Protocol to establish a mouse model for hepatic metastasis of colorectal cancer

Summary

Hepatic metastasis is the leading cause of colorectal-cancer (CRC)-associated death. Here we describe an optimized protocol to establish a more clinically relevant mouse model of CRC metastasis. We detail steps for subcutaneous transplantation of luciferase-expressing CRC cells and subsequent orthotopic transplantation of subcutaneous tumor tissues. This mouse model allows CRC cells to form tumors within the intestinal tract and metastasize to the liver, thereby providing the approach to assess hepatic metastasis of CRC in vivo.

For complete details on the use and execution of this protocol, please refer to Zhang et al. (2021).¹

Graphical abstract

Highlights

• •Optimized protocol to establish a mouse model of CRC hepatic metastasis
• •Sensitive visualization and quantification for small metastases
• •Surgical guidelines for the transplantation of CRC cells or tissues

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Hepatic metastasis is the leading cause of colorectal-cancer (CRC)-associated death. Here we describe an optimized protocol to establish a more clinically relevant mouse model of CRC metastasis. We detail steps for subcutaneous transplantation of luciferase-expressing CRC cells and subsequent orthotopic transplantation of subcutaneous tumor tissues. This mouse model allows CRC cells to form tumors within the intestinal tract and metastasize to the liver, thereby providing the approach to assess hepatic metastasis of CRC in vivo.

Before you begin

Before beginning, we need to prepare the following essential elements.

• 1.Preparation of plasmids: PCR-amplified firefly luciferase was cloned into pCDH-CMV-EF1α-BSD vector. Lentiviral package plasmids (pMD2.G and psPAX2) were bought from Addgene.
• 2.Preparation of animals: 6-week-old female BALB/c nude mice were purchased from SLAC Laboratory Animal Co., Ltd. (Shanghai, China) and maintained in pathogen-free facilities at Shanghai Institute of Biochemistry and Cell Biology (SIBCB).

Institutional permissions

Animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of SIBCB and complied with all relevant ethical regulations. Animals were randomly allocated to experimental groups. Animals had free access to water and food and were housed in a controlled environment with a 12 h–12 h light-dark cycle, constant temperature (21°C) and relative humidity (20%–40%).

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Chemicals, peptides, and recombinant proteins
DMEM	Sigma	D5796
Fetal bovine serum	Excell	FSD500
Penicillin-streptomycin solution	Gibco	15140163
PBS	Meilunbio	MA0008
Trypsin	Gibco	25200072
LipoFiter™ Liposomal Transfection Reagent	Hanbio	HB-TRLF-1000
Polybrene	Sigma	H9268-5G
Blasticidin S	Yeasen	60218ES10
Matrigel	Corning	356237
Surgical tissue adhesive	Compont	N/A
D-luciferin	PerkinElmer	122799
Isoflurane	RWD	R510-22-10
Trypan blue	Thermo	15250-061
Hematoxylin-eosin	Beyotime	C0105S
Experimental models: Cell lines
Human: HEK-293T cells	Cell library of the Chinese Academy of Sciences	SCSP-502
Human: HCT116 cells	Cell library of the Chinese Academy of Sciences	SCSP-5076
Experimental models: Organisms/strains
Mouse: 6 weeks-old female BALB/c nude mice	SLAC	N/A
Recombinant DNA
PCDH-CMV-luciferase-EF1α-BSD	This paper	N/A
Software and algorithms
GraphPad Prism	GraphPad	http://www.graphpad.com/support
Tanon Image	Tanon	N/A
Other
100 mm dish	NEST	704001
35 mm dish	Thermo	150460
1.5 mL centrifuge tube	NEST	615001
15 mL centrifuge tube	NEST	601002
1 mL syringe (with 27G 0.5 inch Needle)	BD Biosciences	309623
0.45 μm filter	Millipore	SLHVR33RB
0.22 μm filter	Millipore	SLGPR33RB

Materials and equipment

Cell Culture medium

Reagent	Final concentration	Amount
FBS	10%	5 mL
Penicillin-Streptomycin Liquid	1%	0.5 mL
DMEM (high glucose)	N/A	44.5 mL
Total	N/A	50 mL

Note: This medium can be stored at 4°C for 1 month.

