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10.1245/ASO.2006.03.076
Annals of Surgical Oncology 13:168-175 (2006)
© 2006 Society of Surgical Oncology
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Original Article

Synergistic Effect of Interstitial Laser Coagulation and Doxorubicin in a Murine Tumor Recurrence Model of Solitary Colorectal Liver Metastasis

Liesbeth M. Veenendaal, MD1, Richard van Hillegersberg, MD, PhD1, Niels Smakman, MD1, Jarmila D. W. van der Bilt, MD1, Paul J. van Diest, MD, PhD2, Onno Kranenburg, PhD1 and Inne H. M. Borel Rinkes, MD, PhD1

1 Department of Surgery, University Medical Center Utrecht, P. O. Box 85500, 3508 GA Utrecht, The Netherlands
2 Department of Pathology, University Medical Center Utrecht, P.O. Box 85500, 3508 GA Utrecht, The Netherlands

Correspondence: Address correspondence and reprint requests to: Inne H. M. Borel Rinkes, MD, PhD; E-mail: i.h.m.borelrinkes{at}chir.azu.nl.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: Interstitial laser coagulation (ILC) is gaining acceptance for treatment of unresectable colorectal liver metastases. However, local recurrence rates are still high. To overcome this problem, we investigated the potential of additional systemic therapy after ILC in a murine model.

Methods: Single C26 colon carcinoma nodules (~1 mm3) expressing firefly luciferase were implanted in the left liver lobe of 32 BALB/c mice. Seven days after implantation, tumors were treated with either ILC alone (neodymium–yttrium aluminum garnet; 6 W/cm; 800 J/cm) or ILC followed by 1 mg/kg of doxorubicin intravenously. Controls received either doxorubicin alone or sham treatment. Tumor load was measured by in vivo bioluminescent imaging.

Results: Solitary colorectal liver metastases developed over 7 days after tumor implantation in the liver. Extrahepatic disease was not observed. The ILC dose was set to ablate the liver metastases with recurrent tumor growth in 9 of 16 mice after 7 days. After ILC plus doxorubicin, complete tumor destruction occurred without recurrence (0 of 14). Sham treatment or treatment with doxorubicin alone showed an exponential increase in tumor load.

Conclusions: A murine tumor recurrence model after local ablative treatment of solitary liver metastasis was developed. The combination of ILC and doxorubicin had a strong synergistic effect that led to complete tumor remission in all animals treated.

Key Words: Liver metastases • Colorectal carcinoma • Interstitial laser coagulation • Doxorubicin • Heat fixation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The liver is the most common site of metastatic disease of colorectal carcinoma.1 The treatment options for patients with colorectal metastases are limited. If possible, surgical resection has been the standard of care, leading to 5-year survival rates of up to 40%.2–4 However, after radical resection, approximately 30% to 50% of patients will develop intrahepatic recurrence.5 In addition, resection is applicable to only a small number of patients because of high tumor stage, unfavorable intrahepatic tumor distribution, comorbidity, or limited residual liver function. For unresectable metastases, radiofrequency ablation or interstitial laser coagulation (ILC) may be an alternative approach.6 These therapies use interstitial application of energy-transmitting sources that result in local destruction of the tumor by heat coagulation. However, a key limitation of these techniques is incomplete ablation, particularly of larger lesions. Foci of viable tumor at the tumor border can persist even after apparently adequate thermal ablation.7 Studies on ILC report local recurrence rates of up to 63%.8–10 Systemic treatment in conjunction with ILC could destroy these remaining tumor foci, thereby leading to a reduction of intrahepatic tumor recurrence. The aim of this study was to investigate this combined strategy in a murine model of solitary liver metastasis. For this purpose, a murine tumor recurrence model after ILC was established and evaluated.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals
Male BALB/c mice (Charles River, Maastricht, The Netherlands), 12 weeks old, were housed under aseptic conditions in filter paper–topped cages and were given standard diet and water ad libitum. Experiments were performed according to the guidelines of the Animal Welfare Committee of the University Medical Center Utrecht, The Netherlands. Mice were monitored daily by using the scoring system provided by the animal facility as described previously.11

