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International Journal of Experimental Pathology logoLink to International Journal of Experimental Pathology
. 2008 Dec;89(6):447–457. doi: 10.1111/j.1365-2613.2008.00605.x

An in vivo rat model for early development of colorectal cancer metastasis to liver

John H P Robertson *, Shi Yu Yang *, Arthur M Iga *, Alexander M Seifalian †, Marc C Winslet *,‡,§
PMCID: PMC2669606  PMID: 19134054

Abstract

At diagnosis of colorectal cancer, approximately 25% of the patients have established colorectal liver metastasis. Optimal management of disseminated disease requires therapies targeting multiple stages in hepatic colorectal cancer metastasis development. To facilitate this, biologically accurate in vivo models are required. Early colonic cancer liver metastases development was studied using BDIX and Sprague–Dawley rat strains with human HT29 and rat DHDK12 colonic cancer cell lines. Different cancer cell–host combinations were used. Rat DHDK12 was previously chemically induced in the BDIX rat. Real-time intra-vital microscopy was employed to analyse the early development of liver metastases in four groups (n = 6 per group) (HT29–BDIX, DHDK12–BDIX, HT29–SD and DHDK12–SD). Data were compared using one-way anova with Bonferroni’s multiple comparison test. The total number of tumour cells visualized, adherent cells within the hepatic sinusoids, extravasated tumour cells and migration rates were significantly higher in the DHDK12–BDIX combination. Maximum number of visualized cells and maximum migration rate were also significantly higher in this group. No significant differences were observed in these experimental parameters among the other three groups or in the haemodynamic parameters among all groups. In conclusion, cancer cell line–host selection has a significant effect on early colonic cancer liver metastasis development.

Keywords: colonic cancer, early liver metastasis development, intra-vital microscopy

Colorectal cancer is the second most prevalent form of cancer diagnosed in the western world. The management of disseminated colorectal cancer remains a therapeutic challenge. In Europe, an estimated 13.2% of 2.9 million cancer cases diagnosed were colorectal cancer and colorectal cancer was responsible for 203,700 deaths in 2004 (Boyle & Ferlay 2005). Globally, almost one million cases of colorectal cancer are registered annually and half a million deaths are attributed to this disease per year (Parkin et al. 2001). At diagnosis, 25% of colorectal cancer patients have established liver metastases (Kavolius et al. 1996). For optimization of oncological management, specific therapeutic targeting of multiple stages of colorectal metastasis development is required.

To develop tailored therapeutic interventions, an in vivo model for early development of liver metastasis must be established. Such an in vivo model should closely mimic the genuine biological cancer metastatic process and allow one to investigate the complex development of metastasis and improve the knowledge of the integral stages involved. This will facilitate the development of novel therapeutic interventions and enable comparison of treated and control groups to analyse the biological effect of the intervention. Numerous models of early colorectal liver metastasis development have been proposed (Table 1). Establishment of an in vivo model requires tumour cell line and host selection, tumour cell introduction and tumour cell labelling. Each of these processes can influence early colorectal liver metastasis development and affect the biological accuracy of the in vivo model (Robertson et al. 2008a).

Table 1.

Summary of evolution of in vivo studies examining early stages of colorectal cancer liver metastasis

