Annals of Surgical Oncology Sign the Guestbook
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
10.1245/ASO.2006.03.096
Annals of Surgical Oncology 13:263-275 (2006)
© 2006 Society of Surgical Oncology
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kollmar, O.
Right arrow Articles by Schilling, M. K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kollmar, O.
Right arrow Articles by Schilling, M. K.

Original Article

Macrophage Inflammatory Protein-2 Promotes Angiogenesis, Cell Migration, and Tumor Growth in Hepatic Metastasis

Otto Kollmar, MD1, Claudia Scheuer, MD2, Michael D. Menger, MD2 and Martin K. Schilling, MD1

1 Department of General, Visceral, Vascular and Pediatric Surgery, University of Saarland, D-66421 Homburg-Saar, Germany
2 Institute for Clinical & Experimental Surgery, University of Saarland, D-66421 Homburg-Saar, Germany

Correspondence: Address correspondence and reprint requests to: Otto Kollmar, MD; E-mail: chokol{at}uniklinik-saarland.de.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: In a mouse model of hepatic metastasis, we herein analyzed whether the CXC chemokine macrophage inflammatory protein (MIP)-2, a functional analogue of the human interleukin 8, stimulates tumor cell migration in vitro and angiogenesis and tumor growth in vivo.

Methods: By using chemotaxis chambers, CT26.WT colorectal tumor cell adhesion and migration were studied under stimulation with different concentrations of MIP-2. To evaluate angiogenesis and tumor growth in vivo, 1 x 105 CT26.WT cells were implanted into the left liver lobe of syngeneic BALB/c mice, and 10, 100, and 1000 nM of MIP-2 or phosphate-buffered saline (controls) was injected into the peritumoral area. After 7 days, angiogenesis, proliferation, tumor growth, apoptosis, cleaved caspase 3, and CXCR-2 expression were analyzed by using intravital fluorescence microscopy, histology, immunohistochemistry, and fluorescence-activated cell sorting.

Results: In vitro, 98.8% of unstimulated CT26.WT cells showed CXCR-2 receptor expression. In the chemotaxis assays, MIP-2 provoked a dose-dependent increase of cell migration and a most pronounced cell adhesion at a dose of 100 nM. In vivo, MIP-2, in particular in a dose of 100 or 1000 nM, induced a significant increase of tumor capillary density and a marked widening of the angiogenic front at the tumor margin. Capillaries of the angiogenic front, but not of the tumor center, showed significant dilation, thus indicating a pronounced action of vascular endothelial growth factor. Tumor volume was significantly increased, in particular after 100 nM of MIP-2 stimulation, when compared with phosphate-buffered saline–treated controls, whereas only 1000 nM of MIP-2–treated animals additionally showed a higher frequency of apoptotic cell death within the tumor margin.

Conclusions: Our study indicates for the first time that the CXC chemokine MIP-2 promotes angiogenesis and growth of colorectal CT26.WT hepatic metastasis.

Key Words: Chemokines • Chemotaxis • Cell migration • Tumor growth • Angiogenesis • Microcirculation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The human chemokine interleukin (IL)-8 has been shown to act as potent neutrophil chemoattractant that contributes to healing processes but also to the manifestation of inflammatory diseases, such as sepsis, inflammatory bowel disease, rheumatoid arthritis, and cerebral and myocardial infarction.1 IL-8 is also thought to be involved in tumor growth and metastasis. Recent studies have indicated that constitutive levels of IL-8 expression by individual tumor cell lines in vitro strongly correlate with in vivo growth and metastatic potential.2,3 IL-8 may stimulate tumor cell motility and migration4,5 and has been shown to act as an autocrine growth factor for tumor cell proliferation.6 In addition, the progression of tumor growth by IL-8 not only may be due to a direct action on tumor cells, but also has been proposed to involve the induction of angiogenesis.7 This view is based on studies that demonstrated a significant correlation between tumor microvessel density and IL-8 levels8 and an inhibition of tumor angiogenesis by neutralization of IL-8.9–11

The biological relevance of IL-8 expression and its role in tumor progression and metastasis, however, is still controversial.2,4 There are metastatic tumor cell lines that do not produce IL-8,12 and others have indicated that there is a lack of correlation between the level of IL-8 expression and tumorigenic and metastatic potential.4 Although in epithelial ovarian cancer, vascular endothelial growth factor (VEGF) and IL-8 expression have been considered to indicate a poor prognosis,13 other studies have shown that IL-8 production by ovarian cancer cells significantly reduces tumor growth.14 Accordingly, in colorectal cancer, serum levels of IL-8 have been shown to be significantly higher in patients with liver metastasis than in those without metastasis,2,15 but it has also been demonstrated that IL-8 is capable of producing an antitumoral effect that is associated with an enhanced infiltration of T lymphocytes.16

The murine CXC chemokine macrophage inflammatory protein (MIP)-2 is a functional analogue of human IL-8.17–23 In general, macrophages, as well as granulocytic and monocytic leukocytes, endothelial cells, and fibroblasts, are capable of producing MIP-2 upon stimulation.24–26 However, MIP-2 is also expressed by a variety of different tumors, including colorectal carcinoma.27 Similar to IL-8, MIP-2 also acts as a potent neutrophil chemoattractant, contributes to wound healing,28 and mediates inflammatory injury.29 Little is known, however, about whether MIP-2 possesses angiogenic properties and whether it is able to induce tumor growth and metastasis. With the use of a murine colon cancer liver metastasis model, we therefore studied the potential of MIP-2 to promote angiogenesis, cell migration, and tumor growth in hepatic metastasis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Experiments were performed after approval by the local governmental ethic committee and were in accordance with the United Kingdom Coordinating Committee for Cancer Research Guidelines for the Welfare of Animals in Experimental Neoplasia and the Interdisciplinary Principles and Guidelines for the Use of Animals in Research (New York Academy of Sciences Ad Hoc Committee on Animal Research).

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 (American Type Culture Collection CRL-2638; LGC Promochem GmbH, Wesel, Germany) was grown in cell culture as monolayers in RPMI-1640 medium with 2 mM of L-glutamine (Sigma Aldrich Chemie GmbH, Taufkirchen, Germany) supplemented with 10% fetal calf serum (FCS Gold; PAA Laboratories GmbH, Cölbe, Germany), 100 U/mL of penicillin, and 100 µg/mL of streptomycin (PAA Laboratories GmbH). The cells were incubated at 37°C in a humidified atmosphere containing 5% carbon dioxide in air. Only cells of the first two serial passages after cryostorage were used. At the day of implantation, CT26.WT cells were harvested from subconfluent cultures (70%–85%) by trypsinization (.05% trypsin and .02% ethylenediaminetetraacetic acid; PAA Laboratories GmbH) and washed twice in phosphate-buffered saline solution (PBS; PAA Laboratories GmbH) before they were resuspended in PBS at 1 x 105 cells per 10 µL.