Cells and Cell Culture Conditions
The murine colon carcinoma cell line C26 was transduced with a lentiviral construct containing the firefly luciferase gene under control of the cytomegalovirus promoter as described previously.12 C26-luciferase cells were routinely cultured in Dulbecco’s modified Eagle medium (ICN Pharmaceuticals, Costa Mesa, CA) supplemented with 5% heat-inactivated fetal calf serum, 2 mM of glutamine, 100 U/mL of penicillin, and .1 mg/mL of streptomycin in a 5% carbon dioxide environment. The cells were washed once with phosphate-buffered saline (PBS) and then harvested after brief trypsinization (.05% trypsin in .02% ethylenediaminetetraacetic acid). The single-cell suspension was then washed and suspended at a density of 1.0 x 106 cells/100 µL in PBS and then kept on ice before injection of cells. Cells were injected into the subcutaneous space of the left and right flank of one mouse in a total volume of 100 µL within 60 minutes of harvesting.

Intrahepatic Tumor Implantation
One mouse with flank tumors was sacrificed, after which the tumors were dissected. Necrotic tissue and noncancerous tissue of the specimen were removed, and the remaining tumor tissue was divided into small pieces of approximately 1 mm in diameter with the help of a template and kept in PBS solution. Anesthesia was induced with 4% isoflurane and maintained by ventilation with a mixture of 1.5% to 2% isoflurane and oxygen. Buprenorphine (3 µg per mouse) was given intramuscularly before surgery to provide sufficient analgesia. A midline incision was made, and the left lobe of the liver was exteriorized. A small incision was made through the liver capsule, and a piece of tumor was implanted into the liver. The liver was repositioned, and the peritoneum and skin were closed in two layers with 5.0 Vicryl sutures (Ethicon, Johnson & Johnson, International, St. Stevens-Woluwe, Belgium).

Measurements of Intrahepatic Tumor Growth
After laparotomy on day 7 after subcapsular implantation, tumor size was measured by using a caliper. In addition, tumor growth was assessed on days 6, 9, 14, 19, and 22 after subcapsular implantation by in vivo bioluminescent imaging with a highly sensitive, cooled charge-coupled device camera (VersArray 1300B; Roper Scientific Inc., Vianen, The Netherlands) mounted in a light-tight imaging chamber (Roper Scientific Inc.). Imaging and quantification of signals were controlled by the acquisition software MetaVue (Universal Imaging Corporation, Downingtown, PA). Before imaging, mice were anesthetized with an intramuscular injection of KXA (60 mg of ketamine, 2 mg of xylazine, and .4 mg of atropine per milliliter; Aescoket Plus; Aesculaap BV, Boxtel, The Netherlands). The substrate D-luciferin sodium salt (Synchem Laborgemeinschaft OHG, Kassel, Germany) dissolved in PBS was injected intraperitoneally at 125 mg/kg.13 Mice were then placed onto the stage inside the light-tight camera box. Approximately 5 minutes after the intraperitoneal injection of D-luciferin, the bioluminescent signal had reached maximum intensity and remained fairly constant for >15 minutes.13 Therefore, all mice were imaged with an integration time of 5 minutes, exactly 10 minutes after the intraperitoneal injection of D-luciferin. Eight mice were imaged simultaneously. Total photon counts were quantified with MetaMorph software measuring the same delineated abdominal region in each mouse; this region was large enough to fit the largest tumor-bearing liver.

Interstitial Laser Coagulation
A neodymium–yttrium aluminum garnet laser (Medilas 4060 N, MBB; Medizin Technik, Munich, Germany) was used in all experiments at a wavelength of 1064 nm. The laser light was delivered in the continuous-wave mode through a 400-µm fiber, which had a diffuser tip applicator (outer diameter, 2.1 mm; active length, 5.0 mm; Trumpf Medizine Systeme, Umkirch, Germany).

Experimental Protocol
A laser dose-effect relationship was established in ex vivo porcine livers. Porcine livers were heated to 37°C with warm water sacs and divided into two separate parts. The diffuser tip of the laser was positioned between the two repositioned parts of the porcine liver. ILC was applied at a power setting of 6 or 8 W/cm of diffuser length for different time periods ranging from 75 to 225 seconds, corresponding to a total energy output of 600 to 1800 J/cm. The diameter of the coagulation lesion was measured by using a caliper. Each measurement was repeated three times.