Study Strain of animal Cancer cell line Molecular target (intervention) Labelling method Tumour inoculation Outcomes
Reinmuth et al. (2003) BALB/c mice Murine CT26 adeno-carcinoma αvβ3 and αvβ5 (S247 and αvβ3/αvβ5) None Spleen S247 prolonged survival in this animal model. S247 impaired both metastatic and angiogenic processes
Steinbauer et al. (2003) BALB/c mice – SCID and wild type Murine CT26 adeno-carcinoma NONE (none) GFP and calcein AM Portal vein GFP stain in longer experiments can trigger an immune reaction
Sturm et al. (2003) BALB/c mice Murine CT26 adeno-carcinoma NONE (none) GFP Spleen Created an animal model for colorectal cancer using murine cancer cells in a murine host
Haier et al. (2003) Sprague–Dawley rats Human HT29 and rat CC531 colorectal cancer cells NONE (none) Calcein AM Intra-arterially, intravenous and extrahepatic portal vein Created animal model without GFP labell, which could cause immune reaction. Showed despite method of colorectal cancer cell inoculation, cells still metastasized
Enns et al. (2004) Sprague–Dawley rats Human HT29 cells (HT29P and HT29LMM) Integrins –α1, α3, α5, α6, β4 and α2β1. VCAM-1 Calcein AM Intra-cardiac Specific integrins play a key role in colorectal cancer cell adhesion to the liver and in tumour migration. ECM of space of Disse is important in metastasis formation
Enns et al. (2005) Sprague–Dawley rats Human HT29 colorectal cancer cells Intergins pan αv, αvβ3 and αvβ5 Calcein AM Intra-arterial αv integrins esp αvβ5 have a key role in colorectal cancer cell adhesion to the liver
Schluter et al. (2006) Sprague–Dawley or nude rats HT29P low, KM-12C intermediate or HT29LMM, KM-12L4 high metastatic colorectal cancer cells Calcein AM Intra-arterial Cell adhesion occurred in metastatic target organs only. Migration into target organs correlated with their metastatic potential
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Arguably, the most important decision in establishing a biologically accurate model of colorectal liver metastasis is the compatibility of the colorectal cancer cell line selected and the host. Extensive work has focused on the influence of cancer cell line–host selection in macroscopic metastasis models. As the initial work by Martin et al. (1983), further work has shown that DHDK12 colonic cancer produces macroscopic metastases in immunocompetent BDIX rats (Benoit et al. 2000, 2006; Favoulet et al. 2002, 2004; Mezhir et al. 2006). Human colorectal cancer liver metastasis development in immunocompetent mice showed considerable variation with regard to tumour propagation and dissemination. Only one of the 12 colorectal cancer cell lines produced liver metastases, which occurred in two of 10 animals (Flatmark et al. 2004). Given these results, it was our sole objective to analyse differences at the early stages of colorectal cancer metastasis to the liver in this work. Recent models (Haier et al. 2003; Enns et al. 2004, 2005; Schluter et al. 2006) have used HT29 human colorectal cancer cells in a Sprague–Dawley rat. Despite statistical analysis of human HT29 and rat CC531 colorectal cancer cell lines (Haier et al. 2003) revealing no differences over the 30-min observation period, the differences between the species in hepatic architecture and homeostatic regulation are likely to influence metastasis development. In vivo models using syngeneic cell lines and host are likely to provide a more accurate representation of metastatic development. This accurate representation is vital. Inhibition of molecules, especially specific integrins (Enns et al. 2004, 2005; Robertson et al. 2008b), has been shown to inhibit crucial stages in the early stages of colorectal cancer development. In vivo models examining these early stages of metastasis development must reduce the amount of experimental variables that could influence metastasis development.

To the best of our knowledge, the early stages of colorectal cancer liver metastasis have never been studied using a syngeneic tumour cell line in a rat host. We establish an in vivo model for colorectal liver metastasis development by introducing DHDK12 as a cancer cell line in a BDIX rat. DHDK12 is a chemically induced rat colon carcinoma cell line induced from BDIX rats (Martin et al. 1983). Many models studying later stages of metastasis development have used this biologically accurate cancer cell line–host combination (Favoulet et al. 2004; Kobaek-Larsen et al. 2004; Sinibaldi et al. 2004; Benoit et al. 2006; Mezhir et al. 2006). Using Calcein acetoxymethylester (Calcein AM) as an intracellular label and an intra-arterial method of tumour cell introduction, we compared early liver metastasis development in the four experimental groups – DHDK12 in BDIX, DHDK12 in SD, HT29 in SD and HT29 in BDIX.

