope Leica N/A
Wound clip remover FST Cat#: 12033-00

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(*) Note: This protocol can be used in immunocompromised mice such as NSG mice for liver
orthotopic implantation of human HCC cell lines such as SK-HEP-1.

MATERIALS AND EQUIPMENT

Maintenance Media

Reagent Final Concentration Stock Concentration Volume (mL)

DMEM - - 440
Glutamine 200 mM 2 mM 5
Penicillin-Streptomycin 1% 100% 5
Fetal Bovine Serum 10% 100% 50

Note: Store DMEM at 4 C and all the other components of the media at 20 C.

STEP-BY-STEP METHOD DETAILS

This protocol is aimed at injecting HCC cells orthotopically in preconditioned liver with high-fat diet
feeding. Before the injection, the HCC cells are trypsinized, resuspended in serum-free DMEM, and
placed in ice until the moment of injection. Following the anesthesia of the mouse, a parallel incision
to the linea alba is made in the abdominal wall to expose the liver. Then, HCC cells are mixed 1:1 with
Matrigel before injection. The injection is done orthotopically into three different sites: twice in the
left-lateral and once in the left-median lobes of the liver. After closing the incision with suture, the
skin is closed with wound clips. We show two different methods for closing the peritoneum after
the incision by continuous or interrupted suture. Mice are monitored for 2 to 6 weeks after implan-
tation. The main improvement of this protocol is to increase the incidence of tumors thanks to a
robust procedure and the injection into three sites in the liver.

Preparation of HCC Cells for Injection

Timing: 1 h

Here, the HCC cells are trypsinized and counted before injection.

1. Prewarm DPBS and maintenance media at 37 C and 0.25% trypsin-EDTA at 20 C–22 C before
use.
2. Remove the medium from the dish of HCC cells at around 80% confluence.

Note: This protocol uses DihXD3 as HCC cells since they are syngeneic and can be injected
and grow in C57BL6 mice, but other HCC cells can also be used.

CRITICAL: Avoid cell passaging for at least 2 days before implantation.

3. Rinse the dish with prewarmed DPBS (6–8 mL per 100 mm dish) and aspirate the DPBS.
4. Add 2 mL of 0.25% trypsin-EDTA to the dish. Place the plates in the 37 C incubator.
5. After 3–5 min, check the cells detaching from the plate by using a bright-field microscope.

CRITICAL: Do not leave the cells in trypsin-EDTA for more than 5 min.

6. When more than 90% of HCC cells are detached, add 6–8 mL per 100 mm dish of maintenance
media to neutralize the trypsin in the dish. Gently mix the HCC cell suspension and collect the
cells in a 15-mL centrifuge tube.

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Figure 1. Surgical Preparation for Orthotopic Implantation

In a disinfected biological safety cabinet, prepare the surgical instruments as follows: (A) Tubes for Matrigel and HCC
cell suspension on ice and 0.3-mL 31-gauge needle insulin syringes, (B) gauzes with 10% povidone-iodine solution, (C)
gauzes with 70% ethanol, (D) sterilized surgical instruments, (E) autoclip applicator, (F) suture, (G) bead sterilizer, (H)
ophthalmic ointment. Missing in the picture but required: an isoflurane chamber and nose cone, and a surgical
microscope.

7. Centrifuge the HCC cell suspension in the 15-mL centrifuge tube for 5 min at 350–400 3 g at
20 C–22 C. Wash the cells with 10 mL of PBS. Centrifuge the cells for 5 min at 350–400 3 g at
20 C–22 C and resuspend the cells in serum-free DMEM medium.
8. Count the number of HCC cells and adjust the cell concentration to 106 cells per 10 mL in serum-
free DMEM (corresponding to the final tumor cell inoculum per injected volume). One injection
requires 106 cells in 10 mL. Prepare 10% more volume than the exact volume in the case of the
loss during the injection. Keep the cell suspension on wet ice.

Orthotopic Implantation of Syngeneic HCC Cells in Fatty Liver

Timing: 20–30 min/mouse

Here, HCC cells are mixed with Matrigel and injected into the liver. HCC cells are mixed 1:1 with Ma-
trigel and kept in ice until the moment of the injection. After anesthetizing the mouse, the liver is
exposed, and cells are injected into three sites of the liver: two injections are done into the left-lateral
lobe, and the third injection is done into the left-median lobe. Each injection is performed very
slowly, and the change of color of the liver is observed during the injection. After injection, the inci-
sion and the skin are closed, and mice are set up for recovery.

9. Place the Matrigel solution (10 mL per 1 injection) on wet ice.

10. Place the required materials in the biological safety cabinet to prepare for orthotopic implanta-
tion (Figure 1).
11. Anesthetize the mice by using isoflurane inhalation (2%–3% to start the flow of isoflurane).
12. Apply pre-operative analgesia subcutaneously. Either meloxicam SR or buprenorphine can be
used, depending on your IACUC protocol.