To develop a murine intrahepatic tumor recurrence model, laser treatment was performed on day 7 after tumor implantation (6 W/cm with 800 J/cm). At different time points after treatment, mice were examined for liver metastasis outgrowth and for possible extrahepatic disease. Mice were killed, and the liver was removed and fixed in 4% neutral buffered formalin for 24 hours and embedded in paraffin for histological examination.

In the experimental protocol, solitary liver metastases were established in 31 mice. Six days after tumor implantation, the tumor load was measured by in vivo bioluminescent imaging, and animals with established liver metastases were randomized into four groups. In the treatment groups ILC (n = 8) and ILC plus doxorubicin (n = 8), laser treatment was performed on day 7 after tumor implantation at a power setting of 6 W/cm of diffuser length for 133 seconds, corresponding to a total energy output of 800 J/cm. Doxorubicin treatment consisted of IV injections of doxorubicin into the lateral tail vein (Pharmacia and Upjohn, Woerden, The Netherlands) in a dose of 10 mg/kg of body weight on days 9 and 14 after implantation. This is in accordance with the experimental protocols in metastatic murine colorectal cancer used by other groups and by us.11,14 In the control group (sham; n = 7) sham ILC and sham treatment with NaCl IV were given. In the doxorubicin group (n = 8) the drug was administered IV combined with sham ILC. Sham ILC consisted of laser fiber placement into the tumor. Tumor load was analyzed on days 9, 14, 19, and 22 after tumor implantation by in vivo bioluminescent imaging. On day 22, all animals were sacrificed, and livers were harvested and either fixed in 4% neutral buffered formalin for 24 hours and embedded in paraffin or snap-frozen in liquid nitrogen.

Histological Analyses
Formalin-fixed pieces of liver were sectioned into 4-µm slices, embedded completely in paraffin, and processed for routine hematoxylin and eosin (H&E) staining. Additional liver sections were frozen in liquid nitrogen for the detection of reduced nicotinamide adenine dinucleotide diaphorase, a marker of mitochondrial and, thereby, cell viability.15 For reduced nicotinamide adenine dinucleotide diaphorase activity analysis, 8-µm cryostat-cut unfixed sections were placed on glass slides. Incubation media consisted of 1.3 to 1.5 mM of reduced nicotinamide adenine dinucleotide (NADH.2Na in a nitroblue tetrazolium solution consisting of 1.5 mM of nitro-blue tetrazolium, 5 mM of MgCl, 5 mM of sodium azide, and 1.7 mM of polyvinylpyrrolidone in a .2 M phosphate buffer. Each slide was covered with 1 mL of incubation media for 25 minutes at 37°C. Each slide was then rinsed with distilled water and post-fixed with 4% buffered formaldehyde, rinsed with water, dehydrated in ethanol, cleared in xylene, and coverslipped.

Statistical Analysis
Statistical analysis was performed with GraphPad Prism version 3.0 for Windows (GraphPad Software, San Diego, CA). Statistical differences between groups were analyzed by Mann-Whitney U-test, and P < .05 was used to denote statistical significance. Values are expressed as mean ± SEM.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Reference Dosimetry ILC in Ex Vivo Liver
To establish reproducible dose-response curves for hepatic ILC, porcine livers were used ex vivo. The relationship between the diameter of the coagulation lesion and the energy applied is illustrated in Fig. 1Go. Lines with different power outputs showed a similar slope for increasing energy levels. The lesion diameter enlarged with increasing power output. The coagulation size reached a plateau at 6 W/cm with a total energy output of 1400 J/cm and at 8 W/cm with 1400 J/cm of energy output.


Figure 1
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  		FIG. 1. Reference dosimetry curves describing the diameter of the coagulation lesion versus the energy applied by interstitial laser coagulation in the porcine liver ex vivo. Each line represents a fixed laser power setting: 6 W/cm for open squares and 8 W/cm for closed triangles. Each point represents the mean ± SEM of 3 experimental results.

 
Tumor Induction and Recurrence After ILC
Tumor diameter on day 7 after implantation ranged from 5.3 to 5.8 mm (Fig. 2Go). We did not observe any intrahepatic or extrahepatic (peritoneum or lungs) tumor spread. These studies show that these solitary liver metastases are induced in a reproducible way with a remarkably constant size 1 week after implantation. Given that the average tumor diameter after day 7 was 5 to 6 mm, ILC was applied at 800 J/cm and 6W/cm, representing the setting that could ablate approximately 5 mm of pig liver tissue (Fig. 1Go). With these settings, tumor recurrence was established in 9 of 16 mice.


