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Liver Resection-Associated Macrophage Inflammatory Protein-2 Stimulates Engraftment but not Growth of Colorectal Metastasis at Extrahepatic Sites
Journal of Surgical Research 145, 295–302 (2008) doi:10.1016/j.jss.2007.02.010
Liver Resection-Associated Macrophage Inflammatory Protein-2 Stimulates Engraftment but not Growth of Colorectal Metastasis at Extrahepatic Sites Otto Kollmar,*,1 Bastian Junker,* Kathrin Rupertus,* Claudia Scheuer,† Michael D. Menger,† and Martin K. Schilling* *Department of General, Visceral, Vascular and Pediatric Surgery and †Institute for Clinical and Experimental Surgery, University of Saarland, Homburg/Saar, Germany Submitted for publication January 6, 2007
Background. Previous studies have shown that liver resection enhances intrahepatic engraftment of CXCR-2expressing colorectal cancer cells by the action of the CXC chemokine macrophage inflammatory protein (MIP)-2. Herein we studied how liver resectionassociated MIP-2 affects extrahepatic tumor cell engraftment and whether MIP-2 also stimulates the growth of already established metastases. Materials and methods. Green fluorescent proteintransfected CT26.WT colorectal cancer cells were implanted into dorsal skinfold chambers of syngeneic BALB/c mice. Additionally, all animals underwent a 30% hepatectomy. To study MIP-2 in extrahepatic tumor cell engraftment, animals were treated with an anti-MIP-2 antibody, starting at the day of tumor cell implantation. To study MIP-2 in established metastases, anti-MIP-2 treatment was initiated at day 5 after tumor cell implantation. Hepatectomized animals without neutralization of MIP-2 served as controls. Tumor vascularization and growth as well as tumor cell migration, proliferation, apoptosis, and CXCR-2 expression were studied over 14 days using intravital fluorescence microscopy, histology, and immunohistochemistry. Results. Functional inhibition of MIP-2 significantly delayed extrahepatic tumor cell engraftment but not the growth of established metastases. The initial delay of engraftment was associated with a compensatory stimulation of vascularization and tumor cell migration when compared to controls (P < 0.05). Further, inhibition of tumor cell engraftment by initial anti1
To whom correspondence and reprint requests should be addressed at Department of General, Visceral, Vascular, and Pediatric Surgery, University of Saarland, D-66421 Homburg/Saar, Germany. E-mail: [email protected].
MIP-2 treatment was associated with a significant (P < 0.05) reduction of CXCR-2 expression and tumor cell apoptosis. Conclusion. Our study indicates that MIP-2 is involved in extrahepatic engraftment of CT.26 colorectal cancer cells. The MIP-2/CXCR-2 signaling pathway may be a promising target for early antitumor therapy in patients undergoing liver resection. © 2008 Elsevier Inc. All rights reserved.
Key Words: colorectal cancer; chemokine; MIP-2; tumor growth; metastasis; liver regeneration; hepatectomy; microcirculation angiogenesis. INTRODUCTION
Colorectal cancer is the second leading cause of cancer-related death in the Western world [1]. The benefit of curative resection of colorectal liver metastases with a 5-year overall survival rate of up to 58% is well recognized [1– 4]. For a long time, extrahepatic metastases have been considered a contraindication for hepatectomy. Over the last few years, however, interesting 5-year survival rates of up to 30% have been reported after resection of liver and extrahepatic colorectal metastases in lung, brain, bone, and other sites [5–7]. In line with this, Elias et al. demonstrated that complete resection of all metastases of colorectal cancer is more important for the overall patient survival than the location and the number of the metastases [5]. Besides parenchymal regeneration, hepatectomy also stimulates tumor growth in the remaining liver [8 –10]. The magnitude of stimulation of tumor growth is thought to be proportional to the volume of the resected liver tissue [11–13]. Furthermore, after liver resection about 20% of patients develop metastases in the lung
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and 2 to 5% of patients develop metastases in the brain, bone, and other sites [6, 7]. Clinical and experimental studies have demonstrated that chemokines can regulate angiogenesis, proliferation, and apoptosis of tumor cells and that they mediate tumor cell invasion and trafficking in an organ-specific manner leading to metastases [14 –16]. Previous experimental studies have demonstrated that the CXC chemokine macrophage inflammatory protein (MIP)-2 promotes engraftment of CXCR-2-expressing colorectal liver metastases [17]. Furthermore, blockade of MIP-2 has been shown to significantly inhibit liver resection-induced acceleration of intrahepatic metastatic tumor engraftment [8]. There is, however, no information on whether liver resection-associated MIP-2 also accelerates engraftment of colorectal cancer cells at extrahepatic sites. In addition, the effect of MIP-2 on the growth of established metastases has not been studied thus far. With the use of an established murine extrahepatic colon cancer metastasis model, we therefore analyzed how liver resection-associated MIP-2 affects extrahepatic tumor cell engraftment and whether MIP-2 also stimulates the growth of already established metastases. MATERIALS AND METHODS Tumor Cell Line and Culture Conditions The CT26 cell line is an N-nitroso-N-methylurethane-induced undifferentiated adenocarcinoma of the colon, syngeneic with the BALB/c mouse. For our studies, the CT26.WT cell line (ATCC CRL2638; LGC Promochem GmbH, Wesel, Germany) was grown in cell culture as monolayers in RPMI 1640 medium with 2 mM L-glutamine (Sigma Aldrich Chemie GmbH, Taufkirchen, Germany) supplemented with 10% fetal calf serum (FCS Gold; PAA Laboratories GmbH, Cölbe, Germany), 100 U/mL penicillin, and 100 g/mL streptomycin (PAA Laboratories GmbH). The cells were incubated at 37°C in a humidified atmosphere containing 5% CO 2. With the use of CLONfectin (Clontech, Palo Alto, CA), the cells were transfected with the enhanced green fluorescent protein (GFP) expression vector pEGFP-N1 (Clontech) according to the manufacturer’s instructions [18]. For the individual experiments, only CT26.WT-GFP cells of the first three serial passages after cryo-storage were used. At the day of implantation, CT26.WT-GFP cells were harvested from subconfluent cultures (70 to 85%) by trypsinization (0.05% Trypsin and 0.02% EDTA, PAA Laboratories GmbH) and washed twice in PBS solution.
Animals Experiments were performed after approval by the local governmental ethic committee and conformed to the United Kingdom Coordinating Committee on Cancer Research (UKCCCR) Guidelines for the Welfare of Animals in Experimental Neoplasia (as described in Br J Cancer 1998;77:1) and the Guide for Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, National Research Council; NIH Guide, Vol. 25, No. 28, 1996). Twelve- to 16-week-old female BALB/c mice (Charles River Laboratories GmbH, Sulzfeld, Germany) with a body weight (BW) of 20 –22 g were used. The animals were housed in single cages at a room temperature of 22–24°C and at a relative humidity of 60 – 65% with a 12-h light/ dark-cycle environment. The mice were allowed free access to drinking water and standard laboratory chow (Altromin, Lage, Germany).
Experimental Model For operative procedures, animals were anesthetized by intraperitoneal injection of 90 mg/kg BW ketamine (Ketavet; Parke Davis, Freiburg, Germany) and 20 mg/kg BW xylazine (Rompun; Bayer, Leverkusen, Germany). To allow repetitive analyses of the microvasculature of growing tumors, the dorsal skinfold chamber model was used for intravital microscopy as described previously in detail [19]. Briefly, the chamber consists of two symmetrical titanium frames, which were positioned to sandwich the extended double layer of the dorsal skin. One layer was completely removed in a circular area of 15 mm diameter. The remaining layers, consisting of the epidermis, subcutaneous tissue, and striated skin muscle, were covered with a glass coverslip incorporated into one of the titanium frames [20]. The animals tolerated the chambers well and showed no signs of discomfort or changes in sleeping and feeding habits. After a 48-h recovery period the animals were re-anesthetized. After laparotomy, animals received a 30% hepatectomy of the left lateral liver lobe. For tumor cell implantation, the coverslip of the chamber was temporarily removed and 1 ⫻ 10 5 CT26.WT-GFP cells were implanted onto the surface of the striated muscle tissue within the chamber. Directly after cell implantation, the chamber tissue was covered again with the coverslip [21].