Materials and methods

Cell culturing

HT29 human colorectal cancer cells and DHD K12 rat colorectal cancer cells were purchased from European Collection of Cell Cultures (Health Protection Agency, Salisbury, UK). Media [McCoy’s 5A, F10 and Dulbecco’s modified Eagle’s media (DMEM)] and foetal bovine serum (FBS) were purchased from GIBCO, Paisley, UK.

HT29 cells were cultured in McCoy’s 5A media containing 10% FBS and 1% penicillin and streptomycin. DHDK12 cells were cultured in DMEM and F10 1:1 containing 2 mM glutamine, 10% FBS and 1% penicillin and streptomycin. Both cell lines were grown and maintained at 37 °C in humidified 5% CO2/95% air.

When cells reached 70% confluence, the medium was discarded and the cells were washed three times with Dulbecco's Phosphate-Buffered Saline (D-PBS). The cells were then incubated with 4 ml of trypsin at 37 °C in humidified 5% CO2/95% air for 5 min. Next, the cells were harvested in 10 ml of their respective media without FBS containing 1% penicillin and streptomycin media. The harvested cell mixture was centrifuged at 300 g for 10 min. Cell pellets were re-suspended in those media and cells were then labelled in 6 μM Calcein AM at 37 °C for 2 h. Labelled cells were then centrifuged at 300 g for 10 min and the label solution was discarded. The cell pellet was finally re-suspended in 1.5 ml PBS for introduction to animals.

Animals

Male SD and BDIX rats (body weight 200–250 g) were used in the experiment. The study was conducted under the Home Office issued project licence and personal licence in accordance with the UK Government Guidance in the Operation of the Animals (Scientific Procedures) Act 1986. The rats were maintained in a temperature-controlled environment with 12-h light–dark cycle and allowed tap water and standard rat chow pellets ad libitum.

Anaesthetic

Animals initially were anaesthetized in an anaesthetic chamber with 4 l/min of isofluorane (4%; Baxter, Norfolk, UK). Anaesthetic maintenance was achieved using 1.5–2% isoflurane and 4 l/min of O2. During anaesthesia, the rats were allowed to breathe spontaneously through a concentric mask connected to an oxygen regulator. Physiological parameters (pulse, oxygen saturation, blood pressure and temperature) were continuously monitored during the experiment. The arterial oxygen saturation (SaO2) and heart rate were monitored with a pulse oxymeter (Datex-Ohmeda, Louisville, Colorado, USA). The animals’ body temperature was maintained at 36–37 °C using a heating pad (Harvard Apparatus Ltd, Kent, UK) and monitored with a rectal temperature probe. Systolic blood pressure was monitored by inserting a polyethylene catheter (0.76 mm inner diameter; Portex, Kent, UK) into the right carotid artery. This was connected to a pressure transducer for monitoring mean arterial blood pressure. The left jugular vein was cannulated with a smaller polyethylene catheter (0.4 mm inner diameter; Portex) for the administration of normal saline (1 ml/100 g body weight, hour) to compensate for intra-operative fluid evaporation.

Experiment

A laparotomy was performed through a midline incision. The ligamentous attachments of the liver were cut and the liver was exposed. The right lobe of the liver was placed on a slide under the intra-vital microscope, while the rest of the liver was delicately exteriorized to minimize anatomical architectural disturbance. During the experiments, the liver was continuously irrigated with isotonic saline solution and the open abdomen wrapped in cling film to reduce heat loss and fluid evaporation.

For intra-vital observation of adhesive interactions between circulating tumour cells and the hepatic microcirculation, single-cell suspensions (2 × 106 cells) were injected intra-arterially into the right carotid artery. The volume of injections was 1.5 ml. Prior to the experiments, sham operations were conducted on three animals from each strain. 1.5 ml of PBS solution was not found to cause any cardiovascular instability.