Figure 2
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  		FIG. 2. Macroscopic appearance of a solitary C26-luciferase colorectal metastasis in the left lobe of the mouse liver 7 days after implantation (arrow).

 
Effect of ILC Plus Doxorubicin
Next we investigated the efficacy of tumor ablation by ILC alone, by ILC plus doxorubicin, or by doxorubicin alone. Seven days after tumor implantation in the liver, tumor load, as measured by bioluminescent imaging, was set at 100% in each animal in all groups. One day later, mice were treated with either ILC or sham, and tumor load was analyzed on days 9, 14, 19, and 22 after tumor implantation. As expected, mice treated with ILC alone showed a massive decrease in tumor load, but tumor recurrence was observed from day 14 onward (Fig. 3Go). Mice treated with ILC plus doxorubicin showed complete remission of tumor load (Fig. 3Go). The graph in Fig. 3Go seems to suggest that the tumor load in the ILC plus doxorubicin group increased after 22 days. However, the increase in detected luminescence was very low (.7%–2%) and not significant. It should be noted that the y-axis of this graph is in log scale. Additionally, after termination of the experiment, all livers were examined for microscopic tumor residue by H&E-stained liver tissue sections. In the ILC plus doxorubicin group, we did not find a single lesion in any of the livers examined. Possibly, the apparent increase in bioluminescence is due to the scattered background signal. In the sham group, all animals showed an exponential increase in tumor load, and a similar pattern was observed after treatment with doxorubicin alone (Fig. 3Go). This experiment was repeated with similar results. The pooled data from both experiments showed that tumor recurrence occurred in 9 of 16 mice treated with ILC alone.


Figure 3
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  		FIG. 3. Relative tumor load (mean ± SEM) at different time points after the establishment of a solitary C26-luciferase colorectal metastasis in the left lobe of the mouse liver. Bioluminescent imaging measurements on days 9, 14, 19, and 22 are shown. Interstitial laser coagulation (ILC) or sham treatment was applied on day 7, and doxorubicin or vehicle was given on days 9 and 14 by intravenous injections. Sham (closed squares), doxorubicin (open squares), ILC (closed circles), and ILC plus doxorubicin (open circles) groups are shown (n = 8 per group).

 
We observed a clear distinction between tumor-related mortality and mortality directly related to the ILC and doxorubicin procedure. In the ILC group, tumor recurrence was observed in 6 of 16 mice (1 on day 19, 1 on day 20, and 4 on day 22; Table 1Go). Autopsy showed that mortality was associated with massive tumor growth in the liver. This growth frequently accompanied by the formation of abscesses and large abdominal lymph nodes. In the ILC plus doxorubicin group, two mice died (days 16 and 22), possibly as a result of the combined adverse effects of ILC, doxorubicin, and anesthesia. Autopsy did not reveal a clear cause of death but did show that (macroscopic) tumor recurrence had not occurred in these mice. Therefore, we concluded that mortality in these mice was not related to tumor growth. In general, we observed that ILC therapy caused morbidity in some of the animals, as shown by decreased activity, untreated fur, and loss of body weight. Mortality, which was invariably attributed to excessive tumor growth, was seen in approximately 50% of all control mice (9 of 16; 1 on day 9, 3 on day 19, 4 on day 20, and 1 on day 22) and doxorubicin-treated mice (8 of 16; 1 on day 15, 3 on day 19, 2 on day 20, and 2 on day 22; Table 1Go).