Experimental Protocol A total of 23 animals received a 30% hepatectomy of the left lateral liver lobe and simultaneous tumor cell implantation in the dorsal skinfold chamber. The animals were assigned to three different groups. One group (Phx⫹Abd0; n ⫽ 7) received 1 mg/kg BW of the monoclonal rat anti-mouse MIP-2 antibody (MAB452, R&D Systems, Wiesbaden, Germany) intraperitoneally on the day of hepatectomy and every second day thereafter. The animals of the second group received the same amount of the MIP-2 antibody; however, the treatment was started only at day 5 after hepatectomy (Phx⫹Abd5; n ⫽ 8). Treatment was also continued every second day thereafter. Hepatectomized animals without neutralization of MIP-2 (Phx; n ⫽ 8) served as controls. All animals underwent repetitive intravital microscopic analyses directly as well as 5, 7, 9, 12, and 14 days after tumor cell implantation. At the end of the experiment (day 14), the chamber with the tumor tissue was harvested for histology and immunohistochemistry.
Intravital Fluorescence Microscopy Intravital fluorescence microscopy was performed in epiillumination technique using a modified Zeiss Axio-Tech microscope (Zeiss, Oberkochen, Germany) with a 100-W HBO mercury lamp. Microscopic images were monitored by a charge-coupled device video camera (FK 6990, COHU; Prospective Measurements Inc., San Diego, CA) and were transferred to a video system (VO-5800 PS; Sony, München, Germany) for subsequent off-line analysis. Migration of tumor cells, tumor size, growth kinetics, and neovascularization were analyzed using blue light epi-illumination (450 to 490 nm excitation wavelength and ⬎520 nm emission wavelengths).
Microcirculation Analysis Microcirculatory parameters were assessed off-line by frame-toframe analysis of the videotaped images using a computer-assisted image analysis system (CapImage; Zeintl Software, Heidelberg, Germany). Data analysis was performed by examiners unaware of the treatment. The fluorescent labeling of the tumor cells allowed precise delineation of the tumor from the surrounding unaffected host tissue. It also enabled for distinct identification of individual tumor cells to study tumor cell migration. At each observation time point the surface of the fluorescently labeled tumor mass within the chamber was first scanned for determination of the tumor size (given as tumor
KOLLMAR ET AL.: MIP-2 AND EXTRAHEPATIC TUMOR GROWTH area in mm 2). Next, eight regions-of-interest (ROIs) were randomly chosen within the tumor margin, and the number of migrated tumor cells was counted within each region. In these ROIs the onset of angiogenesis, i.e., development of buds, sprouts, and blood vessels, was documented and scored 0 to 8, with 0 indicating angiogenesis in none of the ROIs and 8 indicating angiogenesis in all of the ROIs. Functional capillary density [cm/cm 2] of the tumor microvasculature served as an indicator of neovascularization. It was defined as the length of RBC perfused capillaries per observation area and was analyzed within the eight ROIs of the tumor margin and within four additional ROIs of the tumor center. Diameters of the newly formed tumor microvessels were measured perpendicularly to the vessel path and are given in micrometers [18]. To study vascular permeability of the newly formed tumor microvessels, the occurrence of petechial bleedings was documented in each ROI and is given as percentage of all ROIs analyzed.
Histology and Immunohistochemistry At the end of the experiments (day 14), the tumor and the adjacent host tissue was harvested. For light microscopy, formalin-fixed biopsies were embedded in paraffin. Sections of 5 m were cut and stained with hematoxylin and eosin for routine histology according to standard procedures. Tumor growth characteristics were analyzed. Tumor cell invasion of the muscle layer was measured and is given in percent of the length of the tumor basis. To study cell proliferation and apoptotic cell death, proliferating cell nuclear antigen (PCNA) and cleaved caspase-3 were stained using indirect immunoperoxidase techniques. Therefore, deparaffinized sections were incubated with 3% H 2O 2 and 2% goat normal serum to block endogenous peroxidases and unspecific binding sites. A monoclonal mouse anti-pan PCNA antibody (PC10, 1:50; DakoCytomation, Hamburg, Germany) and a polyclonal rabbit antimouse cleaved caspase-3 antibody (Asp175, 1:50; Cell Signaling Technology, Frankfurt, Germany) were used as primary antibodies. The cleaved caspase-3 antibody detects endogenous levels only of the short fragment (17/19 kDa) of activated caspase-3, but not full-length caspase-3. Biotinylated goat anti-mouse and goat anti-rabbit immunoglobulin antibodies were used as secondary antibodies for streptavidin-biotin complex peroxidase staining (1:200, LSAB 2 System HRP; DakoCytomation). 3,3=-Diaminobenzidine (DakoCytomation) was used as chromogen. Sections were counterstained with hemalaun according to Mayer and examined by light microscopy. To assess the expression of the chemokine receptor CXCR-2, tumor slices were embedded in tissue freezing medium (Jung; Leica Microsystems, Nussloch, Germany) for immunohistochemistry, snapfrozen in liquid nitrogen, and stored at – 80°C. Cryostat sections of 5 m were cut, fixed in 4°C cold acetone for 5 s, followed by fixation in formalin 4% for 10 min, and blocked with 2% normal donkey serum. Tissue sections were then incubated with the polyclonal rabbit antimouse CXCR-2 antibody (1:10; Santa Cruz, Heidelberg, Germany). A donkey anti-rabbit IgG horseradish peroxidase-conjugated antibody (1: 500; Amersham, Freiburg, Germany) was used as secondary antibody. 3,3=-Diaminobenzidine was used as chromogen. Sections were counterstained with hemalaun according to Mayer and examined by light microscopy. As a negative control, additional slices from each specimen were exposed to appropriate IgG isotype-matched antibody (Sigma Aldrich Chemie GmbH) in place of primary antibody under the same conditions to determine the specificity of antibody binding. All of the control stainings were found to be negative.
Statistical Analysis All values are expressed as means ⫾ SEM. After proving the assumption of normality and homogeneity of variance across groups, differences between groups were calculated by a one-way analysis of variance followed by the appropriate post-hoc comparison, including the correction of the alpha-error according to Bonferroni probabili-
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ties to compensate for multiple comparisons. Overall statistical significance was set at P ⬍ 0.05. Statistical analysis was performed with the use of the software package SigmaStat (SPSS Inc., Chicago, IL).