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Figure 2. Schematic of Linea Alba and Xiphoid Process in Mouse

(A) Incision in the skin should be parallel to the linea alba. The incision should be 2.0–2.5 cm long.
(B) Incision in the peritoneum should be performed 1 cm below the xiphoid process and along the linea alba. A small
lateral incision perpendicular to the linea alba will improve the view.

13. Place the mouse in a supine position on the heating pad to avoid hypothermia under a surgical
microscope.
14. Use sterile gauzes with a 10% povidone-iodine solution followed by 70% ethanol to disinfect and
clean the shaved abdominal region. Wait until the skin is dry from povidone-iodine and 70%
ethanol.

See Methods Video S1.

15. Gently grab the skin with a pair of straight blunt forceps and perform a midline abdominal inci-
sion of the skin (starting 1 cm under the xiphoid process).
16. Identify the linea alba in the midline of the peritoneum and make a 2.0–2.5 cm incision along the
linea alba. To improve the surgical view, perform a lateral incision, perpendicular to the linea
alba (see Figure 2).

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Figure 3. Liver Color Change during Cell Injection

During injection, the liver surface will change from dark brown to pale at the injection site.

CRITICAL: Proper incision makes the procedure easier with a good surgical view of the in-
jection site.

See Methods Video S2.

17. Hold both sides of the peritoneum with two hemostats and flip them to the cranial side to
expose the left-medial and/or left-lateral lobe (s) of the liver.

CRITICAL: The investigator should see an enlarged fatty liver.

18. Immediately before orthotopic implantation, 10 ml of HCC cell suspension should be mixed with
10 mL of Matrigel (equal volume to cell suspension). Withdraw the total of 20 mL of Matrigel and
the cell suspension with a 0.3-mL insulin syringe attached to a 31-gauge needle. This will ensure
a final cell number of 106 cells per 20 mL of the injected volume of the Matrigel/HCC cells suspension.

CRITICAL: Keep the Matrigel-containing solution on ice to prevent gel solidification.

19. Adjust the focus of the surgical microscope on the injection site. Fix the injection site and
perform counter-traction by using sterile gauze.
20. Gently insert (approximately 2–3 mm) a 31-gauge needle of 0.3-mL insulin syringe with the Ma-
trigel/HCC cells solution under the liver capsule.
21. Slowly inject 20 mL of the Matrigel/HCC cells solution (containing 1 3 106 HCC cells) in the sub-
capsular region of the left-medial and/or left-lateral lobe (s). Successful implantation will change
the color of the liver surface to white (see Figure 3).

CRITICAL: Inject the Matrigel/HCC cells solution very slowly as much as possible to avoid
leakage and to prevent damage to the liver capsule.

CRITICAL: Maximum 3 injections (2 injections in the left-lateral lobe and 1 injection of the
left-median lobe) can be performed.

CRITICAL: This procedure requires surgical skill and practice to obtain reproducible re-
sults. In order to get more reproducible results is critical to inject viable cells and this is
achieved by a shorter time of the procedure.

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Figure 4. Continuous Suture

The continuous suture is one in which a continuous, uninterrupted length of material is used.

22. After injecting the Matrigel/HCC cells solution, keep the needle at least 30 s and slowly retract
the needle.
23. Check the liver surface at the needle track for leakage and bleeding.

See Methods Video S3.

24. Close the peritoneum of the abdominal wall by a continuous (Figure 4), or interrupted (Figure 5)
suture using PERMA-HAND SILK 5-0. Close the skin layer of the abdominal wall by Reflex 9 mm
Wound Clips using the autoclip applier (Figure 6).

See Methods Video S4 and S5.

25. Leave the mouse on the heating pad and allow the mouse to recover from anesthesia.
26. Apply postoperative analgesia if needed according to the specific animal protocol at your insti-
tution.

Figure 5. Interrupted Suture

The interrupted suture is one in which each stitch is made with a separate piece of material.

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Figure 6. Wound Clips

Wound clips can be used for rodent skin closure. They must be applied and removed with a specific applicator.

See Methods Video S6.

See Methods Video S7 for a full overview of the surgery procedure.

Monitoring Mouse Condition

Timing: 2–6 weeks

Here, mice are monitored for tumor development.

27. Wound clips must be removed in 10–14 days once the incision has healed.
28. Closely monitor the mice for tumor progression and continue to assess their health until the
experiment will be finished. HFD will be continued during the whole experiment.

CRITICAL: If severe weight loss occurs (>20% loss of starting body weight), the mouse
should be sacrificed according to your institution’s animal protocol.