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  		TABLE 1. Tumor-related mortality and recurrence
 
Immunohistochemical Analyses
On the first day after ILC treatment, H&E staining showed tumor cells with acidophilic cytoplasm and dark elongated nuclei (Fig. 4A and BGo). Large cavities surrounded by densely coagulated cells with pyknotic nuclei were observed. These cavities are caused by boiled tissue water, which creates bubbles of steam within the tissue. Around the tumor, a sharply demarcated concentric rim of necrotic liver tissue was visible that consisted of two zones. Zone 1 showed hepatocytes with vacuolated acidophilic cytoplasm (Fig. 4CGo). Zone 2 consisted of acidophilic necrosis containing deliquesced cells without nuclei and areas of massive inflammatory cells (Fig. 4DGo). In the experimental protocol, tumor tissue in the sham group, the doxorubicin group, and the group treated with ILC showed no morphological differences on day 28 as judged by conventional histological analysis (H&E). H&E sections of tumor tissue demonstrated poorly differentiated tumor cells with vascular and lymphatic invasion. All tumors in sham-operated mice showed massive outgrowth with infiltration in adjacent liver tissue. H&E sections on day 28 of the liver of mice treated with ILC plus doxorubicin showed sharply demarcated lesions around the area of laser application (Fig. 5AGo). The border between normal and necrotic tissue consisted of a practically straight line.


Figure 4
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  		FIG. 4. Hematoxylin and eosin–stained sections of a solitary metastasis in the liver 1 day after interstitial laser coagulation treatment. (A) Coagulated tumor tissue is surrounded by 2 zones of necrotic liver tissue sharply demarcated from normal tissue. T, tumor tissue; 1, zone 1; 2, zone 2; L, normal liver (original magnification, x 20). (B) Tumor cells sho x characteristically dark elongated nuclei and acidophilic cytoplasm surrounded by large cavities (boiling effect; original magnification, x 100). (C) Zone 1: hepatocytes with vacuolated cytoplasm (original magnification, x 100). (D) Zone 2: deliquesced hepatocytes infiltrated by inflammatory cells (original magnification, x 100).

 

Figure 5
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  		FIG. 5. Hematoxylin and eosin–stained sections of a solitary liver metastasis 22 days after treatment with interstitial laser coagulation (ILC) plus doxorubicin. (A) The coagulation area was divided into 3 different zones: I, zone I; II, zone 2; III, zone 3; L, normal liver (original magnification, x 10). (B) Morphologically intact tumor cells in zone III are in a frozen state caused by heat fixation (original magnification, x 400). (C) Reduced nicotinamide adenine dinucleotide (NADH) staining on day 22 of tumor treated with the combination of ILC plus doxorubicin (original magnification, x 10). No tumor recurrence was encountered. Neither zone 1 nor zone 2 or 3 contained live tumor or liver tissue. (D) NADH staining on day 22 of tumor treated with ILC alone showing tumor recurrence on the border (arrow; original magnification, x 10). C, coagulation area; TR, tumor recurrence.

 
The coagulation area was clearly divided into three zones (Fig. 5AGo). Zone I consisted of necrotic liver tissue. Zone II was a rim of necrotic tumor tissue. Zone III, the inner center, showed morphologically intact tumor cells (Fig. 5BGo). However, enzyme histochemical analysis of cell viability by NADH-diaphorase of this area showed no staining of cells in either of these ILC-ablated zones, and this is consistent with a complete loss of cell viability (Fig. 5CGo). This was seen in all mice treated with ILC plus doxorubicin. Adjacent liver tissue demonstrated normal liver architecture with strong NADH activity. In contrast, H&E sections on day 28 of the liver of mice in the group treated with ILC showed tumor recurrence at the border of the coagulation area in 9 of 16 mice. Cell viability analysis by NADH-diaphorase showed a border of blue-stained viable tumor cells around the ILC-ablated region (Fig. 5DGo).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Because intrahepatic recurrence is common after local ablative therapies for colorectal hepatic metastases, novel strategies are needed to improve the an-titumoral efficacy of such treatment modalities. We established a murine tumor model for solitary liver metastasis in which recurrent tumor growth occurs after ILC. Most mouse models for colorectal liver metastases have traditionally been based on intrasplenic or intraportal injection of tumor cell suspensions, thus rendering small, multifocal liver metastases.11,16–18 There have been a few reports on experimental therapy with intrahepatic tumor implantation models. However, most of these were short-term experiments because of lung metastases, peritoneal tumor seeding, or both.19,20 All other intrahepatic tumor implantation models were established in rats.21,22 In our highly reproducible model for solitary liver metastasis in mice, tumor growth was assessed by using noninvasive bioluminescence measurements. We have previously reported that luciferase imaging is a reliable method for measuring tumor growth in the liver without affecting tumor cell viability or the kinetics of tumor growth.12 The use of C26-luciferase allowed us to asses the effect of ILC and chemotherapy on tumor growth over time in a noninvasive manner.