RESULTS
The general conditions of all animals were not affected by the preparation of the dorsal skinfold chamber, the 30% hepatectomy, and the tumor cell implantation. All animals had an uneventful postoperative recovery and tolerated well the intravital fluorescence microscopic observations during the 14-day period. The take rate of the implanted CT26.WT-GFP cells was 100%. Intravital fluorescence microscopy showed a progressive tumor growth during the 14-day observation period. Quantitative analysis of the tumor area (Fig. 1) showed that only anti-MIP-2 treatment, starting at the day of hepatectomy (Phx⫹ABd0), was capable of significantly decreasing (P ⬍ 0.05) the tumor size during the first 9 days when compared with untreated controls. During the later time points of the 14-day observation period, the continued anti-MIP-2 treatment could not prevent an acceleration of tumor growth, indicating that anti-MIP-2, if given from day 0, delays tumor cell engraftment but does not inhibit further tumor growth. Accordingly, anti-MIP-2 treatment of established extrahepatic tumors, starting at
FIG. 1. Stereo-microscopic photomicrographs of representative tumors in the dorsal skinfold chamber at day 14 after hepatectomy without treatment (A; Phx), with anti-MIP-2 treatment starting at day 5 (B; Phx⫹ABd5), and with anti-MIP-2 treatment starting at day 0 (C; Phx⫹ABd0). Quantitative analysis of the tumor area (D) showed a significantly reduced tumor growth during the initial 9 days after tumor implantation in animals in which anti-MIP-2 was started at day 0 (black squares) when compared with controls (white circles), indicating a delay in tumor engraftment. Further continuous treatment with anti-MIP-2 could not prevent later accelerated tumor growth. Accordingly, anti-MIP-2 treatment starting at day 5 after hepatectomy (gray triangles) was not effective to inhibit tumor growth. Mean ⫾ SEM; *P ⬍ 0.05 versus Control; #P ⬍ 0.05 versus Phx⫹ABd5. Magnification (A to C), ⫻4.
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FIG. 2. Intravital fluorescence microscopy of representative tumors in dorsal skinfold chambers at day 14 after hepatectomy without treatment (A; Phx), with anti-MIP-2 treatment starting at day 5 (B; Phx⫹ABd5), and with anti-MIP-2 treatment starting at day 0 (C; Phx⫹ABd0). Quantitative analysis of angiogenesis, i.e., protrusion of capillary buds and sprouts, showed that at day 12 new vessel formations could be observed in almost all ROIs without significant differences between the three groups studied (D). However, analysis of the functional capillary density revealed a significantly increased neovascularization during the late observation period from day 9 to 14 in tumors with anti-MIP-2 treatment starting at day 0 but not day 5 when compared with solely hepatectomized controls (E). The diameters of the tumor capillaries (F) ranged between 9 and 13 m during the 14-day observation period. This involved a slight increase in controls (white circles) and Phx⫹ABd0 animals (black squares), which was not observed in Phx⫹ABd5 animals (gray triangles). Mean ⫾ SEM; *P ⬍ 0.05 versus Control; #P ⬍ 0.05 versus Phx⫹ABd5; §P ⬍ 0.05 versus Phx⫹Abd0. Magnification (A to C), ⫻40.
day 5 after hepatectomy and tumor cell implantation (Phx⫹ABd5), did not affect tumor growth (Fig. 1). Intravital fluorescence microscopy revealed that the angiogenic response, i.e., the protrusion of buds and sprouts, was initiated at day 5. At day 12, almost all ROIs studied within the tumors exhibited bud and sprout formation (Fig. 2). Anti-MIP-2 treatment, irrespective whether it was started at day 0 or day 5, did not affect the onset of the angiogenic response (Fig. 2). Analysis of the functional neovascularization of the extrahepatic metastases revealed a continuous increase of the functional density of tumor capillaries from day 5 to day 14, reaching values of ⬃250 cm/cm 2. Of interest, anti-MIP-2 treatment starting at day 0 but not at day 5 induced a significant increase (P ⬍ 0.05) of
functional capillary density during the late phase of the 14-day observation period. This was observed in both the tumor margin (Fig. 2) and the tumor center (data not shown). Diameters of tumor capillaries ranged between 9 and 13 m during the 14-day observation period. This involved a slight increase in controls (Phx) and Phx⫹ ABd0 animals, which was not observed in Phx⫹ABd5 animals (Fig. 2). Analysis of capillary diameters in the tumor center did not differ from that obtained within the tumor margin (data not shown). The increased microvascular permeability observed during angiogenesis is known to be associated with petechial bleedings. In animals treated with antiMIP-2 starting at day 0 but not at day 5, pronounced bleedings during the initial period of angiogenesis could be observed, which, however, did not prove to be significantly increased more than that of controls. This is probably due to the heterogeneous response, as indicated by the relatively high SEM values (Fig. 3). There was no difference in petechial bleedings between tumor margin (Fig. 3) and center (data not shown) within the individual groups. The tumor mass as well as the individual CT26.WTGFP cells could easily be distinguished by intravital fluorescence microscopy due to their green fluorescent labeling (Fig. 4). Of interest, migration of the tumor cells at the tumor margin could be observed during the entire 14-day observation period (Fig. 4). Migrating tumor cells showed a spindle-shaped configuration, whereas the cells within the tumor mass presented with
FIG. 3. Intravital fluorescence microscopy of petechial bleedings (arrows) in tumors at day 5 after hepatectomy and tumor cell implantation without treatment (A; Phx), with anti-MIP-2 treatment starting at day 5 (B; Phx⫹ABd5), and with anti-MIP-2 treatment starting at day 0 (C; Phx⫹ABd0). Quantitative analysis of the number of ROIs with bleedings (D, in percent) showed that in animals treated with anti-MIP-2 starting at day 0 but not at day 5 markedly more bleedings could be observed during the initial period of angiogenesis. Mean ⫾ SEM. Magnification (A to C), ⫻16.
KOLLMAR ET AL.: MIP-2 AND EXTRAHEPATIC TUMOR GROWTH
FIG. 4. Intravital fluorescence microscopy of tumor cell migration out of the tumor margin. Whereas no tumor cells out of the tumor margin could be found directly after implantation (A), cell migration could be detected at day 5 after implantation in nontreated controls (B). Anti-MIP-2 treatment starting at day 0 induced an increase of cell migration (C). Quantitative analysis (D) confirmed the increased tumor cell migration with anti-MIP-2 treatment starting at day 0 (black squares) when compared with that observed in animals in which the treatment was started at day 5 (gray triangles), and in solely hepatectomized control animals (white circles). Mean ⫾ SEM; *P ⬍ 0.05 versus Control; #P ⬍ 0.05 versus Phx⫹ABd5. Magnification (A to C), ⫻40.
a more rounded structure. The migrating cells had a mean distance of 290 to 420 m to the tumor margin, which did not differ between anti-MIP-2-treated animals and controls. However, anti-MIP-2 treatment starting at day 0 showed a significantly increased (P ⬍ 0.05) number of migrating tumor cells throughout the whole 14-day observation period when compared with both controls (PHx) and PHx⫹ABd5 animals (Fig. 4). Hematoxylin-eosin staining demonstrated tumor cell infiltration into the neighboring muscle tissue over 35.7 ⫾ 15.9% of the tumor basis in hepatectomized control animals (Phx). Additional treatment with antiMIP-2 did not influence this tumor cell invasiveness, as indicated by an infiltration of 38.5 ⫾ 14.4% (Phx⫹ ABd0) and 42.7 ⫾ 11.7% (Phx⫹ABd5). PCNA as an indicator of cell proliferation displayed strong staining of tumor cells in controls (Phx) at day 14 after liver resection. Overall, more than 80% of the tumor cells were found PCNA positive (Fig. 5). Notably, anti-MIP-2 treatment decreased the fraction of positively stained tumor cells at day 14 after hepatectomy to ⬃40% if the treatment was started at day 5, but only to ⬃60% if the treatment was started at day 0. Although this difference did not prove to be statistically significant, these data indicate a tendency toward a suppressed tumor cell proliferation by anti-MIP-2 treatment. Caspase-3 immunohistochemistry was performed to document apoptotic cell death. Surprisingly, caspase-
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3-positive cells could only rarely be observed in all three groups of hepatectomized animals, never exceeding the value of 0.5% (Fig. 5). Nonetheless, anti-MIP-2 treatment significantly reduced (P ⬍ 0.05) the number of apoptotic tumor cells when compared with that of hepatectomized controls (Phx). In fact, apoptotic cell death was reduced by 60% in Phx⫹ABd5 animals and even by 85% in Phx⫹ABd0 animals (Fig. 5). CXCR-2 immunohistochemistry revealed a marked staining of receptors on tumor cells in hepatectomized controls (Fig. 6). Quantitative analysis of CXCR-2 expression in anti-MIP-2-treated animals showed a significant decrease (P ⬍ 0.05) of the number of positively stained cells, regardless whether the treatment was started at day 0 or day 5. These findings did not differ between tumor margin (Fig. 6) and tumor center (data not shown).