Step-by-step method details

Construction of luciferase-expressing human colorectal carcinoma cells

Timing: 5–7 days

Lentiviral particles were produced and transfected into HCT116 cells for stable expression of luciferase.

Lentiviral vector: PCDH-CMV-luciferase-EF1α-BSD.

• 1.
  Lentivirus packaging and production.

  • a.
    Seed HEK293T cells in a 35 mm dish (2 mL DMEM-10% FBS) of 50%–70% confluence and with good adhesion. Culture the cells in 37°C, 5% CO₂ incubator.
  • b.
    Change the medium of HEK293T cells with 2 mL fresh DMEM-10% FBS before transfection.
  • c.
    Co-transfect lentiviral vector (PCDH-CMV-luciferase-EF1α-BSD), packing vector (psPAX2) and envelope vector (pMD2.G) into HEK293T cells using LipoFiter™ Liposomal Transfection Reagent (Mass ratio of 3 vectors is 4:3:3).

    Note: For the details of lipofection procedure, read the protocols provided by commercial Liposomal Transfection Reagent.

  • d.
    Change the medium with 2 mL DMEM-10% FBS in 6–12 h to remove the liposomes and residual plasmids.
  • e.
    48 h from lipofection, collect the culture medium (lentivirus supernatant) and supplement with the same volume of fresh DMEM-10% FBS, which will be collected in another 24 h.
  • f.
    Filter the lentivirus supernatant with 0.45 μm millipore filter membrane and collect the lentivirus in a 15 mL centrifugal tube (polypropylene resin).

    Note: Lentivirus supernatant can be stored at 4°C for no more than one week and at −80°C for no more than one year.

• 2.
  Lentivirus infection of HCT116 cells.

  • a.
    Mix lentivirus supernatant and DMEM-10% FBS (vol/vol = 1:1) in a 35 mm dish, and passage 0.5 × 10⁶ HCT116 cells. Culture the cells in a 37°C, 5% CO₂ incubator.

    Optional: Add 6–8 μg/mL polybrene to promote efficiency of infection.

  • b.
    Change the medium of HCT116 cells with 2 mL fresh DMEM-10% FBS in 12–24 h.
  • c.
    Culture the cells for 2–3 days, and then add 3–10 μg/mL blasticidin S to screen the resistant cells. Wild-type HCT116 cells can be used as a control.

    Note: Remove the medium with blasticidin S when the wild-type cells are all killed. The virus concentration and infection time are adjustable based on the cell type to be infected. The infection time can vary from 12 h to 24 h, and the ratio of virus/DMEM-10%FBS can vary from 1:9 to 1:1.

    CRITICAL: Make sure the cells are in good state (Cell survival, growth, morphology, etc).

• 3.
  Detect luciferase expression in infected HCT116 cells.

  • a.
    Collect 1 × 10⁶ HCT116 cells stably expressing luciferase (HCT116-luc) in a 1.5 mL centrifugal tube. Centrifuge cell suspension at 1,000 × g for 2 min. Remove the supernatant.
  • b.
    Add 100 μL 7.5 mg/mL D-luciferin into the 1.5 mL centrifugal tube.
  • c.
    Bioluminescence imaging of the cells immediately by Tanon-5200 Chemiluminescent Imaging System with 10 s exposure time (Figures 1A and 1B).

Figure 1.

Bioluminescence imaging of HCT116-luc cells

(A and B) 1 × 10⁶ HCT116-luc cells were collected in a 1.5 mL centrifugal tube and 100 μL D-luciferin were added into the tube. Brightfield imaging (A) and bioluminescence imaging (B) of the cells were performed.