The prognosis of patients with liver metastases from colorectal cancer is poor.23–25 Surgical resection provides the only hope for cure, but only 10%to 20% are eligible for surgery.2,4 In patients not eligible for surgery, locally ablative techniques, such as radio-frequency ablation or ILC, may still offer a survival benefit.6,26 ILC has several advantages over surgical resection, including a minimally invasive approach associated with significantly lower morbidity and mortality rates.2,27–29 However, reported recurrence rates vary from 0% to 63%; these rates are probably related to either the failure to achieve adequate coagulation of the lesion or the presence of micrometastases.8,9,29 These micrometastases may be seeded from primary lesions or may originate from the macrometastatic lesions as satellite lesions.5 Therefore, there is a need for an efficient, additional treatment that destroys any residual disease after tumor ablation.

We postulate that complete remission in the combined treatment group results from synergy between sublethal thermal tissue damage at the rim of the tumor induced by ILC and the doxorubicin chemotherapy. In our experimental protocol, microscopic examination with H&E staining of tumor tissue treated by ILC plus doxorubicin demonstrated well-defined areas of characteristic cautery effects evidenced by acellular coagulum. The observed pathologic features in the coagulated zone are in accordance with our previous work and that of others.21,30 Enzyme histochemical analysis of cell viability by NADH-diaphorase showed no staining of tumor cells in the inner center of the coagulation region, and this is consistent with a complete loss of tumor cell viability. However, morphologically these cells are only subvital and may not become necrotic because of heat fixation. We found that additional treatment with doxorubicin could completely prevent intrahepatic tumor recurrence, whereas after ILC treatment without chemotherapy, tumor recurrence occurred in almost all animals. The combination of ablation therapy with systemic antitumor therapy may be very attractive, because the remaining viable cells in the well-perfused periphery of the tumor are particularly sensitive to chemotherapeutic agents.31–33

Tumor-related mortality was seen in approximately 50% of sham-operated mice irrespective of the use of doxorubicin. After ILC, tumor-related mortality was decreased and was completely abrogated when ILC was combined with chemotherapy. However, we also observed morbidity and mortality that were not attributed to tumor growth. This is possibly due to the strong inflammatory response to tissue and necrosis induced by ILC.

In this model, doxorubicin alone had no effect on tumor growth, probably because the initial tumor was too large for effective treatment. In contrast, in a mouse model of diffuse colorectal liver metastases induced by intrasplenic injection of C26 tumor cells, we have previously shown that doxorubicin (10 mg/kg of body weight) induced a significant decrease of tumor hepatic replacement area.11 This might explain the successful antitumor effect of doxorubicin in minimal residual disease after ILC in this study. The use of an intravenous chemotherapeutic delivery route has several characteristics that would be potentially beneficial for its use in clinical practice. Most importantly, the known increased vascular permeability of tumors treated with hyperthermia is likely responsible for the maintenance of effectiveness for several days after administration.34,35

In conclusion, our study demonstrated a synergistic effect of doxorubicin after ILC treatment. Several mechanisms may underlie the observed synergy between ILC and doxorubicin treatment. First, a smaller tumor volume at the start of doxorubicin treatment (after ILC) is likely to be more effectively eradicated than a large tumor volume (no ILC). This notion is supported by our earlier findings that C26 micrometastases are effectively suppressed by doxorubicin.11 Second, tumor cells exposed to hyperthermia (ILC) may have become sensitized to doxorubicin-induced cell death.36 Third, an increase in vascular permeability after ILC may lead to more efficient targeting of tumor cells by doxorubicin.34,35 Obviously, the above-mentioned possibilities are not mutually exclusive. Future experiments should reveal whether tumor cells in the transition zone surrounding the ablated area are better accessible and/or sensitized to chemotherapeutics.

Our results support the concept that combined ILC and adjuvant chemotherapeutic treatment can increase the extent of tumor destruction. The results of this study may provide a basis for further clinical investigation of combined treatment with local ablation plus chemotherapy in patients with colorectal liver metastases.


   ACKNOWLEDGMENTS
 
The authors thank Andre Verheem for his expert technical assistance.