FIG. 5. PCNA (A and B) and cleaved caspase-3 (C and D) immunohistochemistry of tumors at day 14 after implantation of solely hepatectomized controls (A and C) and of tumors of animals with anti-MIP-2 treatment starting at day 5 (B) and day 0 (D), respectively. Quantitative analysis of the number of PCNA-positive cells (given in percent of all cells visible) indicated a slight decrease of cell proliferation after anti-MIP-2 treatment, in particular, if started at day 5, when compared with that of controls (Phx) (E). Analysis of cleaved caspase-3 expression demonstrated a low rate of apoptotic cell death in the solely hepatectomized controls (F). Cleaved caspase-3 expression in the anti-MIP-2-treated animals was even significantly lower than that of the controls (F). Mean ⫾ SEM; ⴱP ⬍ 0.05 versus Phx. Magnification (A-D), ⫻175. (Color version of figure is available online.)
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FIG. 6. Immunohistochemistry of CXCR-2 expression of tumor cells at day 14 after tumor cell implantation in solely hepatectomized controls (A) and animals with anti-MIP-2 treatment starting at day 5 (B) or day 0 (C). Quantitative analysis (D) revealed a ⬎80% CXCR-2 expression in controls (Phx), which was significantly reduced in tumors with anti-MIP-2 treatment starting at day 5 (Phx⫹ABd5) or day 0 (Phx⫹ABd0). Mean ⫾ SEM; *P ⬍ 0.05 versus Phx. Magnification (A-C), ⫻175. (Color version of figure is available online.)
DISCUSSION
The major finding of the present study is that liver resection-associated engraftment of colorectal cancer cells at extrahepatic sites is mediated by MIP-2, whereas this chemokine does not contribute to the growth of already established extrahepatic metastases. Of interest, neutralization of MIP-2 starting at the day of tumor cell implantation (day 0) provokes a compensatory angiogenic response and a reduction of apoptotic tumor cell death during the later phase of metastatic growth. Liver resection initiates rapid regeneration and growth of the remaining liver to restore the functional capacity. The MIP-2/CXCR-2 system is thought to be involved in the hepatic regenerative process [8, 22, 23]. Ren et al. demonstrated that blockade of the chemokine receptor CXCR-2 inhibits regeneration of the residual liver, while selective neutralization of MIP-2 through anti-MIP-2 antibodies is not affecting the progress of regeneration as indicated by unchanged liver weight to body weight ratios [22]. After liver resection of colorectal metastases, the disease recurs in over two-thirds of the patients [4]. This observation indicates that undetectable residual microscopic tumors remain in the majority of patients undergoing curative hepatectomy [10]. In fact, a variety of studies have shown that liver resection enhances tumor growth during regeneration also at extrahepatic sites [24]. In a previous study, we have already shown that in
the remnant liver MIP-2 significantly promotes liver resection-induced acceleration of metastatic tumor growth [8]. The role of MIP-2 on hepatectomy-induced acceleration of metastatic growth at extrahepatic sites has not been elucidated yet. Herein, we now demonstrate for the first time that neutralization of MIP-2 significantly delays engraftment of colorectal cancer cells at extrahepatic sites, as indicated by a reduced tumor outgrowth during the early phase after hepatectomy. Of interest, MIP-2 neutralization could not prevent tumor growth during the later phase after hepatectomy. In contrast, growth rate, cell migration, and neovascularization were found increased when compared with solely hepatectomized controls, indicating a compensatory stimulation to counteract the delay of engraftment during the early phase of tumor outgrowth. Neutralization of MIP-2 starting at day 0 after hepatectomy did not affect PCNA expression of tumor cells. Thus, the compensatory stimulation of growth during the late observation phase after initial delay of engraftment by anti-MIP-2 is not mediated by an increase of tumor cell proliferation. More probably, it is induced by a reduction of apoptotic cell death. This is indicated by the 85% reduction of cleaved caspase-3 expression after 14 days of anti-MIP-2 treatment when compared to solely hepatectomized controls. We additionally studied the effect of anti-MIP-2 treatment starting at day 5 after hepatectomy and tumor cell implantation to elucidate the role of MIP-2 in stimulating growth of already established metastases. The present data show that endogenously produced MIP-2 does not affect the growth of already established extrahepatic metastases. This lack of effect is observed, although there was a reduction of apoptotic cell death by ⬃60% compared to solely hepatectomized controls. However, this reduction of caspase-3 expression was associated with an almost ⬃50% reduction of PCNA expression, indicating that the reduction of apoptosis is almost completely outweighed by the reduction of cell proliferation. The expression of the CXCR-2 receptor is thought to be required for the proliferative action of chemokines on tumor cells [17, 25, 26]. The fact that in the present study anti-MIP-2 treatment reduced CXCR-2 receptor expression further supports our interpretation that the compensatory increase of growth rate after initial delay of tumor cell engraftment is not due to increased proliferation but rather to the reduction of apoptotic cell death. Chemokines are known to be potent mediators in regulating migration of tumor cells. Our previous in vitro studies showed a dose-dependent increase of migration of tumor cells in response to MIP-2, indicating a MIP-2-associated enhancement of metastatic tumor growth [17]. Surprisingly, in the present study
KOLLMAR ET AL.: MIP-2 AND EXTRAHEPATIC TUMOR GROWTH
neutralization of MIP-2 starting at day 0 stimulated the migration of tumor cells. This may be due to the fact that the compensatory stimulation of tumor growth due to the delay of tumor cell engraftment may be associated with an increased susceptibility of the tumor cells to other chemokines and chemoattractant factors released after hepatectomy and liver regeneration [27, 28]. Of interest, anti-MIP-2 treatment starting only at day 5 inhibited tumor cell migration. Because this treatment did not result in a delay of tumor cell engraftment and thus compensatory stimulation of growth, neutralization of MIP-2 itself is probably responsible for the reduction of cell migration in vivo, similar to previous experiments in vitro [17]. CXC chemokines are thought to be involved in angiogenesis. In inflammation, Scapini et al. demonstrated that MIP-2 induces angiogenesis in vivo via expression of vascular endothelial growth factor (VEGF)-A [29]. In a previous study we showed that exogenous application of MIP-2 accelerates engraftment of hepatic colon cancer metastasis by induction of tumor vessels formation [17]. Furthermore, we have demonstrated that inhibition of endogenous MIP-2 during the early phase of intrahepatic tumor cell engraftment significantly reduces liver resection-associated neovascularization and tumor development [8]. Interestingly, neutralization of MIP-2 starting at day 0 also showed a minor reduction of angiogenesis during the early phase of delay of tumor engraftment. However, the compensatory growth during the later phase was associated with an increased neovascularization, as indicated by a significantly increased capillary density. This compensatory increase of neovascularization is most probably mediated by growth factors that affect the permeability of the microvessels such as VEGF [30-32], because the increase in neovascularization in animals treated with antiMIP-2 from day 0 on was preceded by an increase of petechial bleedings. In conclusion, we demonstrate that liver resectionassociated MIP-2 is involved in engraftment of CT.26 colorectal cancer cells at extrahepatic sites, but does not substantially contribute to the growth of already established tumors. The MIP-2/CXCR-2 signaling pathway may thus not be a promising target for the treatment of established metastases, but for the prevention of tumor cell engraftment in patients undergoing liver resection for tumor therapy.