Flow Cytometric Analysis of CT26.WT Cells
FACScan (Becton Dickinson, Mountain View, CA) analysis was performed to assess the expression of the chemokine receptor CXCR-2 of the CT26.WT cells. After trypsinization, the cells were fixed in 1 mL of Cytofix/Cytoperm (BD Biosciences, Heidelberg, Germany) for 20 minutes at 4°C, washed twice with Perm Wash (BD Biosciences), and incubated at room temperature for 40 minutes with a polyclonal rabbit anti-mouse CXCR-2 antibody (Santa Cruz, Heidelberg, Germany) or an isotype-matched control antibody (Dianova, Hamburg, Germany). A goat anti-rabbit Cy3-conjugated antibody (1/25; Dianova) was used for fluorescence labeling. To remove excess of antibody, cells were washed again and were then maintained in 1% paraformaldehyde in PBS. Flow cytometry was performed within the next 3 hours. The FACScan flow cytometer was calibrated with fluorescent standard microbeads (CaliBRITE Beads; BD Biosciences) for accurate instrument setting. Tumor cells were selectively analyzed for their fluorescence properties by using the CellQuest data-handling program (BD Biosciences) with assessment of 5000 events per sample.

Cell Migration Assay
The migration capability of CT26.WT cells was assessed by using 24-well chemotaxis chambers and polyvinylpyrrolidone-coated polycarbonate filters with an 8-µm pore size (BD Falcon, Heidelberg, Germany). Chemotaxis assays were performed in duplicate. The chemoattractant MIP-2 (R&D Systems, Wiesbaden, Germany), diluted in PBS with .1% bovine serum albumin (Sigma Aldrich Chemie GmbH), was added in concentrations of .1, 1, 10, 100, 200, and 400 nM to 700 µL of RPMI-1640 medium in the lower wells. PBS with .1% bovine serum albumin alone served as a control. A total of 500 µL of a cell suspension containing 1 x 105 cells in RPMI-1640 was added to each of the upper wells, and the chamber was then incubated for 24 hours at 37° C in a humidified atmosphere with 5% carbon dioxide. After incubation, the nonmigrated cells were removed from the upper surface of the filters, and the migrated cells adherent to the lower surface were fixed with methanol and stained with Dade Diff-Quick (Dade Diagnostika GmbH, Munich, Germany). The number of adherent cells was counted in 10 high-power microscopic fields. In addition, the nonadherent cells that had migrated into the lower well were collected and counted by FACScan flow cytometry. Adherent cells are given as number of cells per 10 high-power fields, and migrated cells are given as the number of cells per well.

Operative Procedure
Eight- to 12-week-old female BALB/c mice with a body weight of 18 to 20 g, kept in a temperature- and humidity-controlled 12-hour light/dark cycle environment, were used. The animals were anesthetized by intraperitoneal injection of 25 mg/kg of body weight xylazine hydrochloride (Rompun; Bayer, Leverkusen, Germany) and 125 mg/kg of body weight ketamine hydrochloride (Ketavet; Pharmacia GmbH, Erlangen, Germany). The operative procedure was performed as described previously.30 Briefly, for intrahepatic implantation of CT26.WT cells, the anesthetized animals were placed in the supine position. After laparotomy through a midline incision, the left liver lobe was gently mobilized, and 1 x 105 tumor cells in 10 µL of PBS were implanted under the capsule of the lower surface by using a 25-µL syringe with a 32-gauge needle (Gastight 1702; Hamilton Bonaduz AG, Bonaduz, Switzerland). MIP-2 was applied in a volume of 10 µL locally under the subcapsular space with a distance of 2 to 5 mm close to the tumor cell implants. The puncture site was sealed with acrylic glue (Histoacryl; B. Braun, Aesculap AG, Tuttlingen, Germany), and the left liver lobe was repositioned anatomically into the peritoneal cavity. The abdominal wall was closed in a one-layer technique with a polypropylene suture (Prolene 5–0; Ethicon, Norderstedt, Germany).

Intravital Fluorescence Microscopy
At day 7 after tumor cell implantation, the animals were again anesthetized and relaparotomized. The left liver lobe with the tumor was exteriorized and placed on an adjustable stage, so that the lower surface of the lobe was positioned horizontal to the microscope. This guaranteed adequate focus levels for the in vivo fluorescence microscopy procedure. The surface of the lobe was covered by a coverslip to avoid drying of the tissue and influences of the ambient oxygen.30

In vivo fluorescence microscopy was performed with an epi-illumination technique by using a modified Zeiss Axio-Tech microscope (Zeiss, Oberkochen, Germany).31 Microscopic images were monitored by a charge-coupled device video camera (FK 6990; Prospective Measurements Inc., San Diego, CA) and were transferred to a video system (VO-5800 PS; Sony, Munich, Germany) for subsequent offline analysis. Angioarchitecture and microvascular perfusion were analyzed after intravenous contrast enhancement with sodium fluorescein (2 µmol/kg; Merck, Darmstadt, Germany) by using blue light epi-illumination (excitation and emission wavelengths were 450–490 and >520 nm, respectively). Leukocyte adhesion within the tumor microvasculature was assessed after in vivo white blood cell staining with rhodamine 6G (2 µmol/kg intravenously; Merck) by using green light epi-illumination (excitation and emission wavelengths were 530–560 and >580 nm, respectively).32 Parenchymal cells were visualized by in vivo nuclear staining with bisbenzimide (H33342; 2 µmol/kg; Sigma Aldrich Chemie GmbH) by using a near-UV filter system (excitation and emission wavelengths were 330–380 and >415 nm, respectively).32

Microcirculation Analysis
Assessment of hepatic microcirculatory parameters was performed offline by frame-to-frame analysis of the videotaped images by using a computer-assisted image-analysis system (CapImage; Zeintl, Heidelberg, Germany). Data analysis was performed by examiners unaware of the treatment. Sinusoidal, capillary, and venular diameters (micrometers) were measured perpendicularly to the vessel path. Capillary density (centimeters per square centimeter) was determined within the tumors by measuring the length of red blood cell–perfused capillaries per area of observation. The number of venules draining the blood flow from the tumor was counted as number per tumor margin. The angiogenic front neighboring the tumor margin was characterized by newly developed, chaotically arranged capillaries and was measured in square millimeters. The numbers of leukocytes that adhered to the endothelial lining of newly formed tumor capillaries are given as cells per microscopic field. The number of apoptotic cells was determined by counting the number of cells with condensation of bisbenzimide-stained nuclei per square millimeter of surface area of the tumor margin.32

Morphological Examinations
At the end of the experiment, the left liver lobe was harvested and cut into 1-mm-thick slices by using a tissue slicer (McIlwain Tissue Shopper; Saur Laborbedarf, Reutlingen, Germany). Slices were documented by a video stereomicroscope (Leica M651; Leica Microsystems AG, Heerbrugg, Switzerland), and the size of the tumor was measured according to the equation


Formula

where a, b, and c represent the three perpendicularly oriented diameters of the tumor.

