In vivo model of liver metastasis by splenic injection.

Protocol for Establishing a Mouse Model of Colorectal Cancer Liver Metastasis by Intrasplenic Injection of MC38 Cells Followed by Splenectomy

1. Objective

To establish an experimental model of colorectal cancer liver metastasis (CRLM) by injecting MC38 murine colorectal cancer cells into the spleen of C57BL/6 mice, thereby introducing tumor cells into the liver through the portal circulation. Subsequent splenectomy is performed to minimize tumor growth within the spleen.

This model is used to investigate hepatic colonization, metastatic outgrowth, and changes in the hepatic tumor microenvironment.

2. Experimental Conditions

3. Materials, Reagents, and Equipment

3.1. Cell culture and preparation

• MC38 colorectal cancer cells
• Cell culture medium and associated reagents
• Trypsin–EDTA
• Sterile PBS (Ca²⁺/Mg²⁺-free)
• 15- and 50-mL conical tubes
• Micropipettes and sterile pipette tips
• Cell counter or hemocytometer
• Trypan blue
• Centrifuge
• Ice bucket
• Sterile syringes and appropriately sized needles

3.2. Experimental animals and anesthesia

• C57BL/6 mice
• IACUC-approved anesthetic agent(s)
• IACUC-approved analgesic agent(s)
• Sterile ophthalmic ointment
• Warming device and temperature-monitoring equipment
• Electronic balance

3.3. Surgery and antisepsis

• Surgical clippers
• 70% ethanol
• Povidone–iodine or chlorhexidine
• Sterile surgical scissors and forceps
• Needle holder
• Hemostatic forceps
• Sterile gauze
• Sterile surgical drape
• Sterile gloves
• Sterile saline
• Sterile suture material without a needle for vascular ligation
• Sterile sutures with attached needles for abdominal wall and skin closure
• Recovery cage
• Surgical and postoperative monitoring records

4. Experimental Procedure

Step 1. Preparation of MC38 Cells

1.  Prepare mycoplasma-free MC38 cells and examine their morphology and growth status.

2.  Detach the cells using trypsin–EDTA and collect the cell suspension.

3.  Centrifuge the suspension at 200 × g for 5 min at 4°C.

4.  Remove the supernatant and resuspend the cell pellet in 10 mL of sterile PBS.

5.  Centrifuge at 200 × g for 5 min at 4°C and discard the supernatant.

6.  Wash the cells with PBS one or two times to remove residual medium and trypsin.

7.  Resuspend the final cell pellet in an appropriate volume of sterile PBS.

8.  Determine the cell count and viability using trypan blue and a cell counter.

9.  Adjust the cell suspension to a final concentration of 1 × 10⁶ ~ 1 × 10⁷ viable cells/mL.

10.  Keep the prepared cell suspension on ice until injection.

Cell quality-control criteria:

• Record cell viability; ≥90% may be adopted as an internal quality-control threshold.
• Check for cell clumping.
• Record the passage number.
• Record the interval between cell harvesting and injection.
• Preferably use cells from the same preparation batch across experimental groups.

Step 2. Mouse Preparation and Anesthesia

1.  Prepare C57BL/6 mice in accordance with the approved animal experimental protocol.

2.  Record body weight and assess general health before the procedure.

3.  Administer an IACUC-approved anesthetic at the approved dose and by the approved route.

4.  Apply sterile ophthalmic ointment to both eyes immediately after induction of anesthesia to prevent corneal drying.

5.  Remove hair from the intended surgical site on the left abdomen using surgical clippers.

6.  Disinfect the surgical area using the institutionally approved regimen, such as povidone–iodine or chlorhexidine with 70% ethanol as appropriate.

7.  Position the mouse on the surgical platform and apply a sterile surgical drape.

8.  Use an appropriate warming device to minimize intraoperative hypothermia.

9.  Provide perioperative pain management according to the institutionally approved analgesic plan.

Step 3. Intrasplenic Injection of MC38 Cells and Splenectomy

1.  Maintain anesthesia and body temperature while positioning the mouse to expose the left abdominal region.

2.  Locate the spleen anatomically and incise the skin and abdominal wall using the approved aseptic surgical procedure.

3.  Expose the spleen carefully while avoiding damage to nearby blood vessels and pancreatic tissue.

4.  Place sterile gauze moistened with sterile saline around the spleen to prevent tissue desiccation.

5.  Gently resuspend the MC38 cell suspension and verify the intended dose of 1 × 10⁵ to 1 × 10⁶ viable cells in 100 µL per mouse.

6.  Draw the suspension into a sterile syringe and ensure that no air bubbles are present.

7.  An experienced operator injects the cell suspension into the spleen according to the approved intrasplenic injection SOP.

8.  Assess the injection site for leakage of the cell suspension, bleeding, and splenic tissue damage.

9.  Allow 2–5 min following the injection.

10.  Ligate the splenic vessels and perform splenectomy according to the approved surgical procedure.

11.  Inspect the abdominal cavity for bleeding and confirm adequate hemostasis after splenectomy.

12.  Check for injury to adjacent organs or residual bleeding.

13.  Close the abdominal wall using sterile sutures in accordance with the approved closure technique.

14.  Close the skin using a separate sterile suture or another approved skin-closure material.

15.  Clean and inspect the surgical site and assess the integrity of the closure.

16.  Record injection success, leakage, bleeding, completion of splenectomy, and any intraoperative complications.

Surgical precautions:

• Leakage of tumor cell suspension into the peritoneal cavity may cause ectopic tumor growth and introduce experimental variability; document any suspected leakage.
• Avoid injury to adjacent vessels and the pancreas during splenectomy.

Step 4. Postoperative Recovery and Monitoring

1.  Transfer the mouse to a recovery cage equipped with appropriate temperature support.

2.  Monitor respiration, body temperature, spontaneous movement, and bleeding from the surgical site during anesthetic recovery.

3.  Observe the animal until it has recovered adequately, including return of normal ambulation and activity.

4.  If needed, administer analgesics according to the schedule approved in the animal protocol.

5.  Monitor postoperative recovery, body weight, food and water intake, activity, and general appearance.

6.  Inspect the incision for bleeding, swelling, redness, infection, or wound dehiscence.

7.  Assess and document the animal’s condition daily during the early postoperative period, in accordance with institutional guidelines.

8.  Consult veterinary staff and apply the approved humane endpoint criteria if persistent pain, reduced activity, delayed recovery, bleeding, or rapid weight loss occurs.

5. Evaluation of Liver Metastasis

At the predefined experimental endpoint, evaluate tumor progression, euthanize the animals using an approved method, and harvest the liver.

5.1. Gross Examination

1.  Photograph the entire liver.

2.  Examine the liver surface for metastatic nodules.

3.  Count and record macroscopically visible metastatic nodules.

4.  Measure the liver weight.

5.  Calculate the liver-to-body weight ratio if required.

5.2. Histological Examination

• Hematoxylin and eosin (H&E) staining: presence and area of metastatic lesions.
• Sirius Red staining: liver fibrosis and peritumoral collagen deposition.
• α-SMA immunostaining: activated hepatic stellate cells and cancer-associated fibroblasts.
• Ki-67 immunostaining: tumor-cell proliferation.
• F4/80 immunostaining: macrophage infiltration.
• CD8 immunostaining: cytotoxic T-cell infiltration.

5.3. Quantitative Analysis of Liver Metastasis

Because surface nodule counts may underestimate metastases located within the liver parenchyma, assessment of gross nodule counts together with H&E-based histological tumor area is recommended.