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ACKNOWLEDGMENTS
We appreciate the excellent technical assistance of Christina Marx and Janine Becker. This study was supported by grants of the Research Committee and the Medical Faculty of the University of Saarland (HOMFOR-A/2003/1).
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Liver Resection-Associated Macrophage Inflammatory Protein-2 Stimulates Engraftment but not Growth of Colorectal Metastasis at Extrahepatic Sites Otto Kollmar,*,1 Bastian Junker,* Kathrin Rupertus,* Claudia Scheuer,† Michael D. Menger,† and Martin K. Schilling* *Department of General, Visceral, Vascular and Pediatric Surgery and †Institute for Clinical and Experimental Surgery, University of Saarland, Homburg/Saar, Germany Submitted for publication January 6, 2007
Background. Previous studies have shown that liver resection enhances intrahepatic engraftment of CXCR-2expressing colorectal cancer cells by the action of the CXC chemokine macrophage inflammatory protein (MIP)-2. Herein we studied how liver resectionassociated MIP-2 affects extrahepatic tumor cell engraftment and whether MIP-2 also stimulates the growth of already established metastases. Materials and methods. Green fluorescent proteintransfected CT26.WT colorectal cancer cells were implanted into dorsal skinfold chambers of syngeneic BALB/c mice. Additionally, all animals underwent a 30% hepatectomy. To study MIP-2 in extrahepatic tumor cell engraftment, animals were treated with an anti-MIP-2 antibody, starting at the day of tumor cell implantation. To study MIP-2 in established metastases, anti-MIP-2 treatment was initiated at day 5 after tumor cell implantation. Hepatectomized animals without neutralization of MIP-2 served as controls. Tumor vascularization and growth as well as tumor cell migration, proliferation, apoptosis, and CXCR-2 expression were studied over 14 days using intravital fluorescence microscopy, histology, and immunohistochemistry. Results. Functional inhibition of MIP-2 significantly delayed extrahepatic tumor cell engraftment but not the growth of established metastases. The initial delay of engraftment was associated with a compensatory stimulation of vascularization and tumor cell migration when compared to controls (P < 0.05). Further, inhibition of tumor cell engraftment by initial anti1
To whom correspondence and reprint requests should be addressed at Department of General, Visceral, Vascular, and Pediatric Surgery, University of Saarland, D-66421 Homburg/Saar, Germany. E-mail: [email protected].
MIP-2 treatment was associated with a significant (P < 0.05) reduction of CXCR-2 expression and tumor cell apoptosis. Conclusion. Our study indicates that MIP-2 is involved in extrahepatic engraftment of CT.26 colorectal cancer cells. The MIP-2/CXCR-2 signaling pathway may be a promising target for early antitumor therapy in patients undergoing liver resection. © 2008 Elsevier Inc. All rights reserved.
Key Words: colorectal cancer; chemokine; MIP-2; tumor growth; metastasis; liver regeneration; hepatectomy; microcirculation angiogenesis. INTRODUCTION
Colorectal cancer is the second leading cause of cancer-related death in the Western world [1]. The benefit of curative resection of colorectal liver metastases with a 5-year overall survival rate of up to 58% is well recognized [1– 4]. For a long time, extrahepatic metastases have been considered a contraindication for hepatectomy. Over the last few years, however, interesting 5-year survival rates of up to 30% have been reported after resection of liver and extrahepatic colorectal metastases in lung, brain, bone, and other sites [5–7]. In line with this, Elias et al. demonstrated that complete resection of all metastases of colorectal cancer is more important for the overall patient survival than the location and the number of the metastases [5]. Besides parenchymal regeneration, hepatectomy also stimulates tumor growth in the remaining liver [8 –10]. The magnitude of stimulation of tumor growth is thought to be proportional to the volume of the resected liver tissue [11–13]. Furthermore, after liver resection about 20% of patients develop metastases in the lung
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and 2 to 5% of patients develop metastases in the brain, bone, and other sites [6, 7]. Clinical and experimental studies have demonstrated that chemokines can regulate angiogenesis, proliferation, and apoptosis of tumor cells and that they mediate tumor cell invasion and trafficking in an organ-specific manner leading to metastases [14 –16]. Previous experimental studies have demonstrated that the CXC chemokine macrophage inflammatory protein (MIP)-2 promotes engraftment of CXCR-2-expressing colorectal liver metastases [17]. Furthermore, blockade of MIP-2 has been shown to significantly inhibit liver resection-induced acceleration of intrahepatic metastatic tumor engraftment [8]. There is, however, no information on whether liver resection-associated MIP-2 also accelerates engraftment of colorectal cancer cells at extrahepatic sites. In addition, the effect of MIP-2 on the growth of established metastases has not been studied thus far. With the use of an established murine extrahepatic colon cancer metastasis model, we therefore analyzed how liver resection-associated MIP-2 affects extrahepatic tumor cell engraftment and whether MIP-2 also stimulates the growth of already established metastases. MATERIALS AND METHODS Tumor Cell Line and Culture Conditions The CT26 cell line is an N-nitroso-N-methylurethane-induced undifferentiated adenocarcinoma of the colon, syngeneic with the BALB/c mouse. For our studies, the CT26.WT cell line (ATCC CRL2638; LGC Promochem GmbH, Wesel, Germany) was grown in cell culture as monolayers in RPMI 1640 medium with 2 mM L-glutamine (Sigma Aldrich Chemie GmbH, Taufkirchen, Germany) supplemented with 10% fetal calf serum (FCS Gold; PAA Laboratories GmbH, Cölbe, Germany), 100 U/mL penicillin, and 100 g/mL streptomycin (PAA Laboratories GmbH). The cells were incubated at 37°C in a humidified atmosphere containing 5% CO 2. With the use of CLONfectin (Clontech, Palo Alto, CA), the cells were transfected with the enhanced green fluorescent protein (GFP) expression vector pEGFP-N1 (Clontech) according to the manufacturer’s instructions [18]. For the individual experiments, only CT26.WT-GFP cells of the first three serial passages after cryo-storage were used. At the day of implantation, CT26.WT-GFP cells were harvested from subconfluent cultures (70 to 85%) by trypsinization (0.05% Trypsin and 0.02% EDTA, PAA Laboratories GmbH) and washed twice in PBS solution.
Animals Experiments were performed after approval by the local governmental ethic committee and conformed to the United Kingdom Coordinating Committee on Cancer Research (UKCCCR) Guidelines for the Welfare of Animals in Experimental Neoplasia (as described in Br J Cancer 1998;77:1) and the Guide for Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, National Research Council; NIH Guide, Vol. 25, No. 28, 1996). Twelve- to 16-week-old female BALB/c mice (Charles River Laboratories GmbH, Sulzfeld, Germany) with a body weight (BW) of 20 –22 g were used. The animals were housed in single cages at a room temperature of 22–24°C and at a relative humidity of 60 – 65% with a 12-h light/ dark-cycle environment. The mice were allowed free access to drinking water and standard laboratory chow (Altromin, Lage, Germany).
Experimental Model For operative procedures, animals were anesthetized by intraperitoneal injection of 90 mg/kg BW ketamine (Ketavet; Parke Davis, Freiburg, Germany) and 20 mg/kg BW xylazine (Rompun; Bayer, Leverkusen, Germany). To allow repetitive analyses of the microvasculature of growing tumors, the dorsal skinfold chamber model was used for intravital microscopy as described previously in detail [19]. Briefly, the chamber consists of two symmetrical titanium frames, which were positioned to sandwich the extended double layer of the dorsal skin. One layer was completely removed in a circular area of 15 mm diameter. The remaining layers, consisting of the epidermis, subcutaneous tissue, and striated skin muscle, were covered with a glass coverslip incorporated into one of the titanium frames [20]. The animals tolerated the chambers well and showed no signs of discomfort or changes in sleeping and feeding habits. After a 48-h recovery period the animals were re-anesthetized. After laparotomy, animals received a 30% hepatectomy of the left lateral liver lobe. For tumor cell implantation, the coverslip of the chamber was temporarily removed and 1 ⫻ 10 5 CT26.WT-GFP cells were implanted onto the surface of the striated muscle tissue within the chamber. Directly after cell implantation, the chamber tissue was covered again with the coverslip [21].