Histochemistry and Immunohistochemistry
For light microscopy, formalin-fixed biopsy samples were embedded in paraffin. Sections of 5 µm were cut and stained with hematoxylin and eosin for routine histological examination according to standard procedures.

Proliferating cell nuclear antigen (PCNA) and cleaved caspase 3 were stained by using indirect immunoperoxidase techniques. Therefore, deparaffinized sections were incubated with 3% H2O2 to block endogenous peroxidases and with 2% goat normal serum for blocking unspecific binding sites. A monoclonal mouse anti–pan-PCNA antibody (PC10; 1/50; DakoCytomation, Hamburg, Germany) and a polyclonal rabbit anti-mouse 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 and not of the full-length caspase 3. A biotinylated goat anti-mouse-rabbit immunoglobulin antibody was used as secondary antibody for streptavidin-biotin complex peroxidase staining (1/200; LSAB 2 System HRP; DakoCytomation), and 3,3'-diaminobenzidine (DakoCytomation) was used as a chromogen. Sections were counterstained with hemalaun according to Mayer and examined by light microscopy.

For CXCR-2 immunohistochemistry, tumor slices were embedded in tissue-freezing medium (Jung; Leica Microsystems, Nussloch, Germany), snap-frozen in liquid nitrogen, and stored at –80°C. Cryostat sections of 5 µm were cut, fixed in 4°C cold acetone for 5 seconds followed by fixation in formalin 4% for 10 minutes, and blocked with 2% normal donkey serum. Tissue sections were then incubated with the polyclonal rabbit anti-mouse CXCR-2 antibody (1/100; Santa Cruz). A donkey anti-rabbit immunoglobulin G horseradish peroxidase–conjugated antibody (1/500; Amersham, Freiburg, Germany) was used as a secondary antibody, and 3,3'-diaminobenzidine was used as the 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 immunoglobulin G 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 negative.

Experimental Protocol
A total of 24 animals underwent tumor cell implantation and were assigned to four different groups: MIP-2 in 10 µL of PBS was given in a dose of 10 nM (n = 6), 100 nM (n = 6), and 1000 nM (n = 6), and animals that received PBS alone served as controls (n = 6). Seven days after tumor cell implantation and treatment, all animals underwent intravital microscopic examination, and the liver and tumor tissues were harvested for histological and immunohistochemical analysis.

Statistical Analysis
All values are expressed as means ± SEM. After the assumption of normality and homogeneity of variance across groups was proven, differences between groups were calculated by a one-way analysis of variance followed by a post hoc Student-Newman-Keuls test, which included correction of the {alpha} error to compensate for multiple comparisons. The overall statistical significance was set at P < .05. Statistical analysis was performed with the use of the software package SigmaStat (SPSS Inc., Chicago, IL).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
In Vitro FACScan Analysis and Migration Assay
FACScan analysis showed that 98.8% of the CT26.WT cells were CXCR-2 receptor positive (Fig. 1Go). The adhesion and migration assay indicated that only a few cells adhered and migrated under control conditions, i.e., with PBS stimulation (Fig. 2A and CGo). MIP-2 at a dose of .1 nM induced a 4-fold increase of cell adhesion at the polyvinylpyrrolidone-coated polycarbonate filters (Fig. 2CGo). With 10 and 100 nM of MIP-2, adhesion was most pronounced, as indicated by an almost 6-fold increase compared with controls (Fig. 2B and CGo). It is interesting to note that further increases of the MIP-2 concentration (200 and 400 nM) markedly attenuated the chemokine-induced adhesive response (Fig. 2CGo). Analysis of the number of cells that had migrated through the filter to the lower well indicated a 2- to 4-fold increase after low-dose MIP-2 stimulation (.1 – 100 nM), whereas challenge with higher doses (200 and 400 nM) resulted in an exponential increase (9- and 18-fold) when compared with PBS controls (Fig. 2DGo).


Figure 1
View larger version (39K):
[in this window]
[in a new window]
  		FIG. 1. FACScan analysis of CT26. WT cells, demonstrating positive staining and, thus, expression of the chemokine receptor CXCR-2 by almost all of the cells studied.

 

Figure 2
View larger version (22K):
[in this window]
[in a new window]
  		FIG. 2. Cell adherence and migration assay consisting of a chemotaxis chamber and polyvinylpyrrolidone-coated polycarbonate filters with an 8-µm pore size. Note that only a few cells adhere and migrate under control conditions (A), whereas stimulation with 100 nM of macrophage inflammatory protein (MIP)-2 exerts a marked increase in the fraction of adhering and migrating cells (B). The dose-response curves confirm the stimulation of both adherence (C) and migration (D) after MIP-2 challenge.

 
Tumor Cell Implantation and Tumor Growth
Tumor cell implantation and MIP-2 treatment did not affect the general condition of the animals. All animals had an uneventful postoperative recovery, and, regardless of the group, animals did not show a significant change of body weight during the 7-day experimental period. MIP-2 at a 10 nM concentration significantly (P < .001) increased the tumor volume by 14-fold when compared with that of PBS-treated controls (Fig. 3Go). Although 100 nM of MIP-2 further increased tumor growth to a volume that was 27-fold of that of controls, treatment with 1000 nM of MIP-2 was less effective and resulted in tumors comparable in size to those of animals treated with 10 nM of MIP-2 (Fig. 3Go).


Figure 3
View larger version (23K):
[in this window]
[in a new window]
  		FIG. 3. Analysis of tumor volume at day 7 after tumor cell implantation and local application of phosphate-buffered saline (control) or different doses of macrophage inflammatory protein (MIP)-2. Note that 100 nM of MIP-2 exerts the most pronounced increase in tumor growth compared with control. Data are mean ± SEM. *P < .05 versus control; #P < .05 vs. 10 nM of MIP-2; §P < .05 vs. 100 nM of MIP-2.

 
Angiogenesis and Microangioarchitecture
Intravital fluorescence microscopy revealed that normal liver presents with a regularly arranged architecture of sinusoids and homogeneous staining of parenchymal cell nuclei (Fig. 4A and BGo). In contrast, the center of the tumor showed a density of capillaries that was markedly lower than that of the sinusoids in the normal liver (Fig. 4CGo). Of interest, nuclei of tumor cells did not stain positive with bisbenzimide (Fig. 4DGo). The margin of the tumor showed an angiogenic front with newly developed, chaotically arranged capillaries (Fig. 4EGo) and large draining venules (Fig. 4GGo). The density of the newly formed capillaries within the angiogenic front (Fig. 4EGo) was markedly higher when compared with that of the tumor center (Fig. 4CGo) and was almost comparable to that of the sinusoids in the normal liver (Fig. 4AGo). Beside the center of the tumors, the angiogenic front also partly lacked in staining of tumor and parenchymal cell nuclei with bisbenzimide (Fig. 4FGo). In addition, the angiogenic front was functionally characterized in that the small molecule sodium fluorescein did not extravasate into tissue. This resulted in a dark appearance of the interface that separated the tumor from the adjacent normal liver (Fig. 5A and BGo).