29. See the ‘‘Quantification and Statistical Analysis’’ section for more detail into the analysis of the
results.

EXPECTED OUTCOMES
The tumors take 2 to 6 weeks to engraft and grow enough to be analyzed (Figure 7). See Figure 6F-J
in our original paper (Kudo et al., 2020). By 6 weeks, if no tumor has formed, it is unlikely that a tumor
will form. Our protocol provides an orthotopic model of HCC in which tumor growth is modulated by
the fatty liver. Co-implantation with stellate cells also helps to the tumor formation and enables to
examine the effect of stellate cells on tumorigenesis and tumor progression. Evaluation of lung me-
tastases can be used to assess the metastatic ability based on both morphology and immunohisto-
chemistry to confirm the expression of typical HCC markers.

QUANTIFICATION AND STATISTICAL ANALYSIS

1. We have observed 80%–100% tumor incidence in wild type mice.

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Figure 7. Macroscopic Appearance of an Orthotopic Tumor in the Liver

Successful implantation generates orthotopic liver tumor. Scale bar represents 10 mm.

2. Create an excel file to record data pre-injection and after tumor harvesting. Data to be recorded
should include: sex, date of birth, age, and body weight at the time of liver fatty induction, body
weight before surgery, body weight at the endpoint.
3. At the endpoint, the liver weight and diameters (short and long) for each tumor should be re-
corded. Tumor volume is calculated for each tumor nodule using the following formula: 1/6 3
3.14 3 (short diameter)2 3 (long diameter).

LIMITATIONS
Limitations of liver orthotopic implantation are the long induction time and the difficulty of the sur-
gical procedure. Induction of fatty liver by HFD takes 8 weeks and HCC orthotopic tumor formation
requires 2–6 weeks, therefore, a total timeline for an experiment is 2–3 months. Also, the time effort
for building surgical expertise is needed. We recommend the use of interrupted suture if there is no
previous experience in mouse surgeries. Potential issues for orthotopic tumor implantation are
leakage and ‘‘accidental’’ peritoneal metastasis. To avoid these problems, we recommend (1)
small-volume Matrigel/HCC cell suspensions (20 mL per injection), (2) mixing Matrigel just before in-
jection to increase the viscosity, and (3) 0.3-mL insulin syringes with 31-gauge needles.

TROUBLESHOOTING
Problem 1
Mice show dermatitis as a result of the high-fat diet pre-treatment.

Potential Solution
Place autoclaved pieces of absorbant paper in the cage.

Problem 2
Mice body weight variates widely after HFD.

Potential Solution
Allocate the mice to each group according to their body weight.

Problem 3
Injection makes bubbles in the liver surface.

Potential Solution
Preparing 10 mL aliquots of Matrigel in 1.5 mL Eppendorf tubes helps to resuspend the Matrigel/
HCC cells solution without drawing bubbles.

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Problem 4
Leakage from the injection site.

Potential Solution
Inject Matrigel/HCC cells suspension as slow as possible and hold for at least 30 s. Reduce the injec-
tion volume. Increase the ratio of Matrigel in the Matrigel-HCC cells solution.

RESOURCE AVAILABILITY
Lead Contact
Further information and requests for resources and reagents should be directed to and will be ful-
filled by the Lead Contact, Jorge Moscat (jom4010@med.cornell.edu).

Materials Availability
DihXD3 cells will be available upon request with a completed Materials Transfer Agreement.

Data and Code Availability

This study did not generate datasets.

SUPPLEMENTAL INFORMATION
Supplemental Information can be found online at https://doi.org/10.1016/j.xpro.2020.100185.

ACKNOWLEDGMENTS
This Research was supported by grants by NCI and NIDDK of the National Institutes of Health under
awards numbers R01DK108743 and R01CA211794 to J.M., who is the Homer T. Hirst III Professor
of Oncology in Pathology. We thank the personnel of the Animal Facility at SBP Medical Discovery
Institute for technical assistance.

AUTHOR CONTRIBUTIONS
Conceptualization, H.K., M.T.D.-M., and J.M.; Investigation, H.K., A.D., T.C.-D., and Y.K.; Writing –
Original Draft, H.K. and A.D.; Writing – Review & Editing, H.K., A.D., T.C.-D., Y.K., M.T.D.-M. and
J.M.; Funding Acquisition, M.T.D.-M. and J.M.; Supervision, M.T.D.-M. and J.M.

DECLARATION OF INTERESTS
The authors declare no competing interests.

REFERENCES
Kudo, Y., Sugimoto, M., Arias, E., Kasashima, H., Nakanishi, N., L’Hermitte, A., et al. (2020). phosphorylation, and NRF2 to promote liver cancer
Cordes, T., Linares, J.F., Duran, A., Nakanishi, Y., PKClambda/iota loss induces autophagy, oxidative progression. Cancer Cell 38, 247–262.e11.

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