In vivo observation of metastatic tumour cell adhesion and extravasation

A 20 microscopic field grid was defined on the exteriorized liver surface using the intra-vital microscope. Filming of the field, using the 40× magnification, was performed prior to injection to ensure good images. During filming, images were viewed on a monitor and recorded simultaneously onto DVD. After 1.5 ml of labelled cells was injected intra-arterially over 90 s, 20 microscopic field grids were filmed at subsequent 15-min intervals. At each time interval, pulse, blood pressure, temperature and oxygen saturation were recorded. Filming and recording were conducted for 2 h.

The recordings were analysed postexperiment.The total number of visualized tumour cells, the number of adhering tumour cells and the number of extravasated tumour cells were counted and recorded. Adherent cells were defined as visualized colorectal cancer cells that remained static within the hepatic microcirculation. Migrated cells were defined as colorectal cancer cells that had moved from the hepatic microcirculation into the hepatic parenchyma. At each time point, the migration rates (MRs) were calculated using the following formula:

graphic file with name iep0089-0447-mu1.jpg

where NEC is the number of extravasated cells and TVC is the total number of visualized cells.

Statistical analysis

As all data were collected serially, analysis of serial measurements was employed. This method is statistically reliable and arguably more valid for serial measurements (Matthews et al. 1990). One-way anova (prism version 4, 2004 edition; GraphPad, La Jolla, California, USA) with Bonferrroni’s multiple comparison test was used for statistical analysis and P < 0.01 was considered significant.

Results

Total number of visualized cancer cells

The total number of visualized tumour cells at each time point is shown in Figure 1a. The area under the graph (Figure 1b) for each group was analysed using the previously reported methods (Matthews et al. 1990). The total number of visualized cancer cells in the DHDK12–BDIX group was significantly higher (P < 0.001) compared with the other three groups. There was no significant difference between the other three groups. Analysis of the maximal visualized cells during the experiment was also compared among the four groups (Figure 2). For DHDK12–BDIX group, the number of maximal visualized cells was significantly higher than in the other groups (HT29–BDIX P < 0.01, HT29–SD and DHDK12–SD P < 0.001). There was no significant difference in either total visualized cancer cells or maximum visualized cells among the other three groups.

Figure 1.

Figure 1

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The total number of tumour cells visualized in 4 groups is shown in (a). Analysing the area under the graph for each group showed that the number of tumour cells visualized in the DHDK 12-BDIX group is significantly higher (P < 0.001) (b).

Figure 2.

Figure 2

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Analysing the maximum tumour cells visualized between 4 groups showed that the maximum tumour cells visualized in the DHDK 12-BDIX group is significantly higher compared to HT29-BDIX (P < 0.01), DHDK12-SD (P < 0.001) and HT29-SD (P < 0.01) groups.

Cells adherent to liver sinusoids

The numbers of visualized adherent cells (Figure 3) are graphically depicted (Figure 4a). Analysis of the area under the graph showed that there were significantly higher numbers of visualized adherent cells in DHDK12–BDIX compared with the other three groups (P < 0.001). There was no significant difference among the other three groups (Figure 4b). During the experiment, the number of adherent cells in the DHDK12–BDIX group diminishes markedly in comparison with the other groups. This decrease correlates with the colonic cancer cells moving into the liver parenchyma.

Figure 3.

Figure 3

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This is an example image of liver tissue captured under intravital video microscopy. In the image green fluorescence is liver parenchyma and black parts are sinusoid vessels. There are three colorectal cancer cells (red) in this image. One is adherent cell (marked with white arrow) and the other two have migrated out of the sinusoid vessel and moved to the liver parenchyma (marked with black arrow).

Figure 4.

Figure 4

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The number of adherent tumour cells visualized in 4 groups throughout the two hour experimental period is shown in (a). Analysing the number of adherent tumour cells visualized between 4 groups (b) showed that adherent tumour cells significantly increased in the DHDK 12-BDIX group comparing with the other groups.

Cells extravasated into the liver parenchyma

The extravasation of cancer cells in the four experimental groups was analysed (Figure 5a). The number of extravasated cells increased in all four groups over the 2-h observation period. The increase was more obvious within the DHDK12–BDIX group. Analysis of the area under the graph (Figure 5b) showed a significantly increased tumour cell migration in the DHDK12–BDIX group compared with the other three groups (P < 0.001). There was no significant difference among the other three groups.