Received for publication March 15, 2005. Accepted for publication August 30, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Weiss L, Grundmann E, Torhorst J, et al. Haematogenous metastatic patterns in colonic carcinoma: an analysis of 1541 necropsies. J Pathol 1986; 150:195–203.[CrossRef][Medline]
  2. Steele G Jr, Ravikumar TS. Resection of hepatic metastases from colorectal cancer. Biologic perspective. Ann Surg 1989; 210:127–38.[Medline]
  3. Stangl R, Altendorf-Hofmann A, Charnley RM, Scheele J. Factors influencing the natural history of colorectal liver metastases. Lancet 1994; 343:1405–10.[CrossRef][Medline]
  4. Nakamura S, Suzuki S, Baba S. Resection of liver metastases of colorectal carcinoma. World J Surg 1997; 21:741–7.[CrossRef][Medline]
  5. Nanko M, Shimada H, Yamaoka H, et al. Micrometastatic colorectal cancer lesions in the liver. Surg Today 1998; 28:707–13.[CrossRef][Medline]
  6. Curley SA. Radiofrequency ablation of malignant liver tumors. Ann Surg Oncol 2003; 10:338–47.[Abstract/Free Full Text]
  7. Goldberg SN, Gazelle GS, Compton CC, Mueller PR, Tanabe KK. Treatment of intrahepatic malignancy with radiofrequency ablation: radiologic-pathologic correlation. Cancer 2000; 88:2452–63.[CrossRef][Medline]
  8. Gillams AR, Lees WR. Survival after percutaneous, image-guided, thermal ablation of hepatic metastases from colorectal cancer. Dis Colon Rectum 2000; 43:656–61.[CrossRef][Medline]
  9. Giorgio A, Tarantino L, de Stefano G, et al. Interstitial laser photocoagulation under ultrasound guidance of liver tumors: results in 104 treated patients. Eur J Ultrasound 2000; 11: 181–8.[CrossRef][Medline]
  10. Vogl TJ, Straub R, Eichler K, Sollner O, Mack MG. Colorectal carcinoma metastases in liver: laser-induced interstitial thermotherapy local tumor control rate and survival data. Radiology 2004; 230:450–8.[Abstract/Free Full Text]
  11. te Velde EA, Vogten JM, Gebbink MF, van Gorp JM, Voest EE, Borel Rinkes IHM. Enhanced antitumour efficacy by combining conventional chemotherapy with angiostatin or endostatin in a liver metastasis model. Br J Surg 2002; 89:1302–9.[CrossRef][Medline]
  12. Smakman N, Martens A, Kranenburg O, Borel Rinkes I. Validation of bioluminescence imaging of colorectal liver metastases in the mouse. J Surg Res 2004; 122:225–30.[CrossRef][Medline]
  13. Honigman A, Zeira E, Ohana P, et al. Imaging transgene expression in live animals. Mol Ther 2001; 4:239–49.[CrossRef][Medline]
  14. Wilmanns C, Fan D, O’Brian CA, Bucana CD, Fidler IJ. Orthotopic and ectopic organ environments differentially influence the sensitivity of murine colon carcinoma cells to doxorubicin and 5-fluorouracil. Int J Cancer 1992; 52:98–104.[Medline]
  15. Fang S, Thomas RM, Conklin JL, Oberley LW, Christensen J. Co-localization of manganese superoxide dismutase and NADH diaphorase. J Histochem Cytochem 1995; 43:849–55.[Abstract]
  16. Kuruppu D, Christophi C, Bertram JF, O’Brien PE. Characterization of an animal model of hepatic metastasis. J Gastroenterol Hepatol 1996; 11:26–32.[Medline]
  17. Okuno K, Shirayama Y, Ohnishi H, et al. A successful liver metastasis model in mice with neuraminidase treated colon 26. Surg Today 1993; 23:795–9.[Medline]
  18. Stapfer M, Hu J, Wei D, Groshen S, Beart RW Jr. Establishment of a nude mouse model of hepatic metastasis for evaluation of targeted retroviral gene delivery. J Surg Oncol 2003; 82:121–30.[Medline]
  19. Grady ED, Nolan TR, Craumbley AJ, Cheek WV, Copelan N, Kunzler HC. Cryotherapy of implanted cancer in the rat liver. Oncology 1973; 28:104–9.[Medline]
