We describe in detail a clinically relevant colorectal cancer liver metastases (CRLM) tumor model and the influence of liver ischemia reperfusion (I/R) in tumor growth and metastasis. This model can help to better understand the mechanisms underlying surgery-induced promotion of liver metastatic growth.
Liver ischemia and reperfusion (I/R) injury, a common clinical challenge, remains an inevitable pathophysiological process that has been shown to induce multiple tissue and organ damage. Despite recent advances and therapeutic approaches, the overall morbidity has remained unsatisfactory especially in patients with underlying parenchymal abnormalities. In the context of aggressive cancer growth and metastasis, surgical I/R is suspected to be the promoter regulating tumor recurrence. This article aims to describe a clinically relevant murine model of liver I/R and colorectal liver metastasis. In doing so, we aim to assist other investigators in establishing and perfecting this model for their routine research practice to better understand the effects of liver I/R on promoting liver metastases.
The liver is one of the most common sites for the development of metastatic disease1. Mortality is almost invariably attributable to complications associated with tumor growth in the liver. In patients with metastatic solid tumors in the liver, surgery remains a crucial intervention for disease control and a possible curative approach. However, the vast majority of patients ultimately present with recurrent disease, predominantly in the liver2,3. During hepatic surgery, intraoperative bleeding is common, often necessitating blood transfusion and different technical approaches for control of bleeding, including vascular clamping methods. However, such measures cause hepatic ischemia/reperfusion (I/R) to the liver tissue. The adverse effects of I/R on hepatocellular function have been well documented. The liver I/R insult ignites inflammatory cascades during the restoration of blood flow via inflammatory pathways4. Not only does liver I/R injury contribute to liver failure, but current evidence also shows that I/R injury stimulates tumor cell adhesion, and promotes the incidence of metastases formation and the growth of existing micrometastatic disease5. We have previously reported that surgical stress induces activation of immune cells which not only helps in the growth of the primary tumor, but also facilitates metastases by capturing cancer cells within the circulation6.
Here we describe in detail a technique to establish a liver metastasis mouse tumor model. In this model, we also present a method to induce hepatic ischemia reperfusion injury which acts as a surrogate to the surgical stress present clinically during hepatectomies. The combined methods of cancer injection and hepatic I/R can successfully interpret the development of CRLM in patients who have undergone primary tumor resection.
All animal protocols are approved by the Institutional Animal Care and Use Committee and adhered to the National Institutes of Health (NIH) Guidelines. Instruments used for any surgical procedure were thoroughly sterilized.
1. Initial preparation
2. Cell culture
3. Injecting tumor cells
4. Splenectomy
5. Ischemia reperfusion Injury
6. Assessment of operated mice
7. Assessment of liver ischemia reperfusion injury
All wildtype (C57BL6) mice (n = 20) were subjected to the liver metastases model using the protocol described above. All injected mice with or without ischemia reperfusion injury survived until the date of sacrifice. The schematic diagram Figure 1A of a cancer-injected liver illustrates the clamping of the portal triad (hepatic artery, portal vein, and bile duct) which induces a partial liver ischemic (70%) insult towards the median and left lateral lobes. An increase in the number of liver metastases can be observed within 2-3 weeks post ischemia reperfusion injury. Mice injected with MC38 cancer cells were randomly divided into sham and I/R groups. As shown in Figure 1B, the first group of mice underwent splenectomy 15 min after the cancer injection. Liver ischemia reperfusion surgery was performed 5 days after the injection. This model allows the circulating cancer cells (CCs) to establish within the organs. Figure 2A shows that surgical stress significantly increased the amount of pre-established micrometastases within the liver. The second group (Figure 1C) underwent surgical I/R 15 min after the cancer injection. The reperfusion was induced by removing the microvascular clamp 60 min after the application. The concurrent influence of the surgical stress leads (Figure 2B) to the capture of recently injected cancer cells within the liver establishing a micrometastatic foci. This significantly increased the number of metastatic nodules in the liver.
Figure 1: A schematic representation of the experimental design. (A) The schematic diagram of a cancer injected liver illustrates the clamping of the portal triad (hepatic artery, portal vein, and bile duct) which induces a partial (70%) liver ischemic insult towards the median and left lateral lobes. An increase in the number of liver metastases can be observed in the ischemic lobes within 2-3 weeks after the reperfusion. Initially, mice were subjected to intrasplenic injection of MC38 colorectal cancer in both tumor capture (B) and tumor growth (C) model. Sham mice were also subjected to laparotomy without the application of microvascular clamps. Two-three weeks after the I/R, mice were sacrificed, and liver tissue was harvested. Please click here to view a larger version of this figure.
Figure 2: Representative images of mice injected with murine cancer. (A) Representative liver metastasis image of a tumor growth model (Model-1) showing a significant increase in the gross tumor nodules at the surface of the liver after inducing hepatic I/R compared to non-I/R group. (B) Similarly, in the setting of tumor capture model (Model-2), liver I/R showed a significant increase in tumor nodules 2 weeks after I/R compared to non-I/R group. *, P < 0.05. Results are expressed as the mean ± standard deviation. Group comparisons were performed using Student’s t test (n = 5/group). Please click here to view a larger version of this figure.
The animal model described in this manuscript is based upon two major approaches. The first is to recognize the ability of cancer cells to localize and proliferate in the liver lobes. The second is to study the effect of hepatic ischemia reperfusion injury influencing the tumor growth and metastases. This model permits the relevant study of liver metastases in the absence of secondary metastases in an immunocompetent mouse. The model is useful in addressing the questions of metastatic efficiency, such as cell survival extravasations and proliferation.
