The Influence of Liver Resection on Intrahepatic Tumor Growth

Summary

A high incidence of tumor recurrence after resection of liver metastases remains an unsolved problem. The illustrated mouse model may be useful to investigate the reasons for such recurrences. It combines a liver resection model with intrahepatic tumor cell injection for the first time.

Abstract

The high incidence of tumor recurrence after resection of metastatic liver lesions remains an unsolved problem. Small tumor cell deposits, which are not detectable by routine clinical imaging, may be stimulated by hepatic regeneration factors after liver resection. It is not entirely clear, however, which factors are crucial for tumor recurrence.

The presented mouse model may be useful to explore the mechanisms that play a role in the development of recurrent malignant lesions after liver resection. The model combines the easy-to-perform and reproducible techniques of defined amounts of liver tissue removal and tumor induction (by injection) in mice. The animals were treated with either a single laparotomy, a 30% liver resection, or a 70% liver resection. All animals subsequently received a tumor cell injection into the remaining liver tissue. After two weeks of observation, the livers and tumors were evaluated for size and weight and examined by immunohistochemistry.

After a 70% liver resection, the tumor volume and weight were significantly increased compared to a laparotomy alone (p <0.05). In addition, immunohistochemistry (Ki67) showed an increased tumor proliferation rate in the resection group (p <0.05).

These findings demonstrate the influence of hepatic regeneration mechanisms on intrahepatic tumor growth. Combined with methods like histological workup or RNA analysis, the described mouse model could serve as foundation for a close examination of different factors involved in tumor growth and metastatic disease recurrence within the liver. A considerable number of variables like the length of postoperative observation, the cell line used for injection or the timing of injection and liver resection offer multiple angles when exploring a specific question in the context of post-hepatectomy metastases. The limitations of this procedure are the authorization to perform the procedure on animals, access to an appropriate animal testing facility and acquisition of certain equipment.

Introduction

Colorectal cancer (CRC) accounts for nearly 9% of all malignant tumors. It is the third most common cancer, both in the U.S. and worldwide. Global mortality rates from CRC range from 300,000 to over 500,000 per year¹. Twenty percent of patients suffer from liver metastases upon discovery of their colorectal tumor. Resectable metastases are normally treated by a partial liver resection^2,3. Improved surgical techniques, new multimodal strategies and new definitions of resectable metastases render the therapy of a partial liver resection possible for an increasing number of patients⁴.

Recurrence of secondary liver malignancies, however, is a challenging clinical sequalae in modern gastrointestinal surgery. Patients with CRC who underwent resection of liver metastases have a 30 to 50% chance of developing a new tumor in their remnant liver⁵. Therefore, there is a need for further research on the mechanisms involved in recurrence of liver metastases.

A liver resection of about 70% is normally compensated within a few weeks by the remaining hepatic tissue. This regeneration involves multiple mechanisms, including cytokines like Interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-α), hepatocyte growth factor (HGF), transforming growth factor beta (TGF-β), vascular endothelial growth factor (VEGF), matrix metalloproteases (MMP-2 and MMP-9) and CXC-Chemokines^6-11. These substances support hepatic regeneration and may also be responsible for the high recurrence rates of primary and secondary liver malignancies by inducing the growth of small tumor cell deposits in the remaining liver which are not detected by routine clinical imaging. This causality has not been proven so far.

The following hypothesis was established. After partial liver resection, the proliferation factors that are responsible for liver hypertrophy may also induce the growth of previously undiscovered tumor cells in the liver. A mouse model was designed which combined the techniques of liver resection and tumor induction. Thirty athymic nude-foxn1nu/nu mice were divided into three groups of ten animals each. Each of them was treated with either a laparotomy alone (Group A), a 30% liver resection (Group B) or a 70% liver resection (Group C). Animals in all groups subsequently received a tumor cell injection into a defined remaining part of the liver, to simulate dormant tumor cells. Animals where observed for two weeks and then evaluated for tumor growth and liver hypertrophy.

The objective was to create a model that could be used to search for the molecular and pathogenetic factors that may play a role in post-hepatectomy tumor formation. This method may be helpful in assessing: the origin of endocrine factors involved in liver regeneration; the responsible mechanisms for intrahepatic tumor growth after liver resection; and the liver resection volume necessary for intrahepatic tumor growth induction. The following method has only been performed on animals because they promise to contribute to the understanding of fundamental biological principles and to the development of knowledge that can be expected to benefit humans by improved treatment options. Due to the mechanisms involved in these matters, it had to be examined in vivo, as in vitro methods may not provide a realistic representation of the human pathology.

These investigations may lead to the discovery of relevant targets for prophylactic treatment options for decreasing tumor recurrence.

Protocol

The government of Middle Franconia in Bavaria, Germany, granted permission for the procedures described. Any similar experiments require prior authorization by the appropriate authorities.

Note: The following manual can be used for previously discussed groups A through C. Steps that have to be left out in groups A and B are marked accordingly.