Figure 4
View larger version (124K):
[in this window]
[in a new window]
  		FIG. 4. Intravital fluorescence microscopy of normal liver (A and B), tumor center (C and D), and tumor margin (E–H). Contrast enhancement with sodium fluorescein (A, C, E, and G) displays the microvascular architecture, whereas bisbenzimide (B, D, F, and H) visualizes the nuclei of parenchymal cells. Normal liver presents with an adequate architecture of sinusoids (A) and a regular staining of nuclei within the parenchymal cell cords (A). The tumor center shows a density of capillaries (C) that is significantly lower than that of the sinusoids of the normal liver (A) and lacks staining of tumor cell nuclei (D). The margin of the tumor shows an angiogenic front (E and F) with newly developed and chaotically arranged capillaries (E; arrows) as well as large draining venules (G; arrows). Beside the tumors (asterisks), the angiogenic front also partly lacks in staining of cell nuclei (F; double asterisk). Note that the draining venules are located exactly within the interface between the tumor and normal liver tissue (H). It is interesting to note that cells adjacent to the angiogenic front occasionally display condensed nuclei according to apoptotic cell death (F; arrows). Original magnification, x80.

 

Figure 5
View larger version (84K):
[in this window]
[in a new window]
  		FIG. 5. Analysis of the size of the angiogenic front at the margin of tumors locally treated with phosphate-buffered saline (PBS; control) or different doses of macrophage inflammatory protein (MIP)-2 (10, 100, and 1000 nM). The tumors (A and B; asterisks) are surrounded by an angiogenic front, which appears as a dark rim of tissue, in which the newly developed microvasculature does not allow extravasation of sodium fluorescein. Small PBS-treated control tumors (A and C) show only a narrow angiogenic front (C; space between the triple arrows) that separates the tumor (asterisks) from the normal liver tissue (L). In contrast, tumors treated with 100 nM of MIP-2 demonstrate a wide angiogenic front at their margins (B). The higher magnification of those tumors (D) reveals a dense network of newly formed microvessels (space between triple arrows) between the tumor (asterisks) and the normal liver (L). Quantitative analysis of the area of the angiogenic front (E) indicates a significant increase compared with controls by treatment with 100 and 1000 nM of MIP-2. Data are mean ± SEM. *P < .05 versus control; #P < .05 vs. 10 nM of MIP-2. Original magnifications, (B) x16; (A, C, and D) x40.

 
MIP-2 at a concentration of 100 and 1000 nM, but not 10 nM, significantly (P < .05) increased the area of the tumor surrounding the angiogenic front when compared with PBS-treated controls (Fig. 5Go). Accordingly, 100 nM and, in particular, 1000 nM of MIP-2 also increased the density of venules draining the tumor, as indicated by a 2-fold and 4-fold increase of the number of venules within the tumor margin when compared with PBS-treated controls and 10 nM MIP-2–treated animals (Fig. 6Go). In line with this, the tumor center showed a 5- to 6-fold increased capillary density after MIP-2 treatment compared with PBS-treated controls, but without significant differences between the 10 nM and the 100 and 1000 nM MIP-2–treated groups (Fig. 7Go). Of interest, analysis of leukocyte adherence within the newly formed tumor capillaries revealed a significant increase after MIP-2 treatment, particularly in a dose of 10 and 100 nM (Fig. 7Go).


Figure 6
View larger version (86K):
[in this window]
[in a new window]
  		FIG. 6. Analysis of the number of venules within the margin of tumors (A and B; asterisks) locally treated with phosphate-buffered saline (PBS; control) or different doses of macrophage inflammatory protein (MIP)-2 (10, 100, and 1000 nM). PBS-treated and 10 nM MIP-2–treated tumors showed only a small number of draining venules (A and C), whereas 100 nM and, in particular, 1000 nM of MIP-2 effectively increased the density of those microvessels (B and C). Data are mean ± SEM. *P < .05 versus control; #P < .05 vs. 10 nM of MIP-2; §P < .05 vs. 100 nM of MIP-2. Original magnification (A and B), x40.

 

Figure 7
View larger version (47K):
[in this window]
[in a new window]
  		FIG. 7. Analysis of capillary density within the center of tumors locally treated with phosphate-buffered saline (PBS; control) or different doses of macrophage inflammatory protein (MIP)-2 (10, 100, and 1000 nM). PBS-treated tumors show only a few capillaries (A and C), whereas 10 nM (C), 100 nM (C), and 1000 nM (B and C) of MIP-2 significantly increased the network density of these microvessels. (D) The number of leukocytes that adhered to the endothelial lining of the newly formed tumor capillaries. MIP-2 induced leukocyte recruitment by significantly increasing the number of adherent leukocytes, particularly in animals that received a dose of 10 or 100 nM. Data are mean ± SEM. *P < .05 versus control. Original magnification (A and B), x80.

 
Because angiogenesis is regularly associated with vasodilatation, we analyzed capillary and sinusoidal diameters within the normal unaffected liver, the area of MIP-2 application of the normal liver adjacent to the tumor, the tumor margin, and the tumor center. In normal liver, including the area of MIP-2 application adjacent to the tumor, MIP-2 treatment did not affect sinusoidal diameters. In contrast, within the tumor margin, MIP-2, in particular at a concentration of 100 and 1000 nM, induced a significant capillary dilation, whereas capillaries within the tumor center were only slightly but not significantly enlarged when compared with normal hepatic tissue (Fig. 8Go). Of interest, the enlarged capillaries within the tumor margin and the tumor center showed a significantly increased coefficient of variance when compared with that of normal liver tissue, thus indicating a pronounced heterogeneity of capillary diameters within the tumor tissue (Table 1Go).


Figure 8
View larger version (41K):
[in this window]
[in a new window]
  		FIG. 8. Analysis of liver sinusoidal and tumor capillary diameters within normal unaffected liver (A), the area of macrophage inflammatory protein (MIP)-2 application of normal liver adjacent to the tumor (B), the tumor margin (C), and the tumor center (D) after treatment with phosphate-buffered saline (control) or different doses of MIP-2 (10, 100, and 1000 nM). In normal liver, sinusoidal diameters are not affected by MIP-2 treatment (A and B). Within the tumor margin, MIP-2, in particular in a dose of 100 and 1000 nM, induces significant capillary dilation (C), whereas capillaries within the tumor center are slightly but not significantly enlarged (D) compared with sinusoids in normal liver (A). Data are mean ± SEM. *P < .05 versus control; #P < .05 versus 10 nM of MIP-2; +P < .05 versus the corresponding values of normal liver.

 

View this table:
[in this window]
[in a new window]
  		TABLE 1. Coefficient of variance of sinusoidal and capillary diameter distribution within normal unaffected liver, the area of MIP-2 application of normal liver adjacent to the tumor, the tumor margin, and the tumor center
 
Morphology, Proliferation, and Receptor Expression
Hematoxylin and eosin–stained sections revealed solid growth of the colorectal CT26.WT hepatic metastasis in all animals studied. Tumors were round, however, with aggressive growth characteristics. Satellite metastases were not observed. In particular, there was no additional tumor growth at the site of application of MIP-2.