Figure 5.

Figure 5

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The number of extravasated tumour cells detected for each of the 4 groups during the experiment is shown in (a). Comparing the number of extravasated tumour cells visualized between the 4 groups (b) showed that the DHDK-12-BDIX group has a significantly higher number of extravasated tumour cells (P < 0.001).

Cell migration rate and maximum migration rate

Cell MRs were shown in Figure 6a. Cell MRs increased during the experimental period in all four groups. After 120 min, the mean MR for the DHDK12–BDIX was 75.5%, DHDK12–SD 62.6%, HT29–BDIX 57% and HT29–SD 55%. Statistical analysis of cell migration showed that the DHDK12–BDIX group has a significantly higher cell migration compared with HT29–SD group (P < 0.001) and the other two groups (P < 0.05) (Figure 6b). To compare the maximum migration, the highest MR was recorded in each experiment (Figure 7). The DHDK12–BDIX group showed a statistically significant increase in maximum migration rate when compared with the other three groups – HT29–SD and HT29–BDIX (P < 0.001) and DHDK12–SD (P < 0.05). For cell migration and the maximum cell migration, there was no significant difference detected among the other three groups.

Figure 6.

Figure 6

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The migration rates for each of the four groups during the experiment are shown in (a). The analysis of migration rates shows that the DHDK-12-BDIX group is statistically higher compared to DHDK12-SD (P < 0.05), HT29-BDIX (P < 0.05) and HT29-SD (P < 0.01) groups.

Figure 7.

Figure 7

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The maximum migration rate in DHDK12-BDIX group is significantly higher compared to DHDK12-SD (P < 0.05), HT29-BDIX (P < 0.01) and HT29-SD (P < 0.01) groups.

Systemic physiological parameters

To study the effect of the experimental procedures, particularly the 1.5-ml intra-arterial injection on the systemic parameters, two sham groups (SD and BDIX) (n = 3) were included. The sham operation was the same as the standard procedure, but an intra-arterial injection of PBS was introduced instead of the cancer cell suspension. Animal systemic parameters such as pulse, temperature, oxygen saturation and blood pressure were measured and recorded. Comparison of the measured parameters between the four experimental and two sham groups revealed no significant differences. The results showed that all animals – four experimental and two sham groups (30 rats in total) – were physiologically stable throughout the whole experimental period.

Discussion

Metastasis development is a highly selective process both locally and systemically. The creation of a biologically accurate model of metastasis is difficult, as there are so many introduced variables able to influence metastasis development (Robertson et al. 2008a). Establishment of a biologically accurate model mirroring the early stages of colonic cancer liver metastasis initiation is highly important. This model mimics haematogenous dissemination of colorectal liver cancer in a rodent host. The importance of this rodent in vivo model for the study colorectal cancer cell arrest, adhesion and extravasation is further highlighted by the fact that models in higher primates are yet to be established. There are limitations to this model. Metastasis development requires tumour cells to complete many stages prior to tumour cell arrest. Indeed, many tumour cells enter the circulation and reach the microcirculation of many organs and metastasis fails to develop. However, tumour cell arrest and extravasation are vital steps in the overall process. This model enables highly focussed study of these stages. Indeed, inhibitors of these key stages are being investigated. Colorectal cancer cells within the hepatic microcirculation are vulnerable to therapeutic intervention. Extravasation provides these cells with a degree of protection within the hepatic environment. Specific therapeutic interventions targeted at these stages would hopefully be associated with fewer systemic side effects for the patient. A biologically accurate in vivo model for early colorectal cancer liver metastasis development will allow increased understanding and insight into the crucial stages in metastasis development. Comparing different animal species and cancer cell lines, it was found that the combination of DHDK12 colonic cancer cell line with BDIX rat produced significantly higher numbers of colorectal cancer cell arrest, adherence and extravasation. This suggests that other models using non-syngeneic combinations may underestimate the efficacy of the metastasis process.