1. Preparations

1. Put on gloves, place the polystyrene pad underneath the microscope, and focus the lens on the area slightly above the pad.
2. Split the sterile sheet and place half over the polystyrene pad and the other half just next to it.
3. Sterilize the surgical instruments and place them on a sterile sheet next to the Polystyrene pad.
4. Unbend a large paper clip to form an arch. Turn it upside down and press it into the polystyrene pad at the top end - just next to where the animal's head will lie.
5. Place the mouthpiece of the vaporizer between the two limbs of the arch. (Figure 7)
6. Set up a heating lamp about 40 cm from the place where the animal's head will lie. Make sure the heat at the level of the polystyrene pad does not exceed 40 °C.
7. Prepare a separate cage for operated animals.
8. Prepare a defined quantity and volume (maximum of 50 µl) of tumor cells in a flex tube and store it on ice.

2. Anesthesia

1. Place the mouse into a plexiglas box and begin anesthetic induction on high flow isoflurane (5% isoflurane at a flow of 10 L/min).
2. After going through the different stages of anesthesia, remove the mouse from the plexiglas box just after it has entered the stage of agonal breathing, which can easily be recognized by a drastic decrease in respiratory rate (<20/min) and deep gasps shaking the animal's entire body.
3. Quickly place the animal belly-up onto the sterile drape covered polystyrene pad and continue ventilation on low-flow isoflurane by inserting the mouse's snout into the mouthpiece to maintain anesthesia. Use an inspiratory fraction of 1.8-2.2% at a flow of about 1 L/min.
4. Evaluate the depth of anesthesia during the operation by calculating the respiratory frequency, which is ideally between 45 and 60 breaths per min. Adapt the inspiratory isoflurane fraction accordingly if this is not the case.
   Note: Changes in the inspiratory isoflurane fraction take about 60 sec until they become effective. Avoid carrying out drastic changes rapidly. Instead, modify the anesthetic's application gradually.

3. Operation

1. Disinfect the chest and abdomen with an adequate disinfectant and change gloves afterwards.
2. Inject a weight-adapted volume of carprofen (5 mg/kg bodyweight) into the animal's thigh.
3. Gently pinch the abdominal skin with a forceps to test if the depth of anesthesia is adequate.
4. Perform an incision along the median line from the xiphoid to the mid-abdomen using scissors and forceps.
5. Carefully dissect the area around the xiphoid to expose it from the surrounding tissue.
6. Place a stay suture through the xiphoid (from inside to outside) and attach both threads to the retainer above the animals head using the clamp.
7. Pinch the retractor's limbs together and slowly introduce the "U"-shaped retractor tips along the animal's internal abdominal wall.
   Note: These measures expose the liver to facilitate access to the different lobes. The identification of the liver's median and left lateral lobe should now be possible.
8. Use a saline-soaked cotton swab and gently push the median lobe downwards. Dissect the ventral three quarters of the falciform ligament, which will now be visible between the median lobe's surface and the diaphragm.
9. Now, use two saline-soaked cotton swabs to shift the median lobe and left lateral lobe upwards against the diaphragm.
10. Visualize the thin membrane between the left lateral lobe and the caudate lobe and carefully dissect it.
    Note: When performing this protocol on animals from group A, jump to step 3.18 at this point.
11. Place a size 4-0 ligature diagonally along the left lateral lobe's base.
12. Next, use the cotton swab to return the left lateral lobe to its original position.
13. Carefully tie the ligature as close to the lobe's base as possible and assess for color change in the lobe to test for adequately interrupted blood supply.
14. Resect the left lateral lobe by following the line of the lobe's base and note the resected lobe's weight.
    Note: When performing this protocol on animals from group B, jump to step 3.18 at this point.
15. Place a second ligature between the left lateral lobe's stump and the median lobe's base.
16. Reposition the median lobe as well and tie the ligature. Again, assess for color change in the lobe to test for adequately interrupted blood supply.
17. Resect the median lobe and note its weight.
18. Connect the 1 ml syringe to the 30 G needle and fill it with the tumor cells from the flex tube without tilting the syringe at any time.
19. Use the cotton swabs to move the intestinal loops to the animal's left in order to expose the inferior right lobe.
20. Insert the cell-loaded syringe into the "third-hand" device at a 30° angle to the vertical.
21. Carefully move the device next to the mouse with the needle just above the inferior right lobe.
22. Slowly advance the syringe within the third hand device until the needle's tip is in the inferior right lobe's center part.
23. Inject the entire volume into the lobe's center part over a period of 30-45 sec.
24. Compress the injection site for at least three minutes until bleeding has stopped.
25. Remove the stay suture and the retractor.
26. Close the fascia with a resorbable 5-0 suture using single button knots.
27. Close the skin with a 4-0, non-resorbable suture using single button knots.
28. Start ventilating the mouse on high flow oxygen for about 1 min.