PCNA as an indicator of cell proliferation showed that tumor cells displayed strong PCNA staining, particularly cells that were located within the tumor margin (Fig. 9A–CGo). By this, these positively stained cells sharply demarcated the tumor from the surrounding PCNA-negative liver tissue. Normal liver tissue only occasionally showed single PCNA-positive cells (Fig. 9DGo). Quantitative analysis revealed that, regardless of whether the tumors were treated with MIP-2 or with PBS only, PCNA staining regularly involved 70% to 100% of all tumor cells.


Figure 9
View larger version (73K):
[in this window]
[in a new window]
  		FIG. 9. Proliferating cell nuclear antigen (PCNA) immunohistochemistry of a tumor treated with 100 nM of macrophage inflammatory protein (MIP)-2 (A–C) and of unaffected liver tissue (D). Boxed area in A is presented in B. Tumor cells display massive PCNA staining, particularly those located within the tumor margin. By this, these positive cells sharply demarcate the tumor from the surrounding PCNA-negative liver tissue (A–C). Normal liver tissue only occasionally shows single PCNA-positive cells (D). CXCR-2 immunohistochemistry reveals that within the tumor center, some receptor-positive cells can be found that are frequently associated with mitoses (E). In the tumor margin, most cells express CXCR-2 (F). Quantitative analysis of CXCR-2 expression (G) indicates that MIP-2, particularly in a dose of 10 nM, significantly increases the number of cells with positive receptor staining. Data are mean ± SEM. *P < .05 versus control; #P < .05 vs. 10 nM of MIP-2; +P < .05 versus the corresponding values of normal liver. Original magnifications: (A) x18, (B) x88, (C–F) x175.

 
CXCR-2 immunohistochemistry revealed that within the tumor center, some receptor-positive cells could be found. These cells were frequently associated with mitoses (Fig. 9EGo). In contrast, in the tumor margin, most cells expressed CXCR-2 (Fig. 9FGo). Quantitative analysis of CXCR-2 expression indicated that MIP-2, particularly at a concentration of 10 nM, significantly increased the number of cells with positive receptor staining (Fig. 9GGo).

Apoptotic Cell Death
To study apoptotic cell death, specimens were stained for cleaved caspase 3 products, and nuclear condensation was analyzed by intravital fluorescence microscopy. Immunohistochemistry of cleaved caspase 3 products showed individual positively stained cells within the tumor margin that were clearly delineated from the adjacent normal liver tissue (Fig. 10A and BGo). In the tumor center, caspase 3–positive cells were only rarely observed (Fig. 10CGo). However, necrotic areas within the tumor center displayed, besides cell detritus, a considerable number of cells with positive staining of activated caspase 3 (Fig. 10DGo). Quantitative analysis of apoptosis by assessing nuclear condensation with the use of intravital microscopy demonstrated that only MIP-2 at a dose of 1000 nM induced a slight (but not significant) increase of the number of apoptotic cells in the tumor margin when compared with that of PBS-treated controls (Fig. 10EGo).


Figure 10
View larger version (79K):
[in this window]
[in a new window]
  		FIG. 10. Immunohistochemistry of caspase 3–cleaved products within the margin (A and B) and the tumor center (C and D) of macrophage inflammatory protein (MIP)-2–treated tumors. Note that some single caspase 3–positive cells within the tumor margin are clearly delineated from the adjacent normal liver tissue (A and B). Although in the tumor center, caspase 3–positive cells can only rarely be observed (C), necrotic areas display, apart from cell necrosis, staining of caspase 3 in a considerable number of cells (D). Quantitative analysis of apoptosis, which was achieved by counting the number of cells with nuclear condensation per square millimeter of surface area of the tumor margin, revealed that only MIP-2 at a dose of 1000 nM induced a marked, although not significant, increase of apoptotic cells when compared with phosphate-buffered saline–treated controls (E). Data are mean ± SEM. Original magnifications: (A and B) x175, (C and D) x350.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The major findings of this study are that the murine chemokine MIP-2 promotes dose-dependent migration of colon cancer cells in vitro and angiogenesis and metastatic tumor growth in vivo. There is ongoing debate regarding whether malignant cells metastasize to environments that favor their specific growth or whether different organs are endowed with the ability to arrest or attract specific types of malignant cells through chemotactic factors, such as chemokines. Although in humans the chemokine IL-8 binds with high affinity to CXCR-1 and with lower affinity to CXCR-2,33 CXCR-1 cannot be found in the murine system, and CXCR-2 has to be considered the only signaling receptor for the murine homologue of IL-8, i.e., MIP-2.34 In this study, we demonstrate for the first time that in vitro almost all CT26.WT cells express the CXC receptor CXCR-2. This is in line with the data of another study35 that investigated CXCR-2 expression in some human colon carcinoma cell lines (KM12C, KM12L4, Caco2, HT-29, SW-480, and LS 174T) and demonstrated that CXCR-2 was expressed in all cell lines studied, but by various degrees. In addition, this study revealed that the magnitude of CXCR-2 expression is associated with metastatic potential and modulates colon cancer proliferation and an invasive phenotype.35 The fact that the CT26.WT cell line in this study showed almost 100% CXCR-2 expression would therefore indicate that this cell line has to be considered highly aggressive by the criteria of growth rate and meta-static spread. This view is indeed supported by a previous in vitro and in vivo study36 that demonstrated pronounced tumorigenicity of CT26 versus CT51 and CT36 cells.

In vivo, approximately 40% of the cells of non-treated CT26 tumors showed positive CXCR-2 staining 7 days after tumor cell implantation. The reduction of the number of CXCR-2–positive cells is probably due to the fact that solid tumors in vivo consist, besides the CT26.WT clone, of a variety of other cells, including endothelia, fibroblasts, and stromal cells, that do not necessarily express CXCR-2. Although in the stromal cell–derived factor 1/CXCR-4 system, in vitro exposure of the chemokine to receptor-expressing human peripheral blood mononuclear cells results in an initial down-regulation of the receptor during the first 24 hours,37 our study indicates that over a 7-day period, in vivo exposure of MIP-2 increases the expression of CXCR-2 to 60% to 80%. This can be due to receptor recycling,37 which may provide the basis for efficient MIP-2 signaling. Because receptor expression seems to be required for the proliferative action of chemokines on tumors,38 the increased in vivo expression of CXCR-2 may represent the mechanism of action of the MIP-2–associated increase in tumor growth.

Our in vitro assay showed a dose-dependent increase of migration of tumor cells in response to MIP-2. This finding extends the knowledge from previous studies, which demonstrated that MIP-2 and other chemokines were instrumental in regulating transmigration and extravascular tissue accumulation of inflammatory cells after tumor necrosis factor {alpha} or lipopolysaccharide stimulation.39 The dose-dependent increase of migration of CT26.WT colon cancer cells may indeed have contributed to the MIP-2–associated enhancement of metastatic tumor growth.