Many experimental factors are known to influence the early development of colorectal liver metastasis development (Robertson et al. 2008a,b). The labelling process of colorectal cancer cells can produce high levels of background fluorescence, which can limit accurate analysis of metastasis development (Weissleder et al. 1999; Hoffman 2002). The administration of exogenous reagents (Sweeney et al. 1999), cellular labelling (Steinbauer et al. 2003), surgical techniques, anaesthesia and the intra-vital microscopy procedure can all distort the image of metastasis development (Enns et al. 2004) and affect the accuracy of the in vivo models. Genetically engineered animals that spontaneously develop cancer and subsequent metastases are the most biologically accurate models of metastasis. Smad3 mutant mice were shown to develop metastatic colorectal cancer (Zhu et al. 1998). Subsequent groups (Philipp-Staheli et al. 2002; Domino et al. 2007) have used these mice to examine different factors influencing colorectal cancer development and progression. However, these models are not amenable to the intra-vital microscope – an essential tool for real-time observation of tumour development. Tumour cells must be labelled to allow detection in vivo. The selection of a closely biologically related cancer cell line and host, which will enable tumour cell labelling prior to introduction, is therefore important.

The DHDK12 colonic cancer cell line was chemically induced in the BDIX strain of rat (Martin et al. 1983). Therefore, the in vivo model of metastasis, DHDK12 cell line in the BDIX rat, should be biologically compatible. Comparison of summary results of this group with the other three experimental groups showed a significant increase in every measured experimental parameter of metastasis development – total cancer cells visualized within the hepatic microcirculation, cancer cells adhering to the sinusoids, cancer cells extravasating, MR, maximum MR and maximum total cancer cells visualized. No significant differences were noted among the other three groups. No haemodynamic variables were noted between the two rat strains in the sham experiments. No significant haemodynamic variables were evident between the four experimental groups. These results demonstrate that, in our experiment, the cancer cell line–host selection and compatibility had significant effects on early metastasis development. Previous works (Chambers et al. 2001, 2002; Fidler 2002, 2003; Onn & Fidler 2002) have highlighted the importance of tumour cell interaction with both the organ of metastasis development and local host homeostatic mechanisms. Our work suggests that not only does non-related cancer cell line–host combination affect metastasis development, but also the introduction of colonic cancer from a different strain within the same species alters metastasis development. These results question the biological accuracy of current in vivo models focussing on the early stages of colorectal liver metastasis development, which have predominantly used human HT29 colorectal cancer cells in a rat host (Haier et al. 2003; Enns et al. 2004, 2005; Schluter et al. 2006).