Experimental Protocol A total of 23 animals received a 30% hepatectomy of the left lateral liver lobe and simultaneous tumor cell implantation in the dorsal skinfold chamber. The animals were assigned to three different groups. One group (Phx⫹Abd0; n ⫽ 7) received 1 mg/kg BW of the monoclonal rat anti-mouse MIP-2 antibody (MAB452, R&D Systems, Wiesbaden, Germany) intraperitoneally on the day of hepatectomy and every second day thereafter. The animals of the second group received the same amount of the MIP-2 antibody; however, the treatment was started only at day 5 after hepatectomy (Phx⫹Abd5; n ⫽ 8). Treatment was also continued every second day thereafter. Hepatectomized animals without neutralization of MIP-2 (Phx; n ⫽ 8) served as controls. All animals underwent repetitive intravital microscopic analyses directly as well as 5, 7, 9, 12, and 14 days after tumor cell implantation. At the end of the experiment (day 14), the chamber with the tumor tissue was harvested for histology and immunohistochemistry.
Intravital Fluorescence Microscopy Intravital fluorescence microscopy was performed in epiillumination technique using a modified Zeiss Axio-Tech microscope (Zeiss, Oberkochen, Germany) with a 100-W HBO mercury lamp. Microscopic images were monitored by a charge-coupled device video camera (FK 6990, COHU; Prospective Measurements Inc., San Diego, CA) and were transferred to a video system (VO-5800 PS; Sony, München, Germany) for subsequent off-line analysis. Migration of tumor cells, tumor size, growth kinetics, and neovascularization were analyzed using blue light epi-illumination (450 to 490 nm excitation wavelength and ⬎520 nm emission wavelengths).
Microcirculation Analysis Microcirculatory parameters were assessed off-line by frame-toframe analysis of the videotaped images using a computer-assisted image analysis system (CapImage; Zeintl Software, Heidelberg, Germany). Data analysis was performed by examiners unaware of the treatment. The fluorescent labeling of the tumor cells allowed precise delineation of the tumor from the surrounding unaffected host tissue. It also enabled for distinct identification of individual tumor cells to study tumor cell migration. At each observation time point the surface of the fluorescently labeled tumor mass within the chamber was first scanned for determination of the tumor size (given as tumor
KOLLMAR ET AL.: MIP-2 AND EXTRAHEPATIC TUMOR GROWTH area in mm 2). Next, eight regions-of-interest (ROIs) were randomly chosen within the tumor margin, and the number of migrated tumor cells was counted within each region. In these ROIs the onset of angiogenesis, i.e., development of buds, sprouts, and blood vessels, was documented and scored 0 to 8, with 0 indicating angiogenesis in none of the ROIs and 8 indicating angiogenesis in all of the ROIs. Functional capillary density [cm/cm 2] of the tumor microvasculature served as an indicator of neovascularization. It was defined as the length of RBC perfused capillaries per observation area and was analyzed within the eight ROIs of the tumor margin and within four additional ROIs of the tumor center. Diameters of the newly formed tumor microvessels were measured perpendicularly to the vessel path and are given in micrometers [18]. To study vascular permeability of the newly formed tumor microvessels, the occurrence of petechial bleedings was documented in each ROI and is given as percentage of all ROIs analyzed.
Histology and Immunohistochemistry At the end of the experiments (day 14), the tumor and the adjacent host tissue was harvested. For light microscopy, formalin-fixed biopsies were embedded in paraffin. Sections of 5 m were cut and stained with hematoxylin and eosin for routine histology according to standard procedures. Tumor growth characteristics were analyzed. Tumor cell invasion of the muscle layer was measured and is given in percent of the length of the tumor basis. To study cell proliferation and apoptotic cell death, proliferating cell nuclear antigen (PCNA) and cleaved caspase-3 were stained using indirect immunoperoxidase techniques. Therefore, deparaffinized sections were incubated with 3% H 2O 2 and 2% goat normal serum to block endogenous peroxidases and unspecific binding sites. A monoclonal mouse anti-pan PCNA antibody (PC10, 1:50; DakoCytomation, Hamburg, Germany) and a polyclonal rabbit antimouse cleaved caspase-3 antibody (Asp175, 1:50; Cell Signaling Technology, Frankfurt, Germany) were used as primary antibodies. The cleaved caspase-3 antibody detects endogenous levels only of the short fragment (17/19 kDa) of activated caspase-3, but not full-length caspase-3. Biotinylated goat anti-mouse and goat anti-rabbit immunoglobulin antibodies were used as secondary antibodies for streptavidin-biotin complex peroxidase staining (1:200, LSAB 2 System HRP; DakoCytomation). 3,3=-Diaminobenzidine (DakoCytomation) was used as chromogen. Sections were counterstained with hemalaun according to Mayer and examined by light microscopy. To assess the expression of the chemokine receptor CXCR-2, tumor slices were embedded in tissue freezing medium (Jung; Leica Microsystems, Nussloch, Germany) for immunohistochemistry, snapfrozen in liquid nitrogen, and stored at – 80°C. Cryostat sections of 5 m were cut, fixed in 4°C cold acetone for 5 s, followed by fixation in formalin 4% for 10 min, and blocked with 2% normal donkey serum. Tissue sections were then incubated with the polyclonal rabbit antimouse CXCR-2 antibody (1:10; Santa Cruz, Heidelberg, Germany). A donkey anti-rabbit IgG horseradish peroxidase-conjugated antibody (1: 500; Amersham, Freiburg, Germany) was used as secondary antibody. 3,3=-Diaminobenzidine was used as chromogen. Sections were counterstained with hemalaun according to Mayer and examined by light microscopy. As a negative control, additional slices from each specimen were exposed to appropriate IgG isotype-matched antibody (Sigma Aldrich Chemie GmbH) in place of primary antibody under the same conditions to determine the specificity of antibody binding. All of the control stainings were found to be negative.
Statistical Analysis All values are expressed as means ⫾ SEM. After proving the assumption of normality and homogeneity of variance across groups, differences between groups were calculated by a one-way analysis of variance followed by the appropriate post-hoc comparison, including the correction of the alpha-error according to Bonferroni probabili-
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ties to compensate for multiple comparisons. Overall statistical significance was set at P ⬍ 0.05. Statistical analysis was performed with the use of the software package SigmaStat (SPSS Inc., Chicago, IL).
RESULTS
The general conditions of all animals were not affected by the preparation of the dorsal skinfold chamber, the 30% hepatectomy, and the tumor cell implantation. All animals had an uneventful postoperative recovery and tolerated well the intravital fluorescence microscopic observations during the 14-day period. The take rate of the implanted CT26.WT-GFP cells was 100%. Intravital fluorescence microscopy showed a progressive tumor growth during the 14-day observation period. Quantitative analysis of the tumor area (Fig. 1) showed that only anti-MIP-2 treatment, starting at the day of hepatectomy (Phx⫹ABd0), was capable of significantly decreasing (P ⬍ 0.05) the tumor size during the first 9 days when compared with untreated controls. During the later time points of the 14-day observation period, the continued anti-MIP-2 treatment could not prevent an acceleration of tumor growth, indicating that anti-MIP-2, if given from day 0, delays tumor cell engraftment but does not inhibit further tumor growth. Accordingly, anti-MIP-2 treatment of established extrahepatic tumors, starting at
FIG. 1. Stereo-microscopic photomicrographs of representative tumors in the dorsal skinfold chamber at day 14 after hepatectomy without treatment (A; Phx), with anti-MIP-2 treatment starting at day 5 (B; Phx⫹ABd5), and with anti-MIP-2 treatment starting at day 0 (C; Phx⫹ABd0). Quantitative analysis of the tumor area (D) showed a significantly reduced tumor growth during the initial 9 days after tumor implantation in animals in which anti-MIP-2 was started at day 0 (black squares) when compared with controls (white circles), indicating a delay in tumor engraftment. Further continuous treatment with anti-MIP-2 could not prevent later accelerated tumor growth. Accordingly, anti-MIP-2 treatment starting at day 5 after hepatectomy (gray triangles) was not effective to inhibit tumor growth. Mean ⫾ SEM; *P ⬍ 0.05 versus Control; #P ⬍ 0.05 versus Phx⫹ABd5. Magnification (A to C), ⫻4.