In previous studies, MIP-2 has been shown to dose-dependently induce hepatocyte proliferation in vitro40 and to accelerate liver regeneration after advanced hepatectomy in vivo.41 Little is known, however, about whether MIP-2 is also involved in processes such as tumor growth and metastasis. A recent study indicated that the reduction of fibrosarcoma growth by inhibition of the lymphotoxin ß receptor can be reconstituted through cotransfection with MIP-2.42 In the present study, we demonstrated for the first time that direct MIP-2 application induces a dose-dependent acceleration of growth of hepatic colon cancer metastasis. Of interest, however, the highest dose of MIP-2 used in this study, i.e., 1000 nM, was associated with a reduced tumor volume. This may be because high doses of MIP-2 are capable of inducing pronounced apoptosis.39 The analysis of cleaved caspase 3 products could confirm programmed cell death within the tumor tissue, and in vivo microscopic evaluation of cells that showed apoptosis-associated nuclear condensation and fragmentation indeed indicated a 4-fold increase of apoptotic cells after application of 1000 nM of MIP-2 when compared with cells of nontreated controls.

The progressive growth of a malignant solid tumor depends on the development of new blood vessels that provide oxygen and nutrients to the tumor cells.43 The tumor vasculature has to be considered abnormal,44 inasmuch as tumor vessels are organized in a chaotic fashion and do not follow the hierarchical branching pattern of normal vascular networks.45 In addition, the endothelial cells form an imperfect lining with wide junctions, and this results in a large number of fenestrations that expose cancer cells to the lumen, thus forming so-called mosaic blood vessels.46 As a result of this abnormal organization and ultrastructure, the diameters of those tumor vessels are irregular, the blood flow is chaotic, and the endothelial lining is leaky.45

In this study, MIP-2 was applied at the time point of tumor cell implantation. This early time point was chosen because the main interest of our study was to analyze the effect of MIP-2 on angiogenesis and because angiogenesis is mostly driven by hypoxia when the cells are without an individual blood supply.

It is widely accepted that tumor angiogenesis is determined by an imbalance between positive and negative regulating molecules that are released by tumor cells and host cells into the tumor microenvironment.35,47,48 Next to important cytokines of members of the VEGF family, chemokines are considered to be important and powerful inducers of angiogenesis. CXC chemokines with the three amino acids Glu-Leu-Arg/ELR immediately amino-terminal to the CXC motif (ELR+) are thought to be angiogenic, whereas CXC ELR– chemokines are angiostatic.49

In inflammation, there is increasing evidence that MIP-2 exerts proangiogenic activity. Scapini et al.50 demonstrated that MIP-2 induces angiogenesis in vivo and that this is mediated by neutrophil-derived VEGF-A. In addition, Keane et al.51 showed that neutralization of MIP-2 by monoclonal antibodies inhibits the bleomycin-induced angiogenic activity in lung specimens. However, there is little information on the proangiogenic activity of MIP-2 in tumor growth. We herein demonstrate for the first time that during engraftment of CT26.WT colon cancer cells in the liver, MIP-2 dose-dependently promotes the formation of new blood vessels, as indicated by an increased capillary density within the tumor center and a widening of the angiogenic front within the tumor margin. Because MIP-2 also induced leukocyte recruitment within the tumors by increasing adherence to tumor capillaries, it is highly likely that the angiogenic action of MIP-2 also involves neutrophil-derived VEGF, as has been reported in inflammation.50

Characteristically, the tumor microvasculature presented with a marked heterogeneity of its individual structures, as indicated by the significantly increased coefficient of variance of capillary diameter. This is in line with the results of previous studies that also demonstrated markedly irregular vessel diameters of tumors grown from other colon cancer cell lines at ectopic sites different from that of the liver.52

The study of the diameters of the newly formed capillaries additionally showed a significant dilation in MIP-2–treated animals, but this dilation was restricted to the margin of the tumor, i.e., the angiogenic front. Although we have not studied the mechanism of this selective vasodilation, it may be speculated that it is caused by the action of VEGF, which is capable of inducing distinct vasodilation, particularly in ischemic tissue.53 VEGF is known as the predominant inductor of tumor angiogenesis,54 and its promotor is preferentially activated within the angiogenic front of the tumor margin.55 Because in this study the selective capillary dilation was shown to be dose-dependent on MIP-2 exposure, our results may additionally indicate that MIP-2 exerts its proangiogenic action by increasing the expression of VEGF within the tumor margin.

The angiogenic front at the tumor margin was associated with an increased cell proliferation, as indicated by a more pronounced PCNA staining within the tumor periphery. Although the angiogenic front showed a high vessel density, these vessels lacked in extravasating the low-molecular-weight fluorescent markers sodium fluorescein and bis-benzimide. In the liver, it is well known that vascular remodeling is associated with capillarization of sinusoids and, thus, a loss of vascular permeability.56 This, however, is unlikely the cause of the reduced extravasation of the fluorescent markers seen in this study, because VEGF is well known to act as a permeability factor on the endothelial cells of the newly formed microvessels.57 More probably, the reduced extravasation of the small molecules is induced by the increased interstitial pressure within the tumor tissue,58 which may expand to the tumor margin and correlate with the size of the tumor. Accordingly, the increased size of MIP-2–treated tumors is associated with an increased interstitial pressure and a widening of the area of the tumor margin that shows the reduced microvascular permeability.

In conclusion, we demonstrate that the murine chemokine MIP-2 dose-dependently promotes angiogenesis, as well as hepatic engraftment and tumor growth of CXCR-2–expressing CT26.WT colon cancer cells, most probably by overexpression of VEGF within the angiogenic front of the tumor margin. Thus, the MIP-2/CXCR-2 signaling pathway may be a promising target for constructing novel therapeutic tools directed against colon cancer growth and hepatic metastasis.


   ACKNOWLEDGMENTS
 
The authors thank C. Marx, R. M. Nickels, and J. Becker for excellent technical assistance. Supported by grants from the Research Committee and the Medical Faculty of the University of Saarland (HOMFOR-A/2003/1).