4. Post-op procedure

1. Place the animal into a warm environment (approximately 40 °C) for the next half hour to ensure adequate recovery.
2. Admix the animal's drinking water with 5 mg/ml of metamizole for 72 hr postoperative.
3. Examine the integrity of the sutures closely for at least three days after the procedure.

5. Observation Period and Euthanasia

1. Measure the animal's weight daily for a total of 14 days. Score them regarding their well-being and limitations respectively.
2. After 14 days, anesthetize the animal following steps 2.1 and 2.2 of the operative protocol and proceed with institution's protocol for animal euthanasia.
3. Perform a median laparotomy as explained in step 3.4. To facilitate access to the abdomen, extend the incision 1-1.5 cm caudally. Insert the retractor as pointed out in 3.7.
4. Dissect along the rest of the falciform ligament and cut the inferior vena cava just as it exits the liver cranially.
5. Separate the liver from the diaphragm, by grabbing the diaphragm with the forceps and bluntly dissecting into the space between liver and muscle tissue.
6. Lift the mobilized liver tissue off the retroperitoneum and dissect it off the remaining structures it is still attached to: retroperitoneal fatty tissue, the inferior vena cava and the portal vein.
7. Inspect the liver after extraction for any additional tissue, which would falsely contribute to its actual size and weight. An extracted liver and its tumor are displayed in Figure 6.
8. Dissect the tumor off the inferior right lobe.
9. Measure the size and weight of both the tumor and the liver parenchyma.
10. Conserve additional tissue samples from the peritoneum, lymph nodes or other organs as needed for tissue analysis

Results

In our specific experiment, we included 30 athymic nude-foxn1nu/nu mice. They received a median laparotomy, tumor cell injections of 500,000 MC38 tumor cells (dissolved in 50 µl of saline), and were subsequently treated with either a 70% liver resection, a 30% liver resection, or no further intervention.

After 14 days, almost complete regeneration of the remaining liver following 30% or 70% liver resections (liver hypertrophy-in...

Discussion

Previous experiments performing surgery in rodents have been able to identify certain variables that could serve as sources for bias. In order to obtain reliable and valid results, consider the following precautions.

Routine pre-op fasting can lead to liver steatosis¹², which may inhibit liver regeneration^13,14. It is therefore not recommended. The highest mitotic activity of hepatocytes varies throughout the day¹⁵. If possible, conduct the procedures at a cert...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Equipment
Operation Microscope	Zeiss	OPMI-1 FC S21
Induction Cage (Plexiglas Box)	UNO BV, Netherlands	180000132
Flowmeter + Connection Kit	UNO BV, Netherlands	180000008
UNO Vaporizer Sigma Delta	UNO BV, Netherlands	180000002
Key Filler for Anesthetic	UNO BV, Netherlands	180000010
Activated Charcoal Filter Adsorber	UNO BV, Netherlands	180000140
Gas Exhaust Unit	UNO BV, Netherlands	180000118
Face Mask for mouse	UNO BV, Netherlands	180000065
Vaporizer Stand	UNO BV, Netherlands	180000006
Heat lamp	Physitemp Instruments	HL-1
Styrofoam Pad	RAYHER	30074000
Third Hand Tool	TOOLCRAFT	ZD-10F
Precision Scales	Kern	EW 220-3NM
Scales	Kern	EMB 500-1
Sliding Caliper	MIB	MIB 82026100
Microdissection forceps	Braun/Aesculap	BD195R
Microdissection scissors	Braun/Aesculap	FD100R
Microdissection needle holder	Braun/Aesculap	BM563R
Retractor	Fine Science Tools (F.S.T.)	No. 17001-0	Type: Bowmann
Clamp	Braun/Aesculap	BJ002R
Name	Company	Catalog Number	Comments
Expendable Items (NOTE: Quantities are per animal and procedure)
Foliodrape sterile cover (45 cm x 75 cm)	Hartmann	2775001
Sterile Cotton Swabs (2x)	Hartmann	4700151	Peha
Sterile fluid (0.9% NaCl)	Braun	3570310	PZN=04454809
Disinfectant (Softasept - 250 ml)	Braun	3887138	PZN=0762008808505018
2 x 1 ml syringe (Injekt-F )	Braun	9166017V
26 G canula (Sterican) - for Carprofen injection	Braun	4665457
30 G canula (Sterican)  - for Tumor injection	Braun	4656300
Caprofen (=Rimadyl)	Pfizer	QM01AE91
Metamizole (= Novaminsulfon)	Ratiopharm	16543.00.00
4-0 Vicryl suture	Ethicon	J835G
5-0 Prolene suture	Ethicon	8618G
SafeLock Flex-Tube 1.5 ml	Eppendorf	22363778
4 x 4 Gauze Sponge	Kendall/Covidien	UPC: 728795135355	ASIN: B005BFQTWM
Large paperclip	ACCO	A7072510G
Name	Company	Catalog Number	Comments
Animals
Female athymic nude-foxn1nu/nu	Harlan Laboratories B.V.	Code 069