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FIG. 2. Intravital fluorescence microscopy of representative tumors in dorsal skinfold chambers at day 14 after hepatectomy without treatment (A; Phx), with anti-MIP-2 treatment starting at day 5 (B; Phx⫹ABd5), and with anti-MIP-2 treatment starting at day 0 (C; Phx⫹ABd0). Quantitative analysis of angiogenesis, i.e., protrusion of capillary buds and sprouts, showed that at day 12 new vessel formations could be observed in almost all ROIs without significant differences between the three groups studied (D). However, analysis of the functional capillary density revealed a significantly increased neovascularization during the late observation period from day 9 to 14 in tumors with anti-MIP-2 treatment starting at day 0 but not day 5 when compared with solely hepatectomized controls (E). The diameters of the tumor capillaries (F) ranged between 9 and 13 m during the 14-day observation period. This involved a slight increase in controls (white circles) and Phx⫹ABd0 animals (black squares), which was not observed in Phx⫹ABd5 animals (gray triangles). Mean ⫾ SEM; *P ⬍ 0.05 versus Control; #P ⬍ 0.05 versus Phx⫹ABd5; §P ⬍ 0.05 versus Phx⫹Abd0. Magnification (A to C), ⫻40.
day 5 after hepatectomy and tumor cell implantation (Phx⫹ABd5), did not affect tumor growth (Fig. 1). Intravital fluorescence microscopy revealed that the angiogenic response, i.e., the protrusion of buds and sprouts, was initiated at day 5. At day 12, almost all ROIs studied within the tumors exhibited bud and sprout formation (Fig. 2). Anti-MIP-2 treatment, irrespective whether it was started at day 0 or day 5, did not affect the onset of the angiogenic response (Fig. 2). Analysis of the functional neovascularization of the extrahepatic metastases revealed a continuous increase of the functional density of tumor capillaries from day 5 to day 14, reaching values of ⬃250 cm/cm 2. Of interest, anti-MIP-2 treatment starting at day 0 but not at day 5 induced a significant increase (P ⬍ 0.05) of
functional capillary density during the late phase of the 14-day observation period. This was observed in both the tumor margin (Fig. 2) and the tumor center (data not shown). Diameters of tumor capillaries ranged between 9 and 13 m during the 14-day observation period. This involved a slight increase in controls (Phx) and Phx⫹ ABd0 animals, which was not observed in Phx⫹ABd5 animals (Fig. 2). Analysis of capillary diameters in the tumor center did not differ from that obtained within the tumor margin (data not shown). The increased microvascular permeability observed during angiogenesis is known to be associated with petechial bleedings. In animals treated with antiMIP-2 starting at day 0 but not at day 5, pronounced bleedings during the initial period of angiogenesis could be observed, which, however, did not prove to be significantly increased more than that of controls. This is probably due to the heterogeneous response, as indicated by the relatively high SEM values (Fig. 3). There was no difference in petechial bleedings between tumor margin (Fig. 3) and center (data not shown) within the individual groups. The tumor mass as well as the individual CT26.WTGFP cells could easily be distinguished by intravital fluorescence microscopy due to their green fluorescent labeling (Fig. 4). Of interest, migration of the tumor cells at the tumor margin could be observed during the entire 14-day observation period (Fig. 4). Migrating tumor cells showed a spindle-shaped configuration, whereas the cells within the tumor mass presented with
FIG. 3. Intravital fluorescence microscopy of petechial bleedings (arrows) in tumors at day 5 after hepatectomy and tumor cell implantation without treatment (A; Phx), with anti-MIP-2 treatment starting at day 5 (B; Phx⫹ABd5), and with anti-MIP-2 treatment starting at day 0 (C; Phx⫹ABd0). Quantitative analysis of the number of ROIs with bleedings (D, in percent) showed that in animals treated with anti-MIP-2 starting at day 0 but not at day 5 markedly more bleedings could be observed during the initial period of angiogenesis. Mean ⫾ SEM. Magnification (A to C), ⫻16.
KOLLMAR ET AL.: MIP-2 AND EXTRAHEPATIC TUMOR GROWTH
FIG. 4. Intravital fluorescence microscopy of tumor cell migration out of the tumor margin. Whereas no tumor cells out of the tumor margin could be found directly after implantation (A), cell migration could be detected at day 5 after implantation in nontreated controls (B). Anti-MIP-2 treatment starting at day 0 induced an increase of cell migration (C). Quantitative analysis (D) confirmed the increased tumor cell migration with anti-MIP-2 treatment starting at day 0 (black squares) when compared with that observed in animals in which the treatment was started at day 5 (gray triangles), and in solely hepatectomized control animals (white circles). Mean ⫾ SEM; *P ⬍ 0.05 versus Control; #P ⬍ 0.05 versus Phx⫹ABd5. Magnification (A to C), ⫻40.
a more rounded structure. The migrating cells had a mean distance of 290 to 420 m to the tumor margin, which did not differ between anti-MIP-2-treated animals and controls. However, anti-MIP-2 treatment starting at day 0 showed a significantly increased (P ⬍ 0.05) number of migrating tumor cells throughout the whole 14-day observation period when compared with both controls (PHx) and PHx⫹ABd5 animals (Fig. 4). Hematoxylin-eosin staining demonstrated tumor cell infiltration into the neighboring muscle tissue over 35.7 ⫾ 15.9% of the tumor basis in hepatectomized control animals (Phx). Additional treatment with antiMIP-2 did not influence this tumor cell invasiveness, as indicated by an infiltration of 38.5 ⫾ 14.4% (Phx⫹ ABd0) and 42.7 ⫾ 11.7% (Phx⫹ABd5). PCNA as an indicator of cell proliferation displayed strong staining of tumor cells in controls (Phx) at day 14 after liver resection. Overall, more than 80% of the tumor cells were found PCNA positive (Fig. 5). Notably, anti-MIP-2 treatment decreased the fraction of positively stained tumor cells at day 14 after hepatectomy to ⬃40% if the treatment was started at day 5, but only to ⬃60% if the treatment was started at day 0. Although this difference did not prove to be statistically significant, these data indicate a tendency toward a suppressed tumor cell proliferation by anti-MIP-2 treatment. Caspase-3 immunohistochemistry was performed to document apoptotic cell death. Surprisingly, caspase-
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3-positive cells could only rarely be observed in all three groups of hepatectomized animals, never exceeding the value of 0.5% (Fig. 5). Nonetheless, anti-MIP-2 treatment significantly reduced (P ⬍ 0.05) the number of apoptotic tumor cells when compared with that of hepatectomized controls (Phx). In fact, apoptotic cell death was reduced by 60% in Phx⫹ABd5 animals and even by 85% in Phx⫹ABd0 animals (Fig. 5). CXCR-2 immunohistochemistry revealed a marked staining of receptors on tumor cells in hepatectomized controls (Fig. 6). Quantitative analysis of CXCR-2 expression in anti-MIP-2-treated animals showed a significant decrease (P ⬍ 0.05) of the number of positively stained cells, regardless whether the treatment was started at day 0 or day 5. These findings did not differ between tumor margin (Fig. 6) and tumor center (data not shown).