  20. Ernst M, Bauknecht KJ, Hartmann CA, Herter M, Haring R, Dressler S. Animal experiment studies of cryosurgical treatment of DS sarcomas and Morris hepatomas implanted in the liver. Zentralbl Chir 1988; 113:1111–5.[Medline]
  21. van Hillegersberg R, Kort WJ, ten Kate FJW, Terpstra OT. Water-jet-cooled Nd:YAG laser coagulation: selective destruction of rat liver metastases. Lasers Surg Med 1991; 11:445–54.[Medline]
  22. Yang R, Rescorla FJ, Reilly CR, et al. A reproducible rat liver cancer model for experimental therapy: introducing a technique of intrahepatic tumor implantation. J Surg Res 1992; 52:193–8.[CrossRef][Medline]
  23. Pestana C, Reitemeier RJ, Moertel CG, Judd ES, Dockerty MB. The natural history of carcinoma of the colon and rectum. Am J Surg 1964; 108:826–9.[CrossRef][Medline]
  24. Bengtsson G, Carlsson G, Hafstrom L, Jonsson PE. Natural history of patients with untreated liver metastases from colorectal cancer. Am J Surg 1981; 141:586–9.[CrossRef][Medline]
  25. Goslin R, Steele G Jr, Zamcheck N, Mayer R, MacIntyre J. Factors influencing survival in patients with hepatic metastases from adenocarcinoma of the colon or rectum. Dis Colon Rectum 1982; 25:749–54.[Medline]
  26. Sakakima Y, Inoue S, Takeda S, Kaneko T, Nakao A. Factors in successful radiofrequency ablation therapy for malignant liver tumors. Hepatogastroenterology 2004; 51:1761–5.[Medline]
  27. Geoghegan JG, Scheele J. Treatment of colorectal liver metastases. Br J Surg 1999; 86:158–69.[CrossRef][Medline]
  28. Wei AC, Tung-Ping PR, Fan ST, Wong J. Risk factors for perioperative morbidity and mortality after extended hepatectomy for hepatocellular carcinoma. Br J Surg 2003; 90:33–41.[Medline]
  29. Vogl TJ, Eichler K, Straub R, et al. Laser-induced thermo-therapy of malignant liver tumors: general principals, equipment(s), procedure(s)—side effects, complications and results. Eur J Ultrasound 2001; 13:117–27.[CrossRef][Medline]
  30. van Hillegersberg R, van Staveren HJ, Kort WJ, Zondervan PE, Terpstra OT. Interstitial Nd:YAG laser coagulation with a cylindrical diffusing fiber tip in experimental liver metastases. Lasers Surg Med 1994; 14:124–38.[Medline]
  31. Goldberg SN, Girnan GD, Lukyanov AN, et al. Percutaneous tumor ablation: increased necrosis with combined radio-frequency ablation and intravenous liposomal doxorubicin in a rat breast tumor model. Radiology 2002; 222:797–804.[Abstract/Free Full Text]
  32. Goldberg SN, Kamel IR, Kruskal JB, et al. Radiofrequency ablation of hepatic tumors: increased tumor destruction with adjuvant liposomal doxorubicin therapy. Am J Roentgenol 2002; 179:93–101.[Abstract/Free Full Text]
  33. Ahmed M, Monsky WE, Girnun G, et al. Radiofrequency thermal ablation sharply increases intratumoral liposomal doxorubicin accumulation and tumor coagulation. Cancer Res 2003; 63:6327–33.[Abstract/Free Full Text]
  34. Eddy HA. Alterations in tumor microvasculature during hyperthermia. Radiology 1980; 137:515–21.[Abstract/Free Full Text]
  35. Lefor AT, Makohon S, Ackerman NB. The effects of hyperthermia on vascular permeability in experimental liver metastasis. J Surg Oncol 1985; 28:297–300.[Medline]
  36. Toffoli G, Bevilacqua C, Franceschin A, Boiocchi M. Effect of hyperthermia on intracellular drug accumulation and chemo-sensitivity in drug-sensitive and drug-resistant P388 leukaemia cell lines. Int J Hyperthermia 1989; 5:163–72.[Medline]




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