This work has purely focused on the very early stages of colorectal metastasis to the liver. Other studies have shown the influence of cancer cell line–host selection in macroscopic metastasis models. As the initial work by Martin et al. (1983), further work by Benoit et al. (2000, 2006), Favoulet et al. (2002, 2004) and Mezhir et al. (2006) have shown that DHDK12 colonic cancer introduced into immunocompetent BDIX rats produces macroscopic metastases. Mezhir et al. (2006) introduced DHDK12 cells into BDIX rats using an intrasplenic injection. After injection, a laparotomy was used to determine metastatic burden and histological analysis was used to confirm the presence of metastases from resected tumours. Of the 53 BDIX inoculated with DHDK12, local growth was seen in all animals. Thirty-six (68%) developed detectable metastases, while 32 (60%) developed oligometastases. They concluded that this cancer cell–host combination consistently produced oligometastasis and provided a model of macroscopic disseminated disease that enabled novel treatments to be trialled and mechanisms of metastatic colorectal cancer development to be analysed. Human colorectal cancer liver metastasis development was examined in mice using orthotopic models (Flatmark et al. 2004). Twelve colorectal cancer cell lines were implanted and considerable variation with regard to tumour propagation and dissemination was shown. Only one, of the 12 colorectal cancer cell lines, produced liver metastases. This occurred in only two of 10 animals. While the method of colorectal cancer cell introduction was different – orthotopic vs. haematogenous – cancer cell line–host selection has a significant effect on tumour development. The HT29 cell line was used in this study. 1 × 106 HT29 cells were implanted into 13 female BALB/c mice. In only six of 13 mice, local tumour growth was seen – 46%. Of these six, five developed lymph node metastases, but none developed liver metastases. The median time from implantation until killing was 12 weeks. Further work (Guilbaud et al. 2001) on HT29 human colorectal cancer cells produced similar results in immunocompetent Swiss nude mice postcaecal implantation. Given these results, it was our sole objective to analyse differences at the early stages of colorectal cancer metastasis to the liver in this work, although future work will focus on differences in macroscopic metastasis development between the four experimental groups. The reason for different growths in different hosts is very important. The results obtained in this work suggest that the interaction between the tumour cells and the host microenvironment can influence at least the early stages of liver metastasis development. Host immune status is another important factor. In immunodeficient SCID mice, HCT116 colorectal cancer was highly invasive and metastasized to the lungs and liver (Guilbaud et al. 2001). In immunocompetent female BALB/c mice, all 10 mice implanted with HCT116 tumour cells showed local growth (Flatmark et al. 2004). Of these 10, seven developed lymph node metastases, but none developed liver metastases. The median time from implantation until killing was 8.7 weeks.

Arguments continue whether haematogenous or orthotopic introduction of colorectal cancer cells provides a more accurate model. Orthotopic implantation (Guilbaud et al. 2001; Reinmuth et al. 2003; Sturm et al. 2003; Flatmark et al. 2004) is likely to portray a more accurate picture of the metastasis process. A heterogeneous population of colorectal cancer cells is allowed to establish itself. Selection will occur throughout the dissemination process and tumour microenvironment interactions occur. The disadvantage is that this process of implantation makes analysis of events in early metastasis development hard to analyse with any degree of accuracy. Accurate analysis of the above steps requires large numbers of tumour cells within the circulation at any specific time point. The bolus of cells, introduced in the haematogenous in vivo models, makes analysis of the early steps of metastasis development much easier. With the orthotopic model, the number of tumour cells within the circulation at any given time point will be significantly less than the haematogenous bolus. In addition, in the orthotopic model, the introduction of tumour cells into circulation will be more erratic. Observations and experiments will be prolonged. This not only will increase observation error and variation, thereby reducing statistical validity, but also present problems with preservation of normal physiological parameters in the host.

In our in vivo model, the principal stages of metastasis development were similar to those described by Haier et al. (2003). Tumour cell arrest and adhesion in the hepatic microcirculation were evident in patent hepatic vessels. This further supports the tumour cell-specific adhesion theory (Ding et al. 2001; Haier et al. 2003; Enns et al. 2004, 2005). This theory proposes that certain tumour cells have specific cell adhesion molecules that enable them target an organ to selectively.

Establishment of sustained adhesion was not always successful. Occasionally, tumour cell stabilization was not achieved and the initial bond broke with the tumour cells re-entering the circulation. Formation of successful adhesion was swiftly followed by extravasation. Previous work by Al-Mehdi et al. (2000) had suggested that metastatic cells initially proliferated intravascularly (Ito et al. 2001; Sturm et al. 2003). In our model, cancer cells were found to migrate rapidly into the liver parenchyma. By the end of the 2-h observation period, over 55% of cancer cells in all four groups had extravasated.

In conclusion, DHDK12 colonic cancer cell line haematogenously introduced into a BDIX rat provides a biologically accurate in vivo model of early colonic liver metastasis development. It is hoped that this model will facilitate creation of specific inhibitors of the early stages of colorectal liver metastasis development.

Acknowledgments

We would like to thank Dr Wenxuan Yang for his invaluable help and support during the writing of the article. Tragically, he died before submission.

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