Establishment of cell-derived xenografts in BALB/c nude mice

Timing: 2 weeks

The aim of this session is to gain transplantable tumor tissues. HCT116-luc cells are inoculated subcutaneously into BALB/c nude mice. The xenografts can be used as transplantable tumor tissues. All animal experiments strictly complied with ethics and relevant regulations.

• 4.
  Preparation of cells for the inoculation.

  • a.
    Change the medium of HCT116-luc cells in a 100 mm dish of 70% influence with fresh DMEM-10% FBS the day before inoculation.

    CRITICAL: The cells must be in the exponential growth stage.

  • b.
    Digest the cells with 0.05% trypsin and wash cells twice with PBS.
  • c.
    Count the cells with an automated cell counter (Invitrogen Countess).
  • d.
    Centrifuge cell suspension at 1,000 × g for 2 min, remove the supernatant.
  • e.
    Resuspend the cells in the mixture of serum-free DMEM and matrigel (vol/vol = 1:1) at a density of 2 × 10⁷ cells/mL.

    Note: Put the cell suspension (step 4-e) on ice, and mix the cell suspension intensively with a pipette before inoculation.

• 5.
  Subcutaneous inoculation.

  Note: 6 weeks-old, 18–20 g weight BALB/c nude mice will be used.

  • a.
    Anesthetize one mouse in an induction chamber, filled with 2% isoflurane.
  • b.
    Aspirate 100 μL cell suspension (step 4-e) with a 1 mL syringe with 27G 0.5-inch needle.
  • c.
    Disinfect the mouse dorsal skin with 75% ethanol.
  • d.
    Insert the needle into the skin at disinfected area to reach the subcutaneous pocket.

    Note: Avoid inserting the needle into muscle layer or thorax.

  • e.
    Gently inject the cell suspension into the subcutaneous pocket.
  • f.
    After injection, hold for a few seconds before pulling out the needle gently and vertically.

• 6.Place the mouse back into the cage and monitor until it is awake and mobile.
• 7.Feed the mice under stable raising conditions for two weeks.

Note: Monitor the mice 2–3 times a week. The tumor lump can be observed in 7–10 days after inoculation (Figure 2).

Figure 2.

Establishment of subcutaneous xenografts with HCT116-luc cells in BALB/c nude mice

HCT116-luc cells were subcutaneously implanted into BALB/c nude mice. The subcutaneous xenograft was indicated with red arrow.

Collection and processing of transplantable tumor tissues

Timing: 20–30 min

In this session, the xenografts established in the previous procedure were excised and minced into small pieces. These pieces will be used in orthotopic implantation procedure.

• 8.Euthanize the mice with xenografts.
• 9.
  Remove the xenografts from sacrificed nude mice.

  • a.
    Using the fine-iris scissors, open the skin near the xenograft.
  • b.
    Using the fine-tissue forceps, gently separate the xenograft from adjacent tissues.
• 10.Place the xenograft into a 100 mm dish with serum-free DMEM (Figure 3A).
• 11.Using a sterile scalpel blade, mince the xenograft into 1–2 mm³ pieces (Figure 3B).

CRITICAL: The small pieces should be immersed in serum-free DMEM to retain viability until orthotopic implantation.

CRITICAL: Surgical instruments must be strictly sterilized.

Figure 3.

Preparation of transplantable tumor tissues

(A and B) The subcutaneous xenograft was dissected from BALB/c nude mice (A) and cut into 1–2 mm³ pieces. The shredded tumor tissues were indicated with white arrows (B).

Orthotopic transplantation of colorectal tumor tissues

Timing: 8 weeks

In this session, the small pieces of tumor tissues were used to orthotopically implant into another group of 6 weeks-old female BALB/c nude mice (Figure 4A). The implantation was performed as described previously.²

CRITICAL: It is the most essential procedure.

• 12.
  Preparation for the surgery.

  • a.
    Sterilize surgical instruments before the surgery.
  • b.
    Anesthetize one mouse with 4% chloral hydrate (100 μL/10 g body weight) via intraperitoneal injection.
  • c.
    Place the mouse onto a heating pad.