FIG. 5. PCNA (A and B) and cleaved caspase-3 (C and D) immunohistochemistry of tumors at day 14 after implantation of solely hepatectomized controls (A and C) and of tumors of animals with anti-MIP-2 treatment starting at day 5 (B) and day 0 (D), respectively. Quantitative analysis of the number of PCNA-positive cells (given in percent of all cells visible) indicated a slight decrease of cell proliferation after anti-MIP-2 treatment, in particular, if started at day 5, when compared with that of controls (Phx) (E). Analysis of cleaved caspase-3 expression demonstrated a low rate of apoptotic cell death in the solely hepatectomized controls (F). Cleaved caspase-3 expression in the anti-MIP-2-treated animals was even significantly lower than that of the controls (F). Mean ⫾ SEM; ⴱP ⬍ 0.05 versus Phx. Magnification (A-D), ⫻175. (Color version of figure is available online.)
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FIG. 6. Immunohistochemistry of CXCR-2 expression of tumor cells at day 14 after tumor cell implantation in solely hepatectomized controls (A) and animals with anti-MIP-2 treatment starting at day 5 (B) or day 0 (C). Quantitative analysis (D) revealed a ⬎80% CXCR-2 expression in controls (Phx), which was significantly reduced in tumors with anti-MIP-2 treatment starting at day 5 (Phx⫹ABd5) or day 0 (Phx⫹ABd0). Mean ⫾ SEM; *P ⬍ 0.05 versus Phx. Magnification (A-C), ⫻175. (Color version of figure is available online.)
DISCUSSION
The major finding of the present study is that liver resection-associated engraftment of colorectal cancer cells at extrahepatic sites is mediated by MIP-2, whereas this chemokine does not contribute to the growth of already established extrahepatic metastases. Of interest, neutralization of MIP-2 starting at the day of tumor cell implantation (day 0) provokes a compensatory angiogenic response and a reduction of apoptotic tumor cell death during the later phase of metastatic growth. Liver resection initiates rapid regeneration and growth of the remaining liver to restore the functional capacity. The MIP-2/CXCR-2 system is thought to be involved in the hepatic regenerative process [8, 22, 23]. Ren et al. demonstrated that blockade of the chemokine receptor CXCR-2 inhibits regeneration of the residual liver, while selective neutralization of MIP-2 through anti-MIP-2 antibodies is not affecting the progress of regeneration as indicated by unchanged liver weight to body weight ratios [22]. After liver resection of colorectal metastases, the disease recurs in over two-thirds of the patients [4]. This observation indicates that undetectable residual microscopic tumors remain in the majority of patients undergoing curative hepatectomy [10]. In fact, a variety of studies have shown that liver resection enhances tumor growth during regeneration also at extrahepatic sites [24]. In a previous study, we have already shown that in
the remnant liver MIP-2 significantly promotes liver resection-induced acceleration of metastatic tumor growth [8]. The role of MIP-2 on hepatectomy-induced acceleration of metastatic growth at extrahepatic sites has not been elucidated yet. Herein, we now demonstrate for the first time that neutralization of MIP-2 significantly delays engraftment of colorectal cancer cells at extrahepatic sites, as indicated by a reduced tumor outgrowth during the early phase after hepatectomy. Of interest, MIP-2 neutralization could not prevent tumor growth during the later phase after hepatectomy. In contrast, growth rate, cell migration, and neovascularization were found increased when compared with solely hepatectomized controls, indicating a compensatory stimulation to counteract the delay of engraftment during the early phase of tumor outgrowth. Neutralization of MIP-2 starting at day 0 after hepatectomy did not affect PCNA expression of tumor cells. Thus, the compensatory stimulation of growth during the late observation phase after initial delay of engraftment by anti-MIP-2 is not mediated by an increase of tumor cell proliferation. More probably, it is induced by a reduction of apoptotic cell death. This is indicated by the 85% reduction of cleaved caspase-3 expression after 14 days of anti-MIP-2 treatment when compared to solely hepatectomized controls. We additionally studied the effect of anti-MIP-2 treatment starting at day 5 after hepatectomy and tumor cell implantation to elucidate the role of MIP-2 in stimulating growth of already established metastases. The present data show that endogenously produced MIP-2 does not affect the growth of already established extrahepatic metastases. This lack of effect is observed, although there was a reduction of apoptotic cell death by ⬃60% compared to solely hepatectomized controls. However, this reduction of caspase-3 expression was associated with an almost ⬃50% reduction of PCNA expression, indicating that the reduction of apoptosis is almost completely outweighed by the reduction of cell proliferation. The expression of the CXCR-2 receptor is thought to be required for the proliferative action of chemokines on tumor cells [17, 25, 26]. The fact that in the present study anti-MIP-2 treatment reduced CXCR-2 receptor expression further supports our interpretation that the compensatory increase of growth rate after initial delay of tumor cell engraftment is not due to increased proliferation but rather to the reduction of apoptotic cell death. Chemokines are known to be potent mediators in regulating migration of tumor cells. Our previous in vitro studies showed a dose-dependent increase of migration of tumor cells in response to MIP-2, indicating a MIP-2-associated enhancement of metastatic tumor growth [17]. Surprisingly, in the present study
KOLLMAR ET AL.: MIP-2 AND EXTRAHEPATIC TUMOR GROWTH
neutralization of MIP-2 starting at day 0 stimulated the migration of tumor cells. This may be due to the fact that the compensatory stimulation of tumor growth due to the delay of tumor cell engraftment may be associated with an increased susceptibility of the tumor cells to other chemokines and chemoattractant factors released after hepatectomy and liver regeneration [27, 28]. Of interest, anti-MIP-2 treatment starting only at day 5 inhibited tumor cell migration. Because this treatment did not result in a delay of tumor cell engraftment and thus compensatory stimulation of growth, neutralization of MIP-2 itself is probably responsible for the reduction of cell migration in vivo, similar to previous experiments in vitro [17]. CXC chemokines are thought to be involved in angiogenesis. In inflammation, Scapini et al. demonstrated that MIP-2 induces angiogenesis in vivo via expression of vascular endothelial growth factor (VEGF)-A [29]. In a previous study we showed that exogenous application of MIP-2 accelerates engraftment of hepatic colon cancer metastasis by induction of tumor vessels formation [17]. Furthermore, we have demonstrated that inhibition of endogenous MIP-2 during the early phase of intrahepatic tumor cell engraftment significantly reduces liver resection-associated neovascularization and tumor development [8]. Interestingly, neutralization of MIP-2 starting at day 0 also showed a minor reduction of angiogenesis during the early phase of delay of tumor engraftment. However, the compensatory growth during the later phase was associated with an increased neovascularization, as indicated by a significantly increased capillary density. This compensatory increase of neovascularization is most probably mediated by growth factors that affect the permeability of the microvessels such as VEGF [30-32], because the increase in neovascularization in animals treated with antiMIP-2 from day 0 on was preceded by an increase of petechial bleedings. In conclusion, we demonstrate that liver resectionassociated MIP-2 is involved in engraftment of CT.26 colorectal cancer cells at extrahepatic sites, but does not substantially contribute to the growth of already established tumors. The MIP-2/CXCR-2 signaling pathway may thus not be a promising target for the treatment of established metastases, but for the prevention of tumor cell engraftment in patients undergoing liver resection for tumor therapy.
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ACKNOWLEDGMENTS
We appreciate the excellent technical assistance of Christina Marx and Janine Becker. This study was supported by grants of the Research Committee and the Medical Faculty of the University of Saarland (HOMFOR-A/2003/1).
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