Received for publication March 25, 2005. Accepted for publication August 22, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Tracey KJ. The inflammatory reflex. Nature 2002; 420:853–9.[CrossRef][Medline]
  2. Ueda T, Shimada E, Urakawa T. Serum levels of cytokines in patients with colorectal cancer: possible involvement of interleukin-6 and interleukin-8 in hematogenous metastasis. J Gastroenterol 1994; 29:423–9.[CrossRef][Medline]
  3. Luca M, Huang S, Gershenwald JE, Singh RK, Reich R, BarEli M. Expression of interleukin-8 by human melanoma cells up-regulates MMP-2 activity and increases tumor growth and metastasis. Am J Pathol 1997; 151:1105–13.[Abstract]
  4. Xie K. Interleukin-8 and human cancer biology. Cytokine Growth Factor Rev 2001; 12:375–91.[CrossRef][Medline]
  5. Miller LJ, Kurtzman SH, Wang Y, Anderson KH, Lindquist RR, Kreutzer DL. Expression of interleukin-8 receptors on tumor cells and vascular endothelial cells in human breast cancer tissue. Anticancer Res 1998; 18:77–81.[Medline]
  6. Brew R, Erikson JS, West DC, Kinsella AR, Slavin J, Christmas SE. Interleukin-8 as an autocrine growth factor for human colon carcinoma cells in vitro. Cytokine 2000; 12:78–85.[CrossRef][Medline]
  7. Takeda A, Stoeltzing O, Ahmad SA, et al. Role of angiogenesis in the development and growth of liver metastasis. Ann Surg Oncol 2002; 9:610–6.[Abstract/Free Full Text]
  8. Haraguchi M, Komuta K, Akashi A, Matsuzaki S, Furui J, Kanematsu T. Elevated IL-8 levels in the drainage vein of resectable Dukes’ C colorectal cancer indicate high risk for developing hepatic metastasis. Oncol Rep 2002; 9:159–65.[Medline]
  9. Strieter RM, Polverini PJ, Arenberg DA, et al. Role of C-X-C chemokines as regulators of angiogenesis in lung cancer. J Leukoc Biol 1995; 57:752–62.[Abstract]
  10. Arenberg DA, Kunkel SL, Polverini PJ, Glass M, Burdick MD, Strieter RM. Inhibition of interleukin-8 reduces tumorigenesis of human non-small cell lung cancer in SCID mice. J Clin Invest 1996; 97:2792–802.[Medline]
  11. Huang S, Mills L, Mian B, et al. Fully humanized neutralizing antibodies to interleukin-8 (ABX-IL8) inhibit angiogenesis, tumor growth, and metastasis of human melanoma. Am J Pathol 2002; 161:125–34.[Abstract/Free Full Text]
  12. Balbay MD, Pettaway CA, Kuniyasu H, et al. Highly metastatic human prostate cancer growing within the prostate of athymic mice overexpresses vascular endothelial growth factor. Clin Cancer Res 1999; 5:783–9.[Abstract/Free Full Text]
  13. Kassim SK, El-Salahy EM, Fayed ST, et al. Vascular endothelial growth factor and interleukin-8 are associated with poor prognosis in epithelial ovarian cancer patients. Clin Biochem 2004; 37:363–9.[CrossRef][Medline]
  14. Lee LF, Hellendall RP, Wang Y, et al. IL-8 reduced tumorigenicity of human ovarian cancer in vivo due to neutrophil infiltration. J Immunol 2000; 164:2769–75.[Abstract/Free Full Text]
  15. Ueda T, Sakabe T, Oka M, et al. Levels of interleukin (IL)-6, IL-8, and IL-1 receptor antagonist in the hepatic vein following liver surgery. Hepatogastroenterology 2000; 47:1048–51.[Medline]
  16. Reisser D, Lejeune P, Lagadec P, et al. Interleukin-8 antitumour effect is associated with a local infiltration but not with a systemic activation of T lymphocytes. Anticancer Res 1994; 14:977–9.[Medline]
  17. Oppenheim JJ, Zachariae CO, Mukaida N, Matsushima K. Properties of the novel proinflammatory supergene "inter-crine" cytokine family. Annu Rev Immunol 1991; 9:617–48.[Medline]
  18. Ness TL, Hogaboam CM, Strieter RM, Kunkel SL. Immunomodulatory role of CXCR2 during experimental septic peritonitis. J Immunol 2003; 171:3775–84.[Abstract/Free Full Text]
  19. Mosher B, Dean R, Harkema J, Remick D, Palma J, Crockett E. Inhibition of Kupffer cells reduced CXC chemokine production and liver injury. J Surg Res 2001; 99:201–10.[CrossRef][Medline]
  20. Wang J, Mukaida N, Zhang Y, Ito T, Nakao S, Matsushima K. Enhanced mobilization of hematopoietic progenitor cells by mouse MIP-2 and granulocyte colony-stimulating factor in mice. J Leukoc Biol 1997; 62:503–9.[Abstract]
  21. Saijo Y, Tanaka M, Miki M, et al. Proinflammatory cytokine IL-1 beta promotes tumor growth of Lewis lung carcinoma by induction of angiogenic factors: in vivo analysis of tumor-stromal interaction. J Immunol 2002; 169:469–75.[Abstract/Free Full Text]
  22. Schramm R, Thorlacius H. Staphylococcal enterotoxin B-induced acute inflammation is inhibited by dexamethasone: important role of CXC chemokines KC and macrophage inflammatory protein 2. Infect Immun 2003; 71:2542–7.[Abstract/Free Full Text]
  23. Schramm R, Liu Q, Thorlacius H. Expression and function of MIP-2 are reduced by dexamethasone treatment in vivo. Br J Pharmacol 2000; 131:328–34.[CrossRef][Medline]
  24. Xing Z, Jordana M, Kirpalani H, Driscoll KE, Schall TJ, Gauldie J. Cytokine expression by neutrophils and macrophages in vivo: endotoxin induces tumor necrosis factor-alpha, macrophage inflammatory protein-2, interleukin-1 beta, and interleukin-6 but not RANTES or transforming growth factor-beta 1 mRNA expression in acute lung inflammation. Am J Respir Cell Mol Biol 1994; 10:148–53.[Abstract]
  25. Armstrong DA, Major JA, Chudyk A, Hamilton TA. Neutrophil chemoattractant genes KC and MIP-2 are expressed in different cell populations at sites of surgical injury. J Leukoc Biol 2004; 75:641–8.[Abstract/Free Full Text]
  26. Otto VI, Heinzel-Pleines UE, Gloor SM, Trentz O, Kossmann T, Morganti-Kossmann MC. sICAM-1 and TNF-alpha induce MIP-2 with distinct kinetics in astrocytes and brain microvascular endothelial cells. J Neurosci Res 2000; 60:733–42.[CrossRef][Medline]
  27. Homey B, Müller A, Zlotnik A. Chemokines: agents for the immunotherapy of cancer? Nat Rev Immunol 2002; 2:175–84.[CrossRef][Medline]
  28. Fahey TJ III, Sherry B, Tracey KJ, et al. Cytokine production in a model of wound healing: the appearance of MIP-1, MIP-2, cachectin/TNF and IL-1. Cytokine 1990; 2:92–9.[CrossRef][Medline]
  29. Schramm R, Thorlacius H. Neutrophil recruitment in mast cell-dependent inflammation: inhibitory mechanisms of glucocorticoids. Inflamm Res 2004; 53:644–52.[CrossRef][Medline]