    Note: Avoid overheating and monitor the reflexes and breathing of the mouse continuously.

  • d.
    Position the mouse on its back and use duct tape to fix its forelegs and hind legs in a V-shape.
  • e.
    Disinfect the abdomen of the mouse with 0.5% iodophor.

• 13.
  Open the abdomen of the mouse.

  • a.
    Using the curved fine-tissue forceps, hold the skin in abdomen.
  • b.
    Using the fine-iris scissors, incise a 15 mm vertical midline on the skin.
  • c.
    To isolate the surgical area, place a small sterile surgical gauze (with a rhomboidal incision in the middle) onto the incision of the mouse abdomen (Figure 4B).
  • d.
    Incise the linea alba to separate the rectus abdominis muscles.

Note: During the incision, avoid damaging the large vessels in the abdominal wall and the abdominal organs.

CRITICAL: Drench the gauze with sterile PBS, it is essential to keep the surgical area hydrated.

• 14.Drape out the cecum at the upper left side of the abdomen with a sterile cotton swab drenched in sterile PBS.

Note: Do not use metal surgical tools to hold the intestine. Make sure to avoid damaging the abdominal organs.

• 15.Place the cecum horizontally onto the gauze (Figure 4C).

CRITICAL: During the extra-abdominal surgery, it is essential to keep intestine hydrated with sterile PBS continuously.

• 16.Using the fine-tissue forceps, gently hold and lift the serosa layer of the cecum.
• 17.Using the fine-spring scissors, make a 3–4 mm vertical incision on the serosa layer.

Note: The incision should be made between the large blood vessels to prevent unintentional bleeding.

CRITICAL: It is essential to incise the external layer of the intestinal epithelium but not the entire intestinal wall. Perforating the entire intestinal wall is lethal for the mouse.

CRITICAL: To prevent rupture of the serosa, keep the intestinal epithelium hydrated with sterile PBS.

• 18.Using two pairs of fine-tissue forceps, remove the muscularis externa to separate the serosa from the underlying submucosa layer on both sides of the opening (Figure 4D).

Note: By performing this operation, a pouch is created.

Note: Make sure to avoid perforating the intestinal wall.

• 19.Using curved fine-tissue forceps, gently pick up a piece of graft and push it into the pouch (Figure 4E).

CRITICAL: The graft should fit within the pouch.

• 20.Cover the orthotopic implantation area with a small amount of surgical tissue adhesive (Figure 4F).

CRITICAL: It is essential to prevent post-operative adhesions between the surgical incision and the abdominal wall.

• 21.Using another cotton swab drenched in sterile PBS, gently put the cecum back into the abdomen (Figure 4G).
• 22.Suture the abdominal wall (Figure 4H).
• 23.Suture the skin (Figures 4I and 4J).
• 24.Place the mouse back into the cage, monitor it to be awake and mobile.

Note: Make sure the mice are in good status and the sutures are not pulled off the day after surgery.

• 25.Feed the mice under stable raising conditions for eight weeks.

Figure 4.

Orthotopic transplantation procedures of intestinal tumor tissues

(A) Schematic diagram of orthotopic transplantation of intestinal tumor tissues.

(B) An incision was made on the skin.

(C) The cecum was exteriorized.

(D) A pouch was created under the serosa. The red arrow indicates the epithelial pocket.

(E) A piece of graft was pushed into the pouch.

(F) The orthotopic implantation area was covered with surgical tissue adhesive.

(G) The cecum was put back into the abdomen.

(H) The abdominal wall was sutured.

(I and J) The skin was sutured.

Analysis of the hepatic metastasis of CRC

Timing: 20–30 min

Eight weeks after the orthotopic implantation, the metastatic nodules in the livers can be counted and statistically analyzed.

• 26.Perform intraperitoneal injection of 100 μL 7.5 mg/mL D-luciferin to the mice.
• 27.Euthanize the mice 10 min after injection.
• 28.Remove the livers from the mice immediately and place the tissues in a clean 100 mm dish. Big metastatic nodules can be easily observed and counted (Figure 5A).
• 29.Bioluminescence imaging of the dissected liver tissues by Tanon-5200 Chemiluminescent Imaging System with 1 min exposure time (Figure 5B). The luciferase intensity of metastatic nodules can be measured with Tanon Image software (Figure 5C).

Figure 5.

Analysis of hepatic metastasis of CRC

(A) The mouse with intestinal tumor was euthanized and the liver was dissected.

(B) Bioluminescence imaging of the dissected liver tissue.

(C) The luciferase intensity of dissected liver was measured with Tanon Image software.

(D) The hepatic nodules were validated to be metastatic tumors by HE staining. The scale bar represents 50 μm.

Expected outcomes

This protocol describes a mouse model to investigate hepatic metastasis of CRC in vivo. Usually, it takes 2 weeks for the HCT116-luc cells to form subcutaneous tumors and 8 weeks for the intestinal xenograft to form hepatic metastasis in BALB/c nude mice. Hepatic metastases of CRC can be easily monitored or measured by the bioluminescent imaging of the dissected liver from the euthanized mice (Figures 5A–5C). In addition, the hepatic nodules can be dissected and validated to be metastatic tumors by hematoxylin-eosin (HE) staining (Figure 5D).

Limitations

This protocol describes a detailed procedure to establish a mouse model of hepatic metastasis of CRC. It takes a long time (8 weeks) for orthotopic grafts to develop hepatic metastatic nodules. In this model, tiny metastases that couldn’t be observed by the naked eyes can be imaged by bioluminescent imaging of the dissected liver. However, due to the presence and strong bioluminescent signal of the orthotopic intestinal tumor, the metastases cannot be imaged in vivo.

Troubleshooting

Problem 1

The virus titer is not high enough (step 2 of step-by-step method details).

Potential solution

The HEK293T cells should be in a good state in the whole process of virus packing and producing. If the host tumor cells are not susceptible to infection, concentrate and purify the virus supernatant by ultra-centrifugation.

Problem 2

The cell-derived xenografts fail to grow in BALB/c nude mice (step 4 of step-by-step method details).

Potential solution

The cell viability of luciferase-expressing tumor cells is too low. Before proceeding with step 4, make sure there are no abnormalities of the cell survival, growth and morphology. The virus concentration, infection time and antibiotic concentration can be adjusted to optimize the cell state.

Problem 3

Unintentional bleeding while making the incision on the serosa layer (step 18 of step-by-step method details).

Potential solution

If the blood vessels are damaged, try to stop bleeding using a dry sterile cotton swab, and rehydrate this area by sterile PBS. Another case is that the epithelium is perforated, the orthotopic implantation could be continued and completed. But the animals must be monitored meticulously after the surgical operation. The experiment must be terminated if the animal status is abnormal.

Problem 4

Rupture of the serosa while creating the pouch on it (step 18 of step-by-step method details).

Potential solution

Try to remove the muscularis externa to separate the serosa from the underlying submucosa layer on one side of the opening. It is better to keep the intestinal epithelium well hydrated to prevent this problem.

Problem 5

The piece of graft cannot be pushed into the pouch (step 19 of step-by-step method details).

Potential solution

The graft does not fit into the pouch, repeat step 18 to enlarge the pouch or place the graft back into serum-free DMEM and mince it into proper size.

Problem 6

No or too weak signal of the bioluminescence imaging (step 29 of step-by-step method details).

Potential solution

Firstly, make sure the model has been successfully established, and the hepatic nodules can be observed with the naked eyes. These hepatic nodules can also be validated as metastatic tumor tissues by other methods e.g., HE staining. In this case, the possible reason is the attenuation of bioluminescence. Strictly control the time that the mice should be sacrificed 10 min after injection, and bioluminescence imaging should be completed in another 5–15 min.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Weiwei Yang (wyang@sibcb.ac.cn).

Materials availability

The plasmid generated in this study is available from the authors on reasonable request.

Data and code availability

This study did not generate/analyze [datasets/code].