In the first model, cancer cells are injected first and micrometastatic disease is allowed to form. Subsequently, liver I/R is performed 5 days later. This model is important when studying the effect of surgery on already established micrometastatic disease. Although imaging has significantly improved in the past decade, there is still the possibility of the presence of micrometastatic disease that may not be detected by imaging and is left behind after a planned liver resection with the intent of cure. This residual microscopic disease is affected by the inflammatory changes accompanying surgery, specifically liver I/R, and the growth is exponentially increased. On the other hand, in the second model liver I/R and tumor injection are performed at the same time. This model focuses on the effects of liver I/R on the circulating cancer cells and the establishment of new metastatic foci. During liver surgery, the manipulation of the tumor releases tumor cells into circulation. Although most of the circulating cells are taken care of by the host’s immune surveillance, a number of cells can establish metastatic foci. This second model is designed to study this phenomenon.
Animal models, such as orthotopic liver injection7 and tail vein injection8, may not be anatomically feasible for such studies. It has been shown that the tail vein injection usually results in an increased metastasis of the lung compared to the liver. The orthotopically injected cancer model has an increased risk of liver injury influencing the microenvironment for the tumor to grow. As an alternative to splenic injection of tumors, the portal vein can also be utilized. The portal vein injection has been a well-established metastatic model in studying liver metastases9,10,11. The injection of cancer cells through the portal vein does not compromise the removal of the spleen compared to the model described above. This indeed will avoid the immune consequences. However, portal vein injection has an increased risk of excessive bleeding due venous tearing (at the site of injection) and thrombosis during or after the application of the microvascular clamp at the portal triad. These risks are exponentially increased when both tumor injection and clamping are done on the same day. Our group has performed both methods and we have obtained similar results9, 12. We do acknowledge that the portal vein injection demands higher technical skills when done at the same time as clamping and is associated with higher complications. Both methods are valid to study liver metastases.
There are numerous important aspects that need to be considered before and during the entire procedure. The use of cancer cells specifically with the same species background is recommenced prior to injection. The cell number is also important to consider in this study, as small number of cells may not be sufficient to complete the study in a short period of time (3 weeks). Increasing the number of cancer cells should be avoided since it may cause an embolism effect leading to thrombosis and death of the rodent. The model described in this manuscript with cell concentration of 1 x 106 is specific to the MC38 cell line and has allowed us to observe a significant difference in the tumor growth stimulated by the effect of surgical ischemia reperfusion injury. We highly recommend trying different concentrations of cancer cells depending on the specific experiment with the desired cancer cell line of interest. Similarly, labeling of cancer cells could be very useful in many metastasis studies. This would provide an idea regarding the percentage of cells that are able to seed and proliferate. Furthermore, proper application of the portal clamp to induce hepatic ischemia injury is very important in this model. Inability to completely block the blood flow may lead to less or no impact on the cancer cells. As described in the methods, it is important to make sure that the blanching of the liver lobes occurs after applying the microvascular clamps. Finally, coagulating vessels properly via cautery is crucial to avoid internal bleeding. In our experience, especially with the use of electrocautery, bleeding from the splenic bed is extremely uncommon and since we perform a repeat laparotomy only 5 days after the first operation, the amount of adhesions is minimal. However, if bleeding is encountered, this may pose a more difficult second operation. If bleeding does occur after the splenectomy, this may indicate that circulating cancer cells may also have implanted in the peritoneal cavity and thus may affect the results of the experiments. It is advised to use caution when handling this issue, because it may affect the experiments and results. Careful thought must be given to determine whether or not to proceed with I/R in these mice.
The authors have nothing to disclose.
The authors thank Sara Minemyer and Alexander Comerci for the linguistic revision.
Dulbecco's Modified Eagle Medium | Lonza | 12-614F | |
Fetal Bovine Serum | Lonza | 900-108 | |
L-Glutamin | Gibco | 25030-081 | |
Penicilin | Fisher scientific | 15-140-122 | |
Stretomysin | Fisher scientific | 15-140-122 | |
HEPES | Fisher Scientific | SH3023701 | |
Trypsin | Hyclone | sh30042.02 | |
Cell culture Flask 75cm | 5 Cells Star | 658170 | |
15ml PP Conical Tubes | BioExcell | 41021037 | |
Trypan Blue Stain | Giibco | 15250-061 | |
Gauze | Fisherbrand | 1376152 | |
Cautry | Bovie | AA01 | |
Microvascular clamp | Finescience tools | 18055-03 | |
Micro-Serrefine clamp applicator with lock | Fine science toosl | FST-18056-14 | |
Spring scissor | Fine science toosl | FST-15021-15 | |
Vessel Dilator | Fine science toosl | FST-00276-13 | |
Magnetic fixator Retraction system | Fine science toosl | FST-18200020 | |
Micro-Adson Forceps | Fine science toosl | FST-11019-12 | |
Micro-Adson Forceps | Fine science toosl | FST-11018-12 | |
4-0 polypropylene suture | Ethicon | K881H | |
Needle holder | Harvard Apparatus | 72-8826 | |
Heating Pad | Fisher scientific | 1443915 | |
Clipper | Oster | 559A | |
Povidone-Iodine solution | Medline | MDS093945 | |
Syringe 1ml 25G | BD safety Glide | 305903 | |
Insulin syringe 0.5 ml | BD insulin Syringes | 32946 | |
Cotton -Tipped Applicator | Fisher Scientific | 23-400-101 | |
Surgical Microscope | Leica | LR92240 | |
Mycoplasma Elisa Kit | Roche | 11663925910 | |
Ketamine | Putney | #056344 | |
Xylazine | NADA | #139-236 | |
ALT strip | Heska | 15809554 | |
AST strip | Heska | 15809542 | |
LDH strip | Heska | 15809607 |