  30. Kollmar O, Schilling MK, Menger MD. Experimental liver metastasis: standards for local cell implantation to study isolated tumor growth in mice. Clin Exp Metastasis 2004; 21:453–60.[CrossRef][Medline]
  31. Menger MD, Marzi I, Messmer K. In vivo fluorescence microscopy for quantitative analysis of the hepatic microcirculation in hamsters and rats. Eur Surg Res 1991; 23:158–69.[Medline]
  32. Schäfer T, Scheuer C, Roemer K, Menger MD, Vollmar B. Inhibition of p53 protects liver tissue against endotoxin-induced apoptotic and necrotic cell death. FASEB J 2003; 17:660–7.[Abstract/Free Full Text]
  33. Wuyts A, Van Osselaer N, Haelens A, et al. Characterization of synthetic human granulocyte chemotactic protein 2: usage of chemokine receptors CXCR1 and CXCR2 and in vivo inflammatory properties. Biochemistry 1997; 36:2716–23.[CrossRef][Medline]
  34. Rempel SA, Dudas S, Ge S, Gutierrez JA. Identification and localization of the cytokine SDF1 and its receptor, CXC chemokine receptor 4, to regions of necrosis and angiogenesis in human glioblastoma. Clin Cancer Res 2000; 6:102–11.[Abstract/Free Full Text]
  35. Li A, Varney ML, Singh RK. Constitutive expression of growth regulated oncogene (gro) in human colon carcinoma cells with different metastatic potential and its role in regulating their metastatic phenotype. Clin Exp Metastasis 2004; 21:571–9.[CrossRef][Medline]
  36. Brattain MG, Strobel-Stevens J, Fine D, Webb M, Sarrif AM. Establishment of mouse colonic carcinoma cell lines with different metastatic properties. Cancer Res 1980; 40:2142–6.[Abstract/Free Full Text]
  37. Tilton B, Ho L, Oberlin E, et al. Signal transduction by CXC chemokine receptor 4. Stromal cell-derived factor 1 stimulates prolonged protein kinase B and extracellular signal-regulated kinase 2 activation in T lymphocytes. J Exp Med 2000; 192:313–24.[Abstract/Free Full Text]
  38. Zeelenberg IS, Ruuls-Van Stalle L, Roos E. The chemokine receptor CXCR4 is required for outgrowth of colon carcinoma micrometastases. Cancer Res 2003; 63:3833–9.[Abstract/Free Full Text]
  39. Li X, Klintman D, Liu Q, Sato T, Jeppsson B, Thorlacius H. Critical role of CXC chemokines in endotoxemic liver injury in mice. J Leukoc Biol 2004; 75:443–52.[Abstract/Free Full Text]
  40. Colletti LM, Green M, Burdick MD, Kunkel SL, Strieter RM. Proliferative effects of CXC chemokines in rat hepatocytes in vitro and in vivo. Shock 1998; 10:248–57.[Medline]
  41. Ren X, Carpenter A, Hogaboam C, Colletti L. Mitogenic properties of endogenous and pharmacological doses of macrophage inflammatory protein-2 after 70% hepatectomy in the mouse. Am J Pathol 2003; 163:563–70.[Abstract/Free Full Text]
  42. Hehlgans T, Stoelcker B, Stopfer P, et al. Lymphotoxin-beta receptor immune interaction promotes tumor growth by inducing angiogenesis. Cancer Res 2002; 62:4034–40.[Abstract/Free Full Text]
  43. Liotta LA, Kohn EC. The microenvironment of the tumour-host interface. Nature 2001; 411:375–9.[CrossRef][Medline]
  44. Jain RK, Munn LL, Fukumura D. Dissecting tumour pathophysiology using intravital microscopy. Nat Rev Cancer 2002; 2:266–76.[CrossRef][Medline]
  45. Jain RK. Molecular regulation of vessel maturation. Nat Med 2003; 9:685–93.[CrossRef][Medline]
  46. Chang YS, di Tomaso E, McDonald DM, Jones R, Jain RK, Munn LL. Mosaic blood vessels in tumors: frequency of cancer cells in contact with flowing blood. Proc Natl Acad Sci USA 2000; 97:14608–13.[Abstract/Free Full Text]
  47. Coussens LM, Werb Z. Inflammation and cancer. Nature 2002; 420:860–7.[CrossRef][Medline]
  48. Bergers G, Benjamin LE. Tumorigenesis and the angiogenic switch. Nat Rev Cancer 2003; 3:401–10.[CrossRef][Medline]
  49. Homey B, Muller A, Zlotnik A. Chemokines: agents for the immunotherapy of cancer? Nat Rev Immunol 2002; 2:175–84.[CrossRef][Medline]
  50. Scapini P, Morini M, Tecchio C, et al. CXCL1/macrophage inflammatory protein-2-induced angiogenesis in vivo is mediated by neutrophil-derived vascular endothelial growth factor-A. J Immunol 2004; 172:5034–40.[Abstract/Free Full Text]
  51. Keane MP, Belperio JA, Moore TA, et al. Neutralization of the CXC chemokine, macrophage inflammatory protein-2, attenuates bleomycin-induced pulmonary fibrosis. J Immunol 1999; 162:5511–8.[Abstract/Free Full Text]
  52. Leunig M, Yuan F, Menger MD, et al. Angiogenesis, microvascular architecture, microhemodynamics, and interstitial fluid pressure during early growth of human adenocarcinoma LS174T in SCID mice. Cancer Res 1992; 52:6553–60.[Abstract/Free Full Text]
  53. Takeshita S, Isshiki T, Ochiai M, et al. Endothelium-dependent relaxation of collateral microvessels after intramuscular gene transfer of vascular endothelial growth factor in a rat model of hindlimb ischemia. Circulation 1998; 98:1261–3.[Abstract/Free Full Text]
  54. Plate KH, Breier G, Weich HA, Risau W. Vascular endothelial growth factor is a potential tumour angiogenesis factor in human gliomas in vivo. Nature 1992; 359:845–8.[CrossRef][Medline]
  55. Fukumura D, Xavier R, Sugiura T, et al. Tumor induction of VEGF promoter activity in stromal cells. Cell 1998; 94:715–25.[CrossRef][Medline]
  56. Vollmar B, Siegmund S, Menger MD. An intravital fluorescence microscopic study of hepatic microvascular and cellular derangements in developing cirrhosis in rats. Hepatology 1998; 27:1544–53.[CrossRef][Medline]
  57. Hippenstiel S, Krull M, Ikemann A, Risau W, Clauss M, Suttorp N. VEGF induces hyperpermeability by a direct action on endothelial cells. Am J Physiol 1998; 274:L678–84.[Medline]
  58. Boucher Y, Leunig M, Jain RK. Tumor angiogenesis and interstitial hypertension. Cancer Res 1996; 56:4264–6.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Int ImmunolHome page
L. Chen, S.-x. Lin, R. Agha-Majzoub, L. Overbergh, C. Mathieu, and L. S. Chan
CCL27 is a critical factor for the development of atopic dermatitis in the keratin-14 IL-4 transgenic mouse model
Int. Immunol., August 1, 2006; 18(8): 1233 - 1242.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kollmar, O.
Right arrow Articles by Schilling, M. K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kollmar, O.
Right arrow Articles by Schilling, M. K.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS