Cancer Research Aziza Shad Jordan
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
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 Email this article to a friend
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
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Arlt, M.
Right arrow Articles by Krüger, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Arlt, M.
Right arrow Articles by Krüger, A.
[Cancer Research 62, 5543-5550, October 1, 2002]
© 2002 American Association for Cancer Research

Tumor Biology

Increase in Gelatinase-specificity of Matrix Metalloproteinase Inhibitors Correlates with Antimetastatic Efficacy in a T-Cell Lymphoma Model1

Matthias Arlt, Charlotte Kopitz, Caroline Pennington, Katrina L. M. Watson, Hans-Willi Krell, Wolfram Bode, Bernd Gansbacher, Rama Khokha, Dylan R. Edwards and Achim Krüger2

Institut für Experimentelle Onkologie und Therapieforschung, Technische Universität München, D-81675 München, Germany (M. A., C. K., B. G., A. K.); University of East Anglia, School of Biological Sciences, Norwich, Norfolk NR4 7TJ, United Kingdom (C. P., D. R. E.); University of Toronto, Ontario Cancer Institute, Toronto, Ontario M5G 2M9, Canada (K. L. M. W., R. K.); Max-Planck-Institut für Biochemie, D-82152 Martinsried, Germany (W. B.); and Roche Diagnostics GmbH, D-82377 Penzberg, Germany (H. W. K.)


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The recognition that matrix metalloproteinases (MMPs) facilitate tumor cell invasion and metastasis has led to the development of synthetic MMP inhibitors (MMPIs) as cancer therapeutic agents. Because several Phase III trials failed recently to show efficacy of broad-spectrum MMPIs in advanced cancer, the feasibility of MMPs as therapeutic targets has been challenged. However, it has not yet been determined whether MMPIs that have increased specificity may have greater benefit. We show that MMP-9 expression closely correlates with the progression of liver metastasis in a T-cell lymphoma model. MMPIs with greater selectivity/specificity for MMP-9 in vitro showed greater efficacy against liver metastasis in vivo. These data demonstrate a link between increased specificity of MMPIs and enhanced anticancer activity.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Proteolytic degradation of components of the extracellular matrix such as collagens, proteoglycans, laminin, elastin, and fibronectin is considered to be a prerequisite for tumor invasion and metastasis. MMPs3 are able to degrade essentially all of the protein components of the extracellular matrix, including fibrillar collagens. In addition, MMPs substantially contribute to other steps in the metastatic cascade, such as angiogenesis, differentiation, proliferation, and apoptosis (1 , 2) . Therefore, MMPs are important regulators of tumor growth, both at the primary site and in distant metastases. This status is affirmed by the association of increased expression of some MMPs with poor prognosis in several tumor types (1) .

Synthetic MMPIs have been developed and administered to patients to reduce cancer spread (3) . However, over the past 2 years clinical studies with MMPIs have been discontinued without reaching their primary end point, i.e., proving a reduction in mortality or extending the disease-free interval (4) . In contrast to the many reports on the implication of MMPs in tumor progression and the potential benefit of the use of MMPIs (5) , recent studies have also revealed detrimental effects of MMPIs on tumorigenesis. Broad spectrum MMPIs prevent MMP-mediated generation of the angiogenesis inhibitor angiostatin (6) , which could lead to facilitation of tumor progression and promotion of metastasis (7) . Promotion of metastasis has also been demonstrated experimentally as batimastat treatment increased metastasis to the liver of several human carcinomas in nude mice (8 , 9) . Additionally, liver metastasis of a syngeneic T-cell lymphoma in DBA/2 mice was promoted on treatment with the broad spectrum MMPI batimastat, which additionally led to liver-specific up-regulation of angiogenesis-promoting genes, as well as MMP-2 and -9 (9) . Stimulation of MMP-9 expression was also reported for a fibrosarcoma cell line treated with another broad-spectrum MMPI (10) .

Given the clear implications of MMPs in many human cancers, MMPs remain important targets for cancer therapy (5) . Therefore, it is necessary to obtain an in-depth understanding of the specific roles of MMPs in tumor progression, to identify the right time point of treatment with MMPIs, and also to test whether increased efficacy in cancer treatment can be achieved with more specific MMPIs (4) . We have addressed the latter point by comparison of four different MMPIs, batimastat and AG 3340 (both hydroxamate-type), as well as Ro 28–2653 and Ro 206-0222 (two structure-related pyrimidine-2,4,6-trione-type inhibitors), in one metastasis model. As MMP-1 structurally differs from MMP-2, -8, -9, and -14 in the active site cleft, in that it exhibits a much smaller S1' specificity pocket (11 , 12) , it provides a model for the design of specific MMPIs. Initially, MMP-8 served as a model system for gelatinase inhibition because of spatial similarity of the enzyme to gelatinases MMP-2 and -9 (13) . The lead structures of the Ro-inhibitors were chemically further optimized based on the IC50 ratios between MMP-1 and -9 to achieve greater selectivity toward MMP-9 (13) . The aggressive lacZ-tagged T-cell lymphoma model offers a quantitative system allowing the study of liver metastasis within 7 days as cells colonize the liver on i.v. injection without forming lung metastases (14) . Clinical studies on lymphomas have reported MMP-9 to be a marker of tumor progression (15) . Here we show that MMP-9 expression closely correlated with the progression of liver metastasis in this model. Furthermore, we found that inhibitors for MMPs with greater selectivity/specificity for MMP-9 in vitro showed greater efficacy against liver metastasis in vivo. These data provide the first demonstration of the linkage between increased specificity of MMPIs and enhanced anticancer activity, and have implications for the clinical use of this class of inhibitors.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Experimental Metastasis Assay.
Pathogen-free female DBA/2 mice (6–8 weeks old; Charles River, Sulzfeld, Germany) were inoculated with 5 x 103 DBA/2-syngeneic lacZ-tagged murine L-CI.5s cells (16) into the tail vein of each mouse. Treatment of mice with the respective inhibitor or the vehicle control began the day after tumor cell inoculation and continued until the day before organ removal. Seven days after tumor cell inoculation, when macrometastases (>0.2 mm) are already formed in this model (14) , mice were sacrificed, livers removed, and stained with X-gal (Ref. 16 ; Roche-Diagnostics, Penzberg, Germany) or flash frozen for protein or RNA isolation. Blue metastatic foci on the surface of the organs were counted. This method allows assessment of the metastatic pattern even of lymphoma cells (14) . [4-(N-hydroxyamino)-2R-isobutyl-3S-(thiopen-2-ylthiomethyl)-succinyl]-L-phenylalanine-N-methyl-amide, which is identical to batimastat and will be referred to as batimastat in this study (synthesized according to the PCT Patent Application WO 90/05719), was suspended in sterile PBS/0.01% Tween 80 (Sigma, Munich, Germany) and administered i.p. at a daily dose of 30 mg/kg (shown previously to be tolerable and effective; Ref. 17 ). The following inhibitors were used at the highest tolerable and effective dose (data not shown). AG3340, (3S)-N-hydroxy-2,2-dimethyl-4-{[4-(4-pyridinyloxy)phenyl]sulfonyl}-3-thiomorpholinecarbox-amide (synthesized according to United States Patent 5,753,653), was dissolved under stirring at 35°C in N,N-dimethylacetamide (Merck, Darmstadt, Germany). Polyethylenglycol 400 solution (50%; Merck) was added under stirring to give a final concentration of 10% N,N-dimethylacetamide. The compound was administered p.o. at a daily dose of 400 mg/kg. 5-Biphenyl-4-yl-5-[4-(4-nitro-phenyl)-piperazin-1-yl]pyrimidine-2,4,6-trione (Ro 28–2653) or 5-(4-phenoxy-phenyl)-5-(4-pyrimidin-2-yl-piperazin-1-yl)-pyrimidine-2,4,6-trione (Ro 206-0222), barbiturate (pyrimidine-2,4,6-trione)-derivates (13) , were synthesized according WO 9858925, and were suspended in a solution of 0.2 mg/ml Na-carboxymethyl-cellulose, 0.09 mg/ml methyl-parabene, and 0.01 mg propyl-parabene, and administered p.o. at a daily dose of 50 mg/kg. The respective vehicle was administered to the animals of the control groups by the same route.

Enzymatic Studies.
IC50 of inhibitors were tested as described previously (13) .

Zymography and Western Analysis.
Snap-frozen tissue was immediately homogenized as described previously (9) . Tissue homogenates were stored for no more than 1 h at -80°C to be able to detect active forms of MMP-9 in the zymography. Total protein of homogenates was quantified by bicinchoninic acid protein assay (Pierce, Rockford, IL). Gelatinolytic activity was detected by 10% SDS-PAGE of 30–35 µg of each homogenate containing 1 mg/ml gelatin under nonreducing conditions. After electrophoresis, proteins were renatured and the gel was incubated at 37°C for 19 h in 500 mM Tris/HCl, 5 mM CaCl2, and 200 mM NaCl (pH 7.5) and stained as described previously (18) . Amino-phenyl mercuric acetate (Sigma)-activated recombinant MMP-2 and -9 (Roche Molecular Biochemicals, Penzberg, Germany), and prestained molecular weight marker (Kaleidoscope Prestained Standards; Bio-Rad Laboratories, Hercule, CA) served as standards.

For the Western blot, 40 µg of protein were electrophoresed through 8% SDS-PAGE and electroblotted at 25 V for 90 min. Blots were blocked with 5% skim milk in TBST overnight at 4°C. Mouse monoclonal anti-PCNA (Novocastra Laboratories Ltd., Newcastle, United Kingdom; diluted 1:500 in 5% skim milk/TBST) and anti-{alpha}-tubulin (Oncogene, Boston, MA; diluted 1:4000 in 5% skim milk/TBST) antibodies were incubated for 1 h, detected with antimouse horseradish peroxidase-conjugated antibody (Cell Signaling, Beverly, MA; diluted 1: 1000 in 5% skim milk/TBST), and visualized by enhanced chemiluminescence reagent (Amersham Pharmacia Biotech, Little Chalfont, United Kingdom). Quantification in relation to {alpha}-tubulin was performed using a densitometer by Molecular Dynamics, (Sunnyvale, CA). The software used was Image Quant Software by Molecular Dynamics.

Immunostaining.
Tissue sections were dewaxed in toluene and rehydrated. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide. Antigen retrieval was performed by microwaving the slides in 10 mM citrate buffer (pH 6.0) for PCNA or by pepsin digestion (0.4% for 5 min at 42°C) for CD31 immunostaining. Endogenous biotin was blocked with Vector biotin blocking kit (Vector Laboratories, Burlingame, CA). The PCNA antibody (Novocastra Laboratories Ltd.) was diluted 1:1000 in antibody diluting buffer (DAKO Corporation, Carpinteria, CA) and incubated for 1 h at room temperature, and detected using biotinylated secondary antibody (Signet Multi-link; Signet Laboratories Inc., Dedham, MA), a Signet Ultrastreptavidin kit, and a Nova Red substrate (Vector Laboratories). Tissue was counterstained with Mayers Hematoxylin, dehydrated, cleared, and mounted with Permount. CD31 antibody (PharMingen, Mississauga, Ontario, Canada) was diluted 1:100 and incubated overnight at room temperature. Secondary antibody was antirat mouse absorbed (Vector Laboratories) at 1:200 dilution, and signal was detected using Ultrastreptavidin as described above.

RNA Isolation and Reverse Transcription.
Isolation of total tissue RNA was performed as described (19) . One µg of total RNA was reverse transcribed in a 20-µl reaction using 2 µg of random hexamers and Superscript II reverse transcriptase (Life Technologies, Inc. Paisley, United Kingdom) according to the manufacturers instructions.

Design of Primers and Probes for TaqMan PCR.
Oligonucleotide primers and fluorescent-labeled TaqMan probes specific for murine MMPs, TIMP-1, and VEGF-A were designed using Primer Express 1.0 software (PE Applied Biosystems, Warrington, United Kingdom). To control against amplification of genomic DNA and to ensure that the PCR signal was generated from cDNA, primers were placed within different exons close to intron/exon boundaries. BLASTN searches were conducted on all of the primer/probe nucleotide sequences to ensure gene specificity. The identity of PCR products was confirmed by direct sequencing of amplicons. The 18S RNase gene was used as an endogenous control to normalize for differences in the amount of total RNA in each sample. TaqMan 18S RNase primers and a 5' VIC-labeled probe were used according to the manufacturers instructions (PE Applied Biosystems). Primers and probes for the murine MMPs, TIMP-1, and VEGF-A are shown in Table 1Citation .


View this table:
[in this window]
[in a new window]

  		Table 1 TaqMan primer and probes

 
TaqMan PCR.
Relative quantification of gene expression was performed using the Applied Biosystems ABI prism 7700 sequence detection system (TaqMan). PCR reactions for all of the samples were performed in duplicate in 96-well optical plates using 0.5% of cDNA, 12.5 ml 2x TaqMan Universal PCR mastermix (PE Applied Biosystems), 100 nM probe, 200 nM of each primer, and water to a 25 µl final volume. Thermocycler conditions comprised an initial holding at 50°C for 120 s then 95°C for 10 min. This was followed by a two-step TaqMan PCR program consisting of 95°C for 15 s and 60°C for 60 s for 40 cycles. Data are presented graphically as relative levels of mRNA for each primer/probe set.

Statistical Analysis.
Data were analyzed using Student’s t test or Mann-Whitney rank sum test where the normality test failed.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Defining the MMPI Specificity/Selectivity.
First we calculated the inhibitory efficacy of the MMPIs batimastat, AG3340, and the newly developed inhibitors Ro 28–2653 and Ro 206-0222 against MMPs -1, -2, -9, -13, and -14 (Table 2)Citation . The selectivity of the inhibitors for MMP-9 was expressed by dividing the IC50 against MMP-1 by the IC50 against MMP-9 (Table 2)Citation . Accordingly, AG3340 is the most effective inhibitor against MMP-9 based on the IC50 values, whereas Ro 206-0222 ranked first (~2000-fold more selective than the broad-spectrum MMPI batimastat) in terms of selectivity, followed by Ro 28-2653, AG3340, and the unselective MMPI batimastat (Table 2)Citation . The same ranking occurred, when the Ki against MMP-1 was divided by the Ki against MMP-9, or when the Ki against MMP-7, which is similar to MMP-1 in terms of the smaller S1' specificity pocket, was divided by the Ki against MMP-9 (data not shown). Because MMP-13 is one of the main fibrillar collagenases in murine tissue (and the murine orthologues of MMP-1 are not expressed in the adult mouse; Ref. 20 ), we also calculated the selectivity of the MMPIs for MMP-9 in relation to MMP-13 by dividing the IC50 against MMP-13 by the IC50 against MMP-9 (Table 2)Citation . The same ranking of specificities was obtained.


View this table:
[in this window]
[in a new window]

  		Table 2 Definition of the MMPI specificity/selectivity

Concentration of each inhibitor at which 50% inhibition of proteolysis ocurred (IC50) are indicated. The ratio of the IC50 values defines the selectivity of the respective inhibitor for MMP-9 as opposed to MMP-1 (IC50MMP-1:IC50MMP-9), and opposed to MMP-13 (IC50MMP-13:IC50MMP-9).

 
Comparative Analysis of Antimetastatic Efficacy.
Injection of 5,000 lacZ-tagged L-CI.5s murine T-cell lymphoma cells resulted in the formation of ~140 macrometastatic foci (diameter >0.2 mm) in the liver. In addition, micrometastases (diameter <0.2 mm) were visible in the parenchyma surrounding the macrometastatic foci, in numbers (>10,000) that by far exceeded the number of inoculated cells, representing secondary infiltration of dividing and spreading lymphoma cells from the initial sites of metastasis, as is known for this model (21) . Whereas batimastat significantly increased liver metastasis (Fig. 1, A and B)Citation , treatment with hydroxamate-type inhibitor AG3340 led to a significant reduction of the number of macroscopic lymphoma liver metastases by >20% (Fig. 1A)Citation , as well as of the micrometastatic infiltration of the liver parenchyma (Fig. 1C)Citation . Treatment with Ro 28-2653 and Ro 206-0222 led to a substantial reduction of the number of macroscopic lymphoma liver metastases by ~54% or 68%, respectively (Fig. 1A)Citation . Again, a decreased micrometastatic infiltration of the parenchyma was visually evident (Fig. 1, D and E)Citation . In the control groups 12% of the macrometastases exceeded a diameter of 1 mm and had necrotic centers, whereas only 8, 2, and 6% exceeded this diameter in the batimastat, AG3340, and Ro 28-2653 treatment groups, respectively. In the Ro 206-0222 treatment group, attenuation of foci growth was not significant (11%).



View larger version (53K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 1. Antimetastatic efficacy of MMPIs. A, proportion of X-gal-stained macrometastases (>0.2 mm) on the surface of mouse livers present 7 days after inoculation of 5 x 103 L-CI.5s lymphoma cells as compared with the number of macrometastases in the vehicle groups. Vehicle controls (consisting of batimastat-vehicle, AG3340-vehicle, and Ro-vehicle controls): n = 26, mean = 100 ± 7.1%; batimastat: n = 9, mean = 184.6 ± 60%; AG3340: n = 9, mean = 75.2 ± 19.4%; Ro 28–2653: n = 10, mean = 45.8 ± 7.94%; and Ro 206-0222: n = 12, mean = 32.2 ± 6.9%. Comparison of the treatment groups with the vehicle control revealed statistically significant differences in each case (AG3340: P = 0.02; all others P < 0.0001) as determined by Mann-Whitney rank sum test. The differences between the treatment groups are also statistically significant (Ro 28–2653 compared with Ro 206-0222: P = 0.0012; all other comparisons: P < 0.0001). Bars, ± SD. B, close-up of the surface of representative X-gal-stained livers of the respective vehicle control group (left) or treatment group (right). In addition to macrometastases, infiltration of the parenchyma by numerous micrometastases consisting of a few or single cells is visible. Bars = 0.5 mm.

 
MMP Expression Profiles upon Metastasis and in MMPI-treated Livers.
MMP-9 appears to be a marker of progression in our mouse lymphoma model, because we found a close correlation between metastasis load and MMP-9 expression as determined by zymographic analysis or quantitative RT-PCR (Fig. 2ACitation and Fig. 3Citation ). mRNAs of all other tested MMPs and TIMP-1 were also significantly induced upon metastasis (Fig. 3)Citation . This induction was a result of tumor-host interaction, as both tumor cells (19) and normal livers individually (Figs. 2Citation and 3Citation ) express relatively low levels of MMPs or TIMP-1. As the increase of MMP-9 expression induced by batimastat treatment correlates with promotion of metastasis (9) , we tested the possible modulation of gene expression of gelatinases by the other inhibitors. We treated tumor-free mice with the four MMPIs and found by zymographic analysis that only batimastat, and not AG3340, Ro 28-2653, or Ro 206-0222, led to induction of pro-MMP-9 as well as active MMP-9 (Fig. 2B)Citation . MMP-2 protein levels remained unaffected by all of the MMPIs (Fig. 2)Citation . Homogenates of metastases-bearing livers of untreated mice showed a tumor-host interaction-related increase of MMP-9 expression (Fig. 2)Citation . The levels of MMP-9 present in the lysates of livers from metastasis-bearing mice approximately correlated with the increase (treatment with batimastat, Fig. 2BCitation ) or decrease of metastasis (treatment with the other MMPIs, Fig. 2, C–ECitation ). To get a preliminary insight into possible alterations of gene expression of MMPs of different subclasses or TIMPs induced by treatment with inhibitors, we used quantitative RT-PCR (Fig. 3)Citation . In tumor-free mice, AG3340, Ro 28-2653, and Ro 206-0222 did not alter the expression of transcripts for MMPs -2,-7, -8, -9, -12, -13, and -14, as well as TIMP-1 (data not shown). In metastasis-bearing livers, only expression of MMP-9 mRNA correlated with metastasis-load. Expression of MMP-2, -7, -8, -12, -13, -14, and TIMP-1 mRNAs varied considerably depending on the treatment, but there was no association with the tumor burden. Moreover, in batimastat-treated mice that had the highest metastasis, load transcript levels for MMP-7, MMP-13, and TIMP-1 approximated those found in the control without metastases (Fig. 3)Citation . mRNAs of the murine orthologs of MMP-1 (Mcol-A and Mcol-B; Ref. 20 ) were not detectable by RT-PCR.



View larger version (88K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 2. Modulation of gelatinase expression and activation by metastasis and MMPIs. Liver tissue was isolated from MMPI-treated or vehicle-treated tumor-free or metastasis-bearing mice, respectively. A, dose dependency of MMP-9 expression in the liver with increasing number of inoculated lymphoma cells. Typically, in livers of untreated control mice we found 140–180 macrometastases after inoculation of 5 x 103 lymphoma cells. Lanes were equally loaded with 35 µg protein each lane. B, zymography of proteins of livers without metastases or of metastases-bearing livers of mice. Both groups of mice either received the respective vehicle or the MMPI for 6 consecutive days before analysis. Lanes were equally loaded with 30 µg protein each. Data shown are representative of at least seven different mice from each group.

 


View larger version (30K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 3. Quantitative RT-PCR of selected MMPs and TIMP-1. Data were obtained from three mice in each group. Relative values (normalized to 18S rRNA) are expressed for each RNA. Bars, ± SE.

 
Possible Relevance of Antiangiogenetic Effects of MMPIs in this T-Cell Lymphoma Model.
While investigating the metastatic pattern in the liver (see above), we noted a higher proportion of large metastatic foci in the control group, which contained central necrosis, when exceeding a diameter of 1 mm. However, in all of the treatment groups these foci were smaller. MMPIs, in principle, can exert an antiangiogenic effect (1 , 3 , 7) . In the frame of this study, we could not rule out this possibility, but obtained some preliminary indications that angiogenesis may not be an important therapeutic target in our tumor model. Immunohistochemical staining for CD31 revealed no notable differences between livers with and without metastases (Fig. 4, A and B)Citation . Also, H&E staining revealed central necrosis in virtually all of the metastatic foci (Fig. 4, C and D)Citation , indicating outgrowth of blood supply. Consistent with these findings, the level of mRNA of VEGF-A (as another marker of angiogenesis) remained unaltered when normal livers were compared with metastasis-bearing livers in RT-PCR analysis (Fig. 4E)Citation . However, VEGF-A mRNA expression seemed to be reduced in the presence of all of the MMPIs, even in livers from the batimastat-treated group, which exerted more metastases than the control (Fig. 4E)Citation .



View larger version (80K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 4. Involvement of angiogenesis in liver metastasis of the T-cell-lymphoma model. A, in metastasis-free livers, CD31-positive endothelial cells (arrows) were present in sinusoids and blood vessels (bar = 50 µm). B, in metastasis-bearing livers, CD31 positivity (arrows) was similarly low within the normal tissue and the viable periphery (v) of the metastatic foci (bar = 50 µm). Unspecific staining occurred in the necrotic center (n) of the metastatic foci (arrow with star). Insets in A and B show CD31 staining in detail at higher magnification. C, H&E-stained sections of metastasis-bearing livers revealing necrosis (n) in the center of the viable peripheries (v) of virtually all metastatic foci >1 mm (bar = 1 mm). D, magnification of a metastatic foci with a central necrotic area (n; bar = 0.5 mm). E, quantitative RT-PCR of VEGF. Data were obtained from three mice in each group. Relative values (normalized to 18S rRNA) are expressed for each RNA. Bars, ± SE.

 
Proliferation in Livers of MMPI-treated Mice.
Decreased tumor cell proliferation is one potential mechanism for reduced metastasis load and for the higher proportion of smaller metastatic foci found in all of the MMPI-treated groups. PCNA is expressed during S phase of the cell cycle and is normally absent in the quiescent liver. We examined PCNA expression in metastasis-bearing livers. Immunohistochemical analysis indicated that PCNA is restricted within metastatic foci (Fig. 5A)Citation . A substantial reduction of PCNA on protein level was evident in the livers of mice treated with AG 3340, Ro 28-2653, and Ro 206-0222 that had significantly fewer metastases (Fig. 5B)Citation . This was not evident in livers of batimastat-treated mice, as these had vastly increased numbers of metastases, which all contributed to the PCNA signal (data not shown). Mice treated with Ro 28-2653 and Ro 206-0222 had a significant reduction in PCNA protein levels compared with those treated with AG 3340 (Fig. 5C)Citation .



View larger version (67K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 5. PCNA expression in livers with and without metastases. A, immunohistochemical analysis of liver sections. Left, absence of PCNA staining in a liver section without metastases (bar = 50 µm). The arrow indicates enlarged area shown in the inset. Right, PCNA staining restricted to the areas of metastatic foci and absent in the liver parenchymal cells (bar = 50 µm). The arrow indicates enlarged area shown in the insert. B, Western analysis of livers treated with the metastasis-inhibiting MMPIs. PCNA protein could not be detected in tumor-free livers in the presence or absence of the MMPIs. Upon metastasis, PCNA expression was induced. In MMPI-treated livers expression of PCNA was found to be decreased. {alpha}-Tubulin was used as a loading control. C, quantification of the bands in the Western revealed a significant (P < 0.005) reduction of PCNA expression in livers treated with Ro 28–2653 (15.6 ± 1.5% of control) and Ro 206-0222 (12.1 ± 1.4% of control), and a substantial decrease in AG 3340 (56.5 ± 13.4% of control)-treated livers. Bars, ± SE.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study we found a correlation between increased MMP-9 selectivity of MMPIs and their efficacy to reduce metastasis in a T-cell-lymphoma model, where MMP-9 expression was identified as closely correlating with the number of liver metastases. MMPIs can be effective at several steps of the metastatic cascade, as MMPs play an important role in extravasation and growth of secondary tumors, the latter by promoting angiogenesis or the release of growth factors (2) . Several studies have suggested an important role for MMP-9 in the clinical aggressiveness of lymphomas (15 , 22 , 23) . In our model, MMP-9 expression is a consequence of the interaction between lymphoma cells and host stroma or vasculature (Fig. 2ACitation ; Ref. 22 ). Therefore, MMP-9 can be considered as an important target. Treatment started 1 day after colonization of individual cells to the liver, i.e., at a time point when extravasation of this aggressive T-cell lymphoma has most likely already occurred, as is also the case for larger cells from solid tumors (24) . Nevertheless, the number of macrometastatic foci is significantly smaller in the livers of mice treated with the MMP-9-selective inhibitors AG 3340, Ro 28-2653, and Ro 206-0222. This is likely a reflection of less efficient growth, supported by the observed PCNA expression levels. Those were far lower than could be expected from the lower metastasis load in these groups of mice. Also, diameters of macrometastases were found to be decreased in all of the treated livers. Although inhibition of angiogenesis is a possible mechanism by which the MMPIs may exert their effect (1 , 3 , 7) , we obtained some preliminary indications that angiogenesis may not be as relevant in our tumor model, because CD31 expression was not induced, the metastatic foci outgrew the blood supply, and VEGF-A mRNA expression was not modified during metastasis. This minor role of angiogenesis can be explained by the efficient blood supply in the sinusoidal system of the liver. Lower VEGF-A mRNA expression in the treatment groups is possibly a function of MMP-14 inhibition by the MMPIs, as this protease has been linked with the transcriptional regulation of VEGF (25) . It is also possible that reduced T-cell-lymphoma growth is a consequence of reduced growth factor release within the cellular microenvironment. The influence of MMP inhibition on outgrowth of micrometastases has also been described for experimental liver metastasis of melanoma cells (26) and may be one important mechanism of MMPI efficacy (2 , 27) .

The total number of macro- and micrometastatic foci, visible on the surface of the livers at the end of the experiment, by far exceeded the number of injected cells (5,000). This suggested that secondary invasion from the original foci into the liver parenchyma takes place, leading to the scattered metastatic phenotype typical for this lymphoma model (21) and for clinical lymphomas (28) . The more selective MMPIs AG 3340, Ro 28-2653, and Ro 206-0222 drastically inhibited this secondary invasion whereas it was promoted by batimastat in the liver in this and other tumor models (9) . Interestingly, another broad-spectrum hydroxamate MMPI also led to up-regulation of pro-MMP-9 in HT1080 fibrosarcoma cells, although an in vitro test revealed down-regulated invasive ability (10) . We propose that the adverse effects of this MMPI are likely not related to the hydroxamate moiety involved in the chelation of the active site Zn2+, as another hydroxamate-based inhibitor AG3340 significantly reduced lymphoma liver metastasis (Fig. 2, A and C)Citation , but likely relate to its ability to induce MMP-9 expression. The mechanism of induction of MMP-9 by batimastat or its metabolites and the basis for the inactivity of AG3340 in this regard are as yet unknown. It is possible that the peptidomimetic nature of batimastat (AG3340 is a nonpeptidomimetic hydroxamate-type inhibitor) plays a role here, or that in contrast to the oral administration of AG3340, direct administration of the batimastat suspension into the peritoneal cavity may have led to liver irritation or damage resulting in up-regulation of MMP-9. It is not surprising that expression of all of the tested MMPs and TIMP-1 is induced upon metastasis, as all have been shown to correlate with tumor progression in different tumors (29) . However, the different MMPIs are able to modulate gene expression in different ways, providing insight into the roles of individual genes in liver metastasis in our lymphoma model. Up-regulation of MMP-9 and simultaneous down-regulation of TIMP-1 by batimastat correlated with additional increase of metastasis, the latter being in accordance with the antimetastatic role that baseline levels of host TIMP-1 have in this model (19) . However, at least at the mRNA level, expression of all of the tested MMPs (and TIMP-1) was increased in metastasis-bearing livers. Among these MMPs, only MMP-9 expression closely correlated with the number of metastases. Although batimastat treatment led to down-regulation of MMP-7, -12, and -14 in metastasis-bearing livers, this treatment led to the highest metastasis load. Moreover, expression of MMP-2, MMP-14, and TIMP-1 was not related to the metastasis load in the different treatment groups, i.e., even in groups of mice with >50% reduction of metastasis, levels remained significantly elevated compared with tumor-free control. In contrast, mRNA levels of MMP-7 and -12 were drastically reduced in all of the treatment groups, even in the batimastat treatment group that had a much higher metastasis-load. Additional MMP/TIMP genes may contribute to lymphoma metastasis capability, but this has not emerged from quantitative real-time RT-PCR analysis for MMP-3, -10, -11, -15, and -19 genes, and the remaining 3 TIMP family members, where no correlation with the progression of metastasis could be seen (data not shown), additionally supporting the dominance of MMP-9 in this system. There may be additional metalloproteinase targets that play important roles in this model, conceivably including members of the MMP-related adamalysin family, many of which are known to be inhibited by MMPIs (30) . However, such targets would have to show the same characteristics of inhibition by the MMPIs used in this study as MMP-9, if they are connected with metastasis in this model. Therefore, this study underscores the advantage of the use of specific rather than broad-spectrum inhibitors, especially in the light of side effects, which need to be avoided in the clinic. Because it has been discussed that inhibition of MMP-1 may cause clinically relevant side effects (e.g., arthralgias; Refs. 3 , 31 ), selectivity of inhibitors for tumor progression-associated MMPs, while sparing MMP-1, is likely of importance in the clinic. Also, the complexity of the MMP family and the as yet incompletely understood mechanisms of MMP regulation argue in favor of the development of more specific MMPIs for cancer therapy on the grounds of safety, as the possible dual function of MMPs in the process of metastasis can be an obstacle for the use of unselective MMPIs (6 , 32) . Whereas MMP-inhibition studies have clearly lost momentum in regard to clinical studies, our study demonstrates that more specific MMPIs are worth consideration for the therapy of aggressive lymphomas.


   ACKNOWLEDGMENTS
 
We thank Katja Honert and Elisabeth Popescu for expert technical assistance.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by the Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 469, project B13 (to A. K.), the Medical Research Council (United Kingdom), the Norfolk and Norwich Big C Appeal (to D. R. E.), and the United States Army (to R. K.). Back

2 To whom requests for reprints should be addressed, at Institut für Experimentelle Onkologie und Therapieforschung, der Technischen Universität München, Ismaninger Str. 22, D-81675 München, Germany. Phone: 49-89-4140-4463; Fax: 49-89-4140-6182; E-mail: achim.krueger{at}lrz.tu-muenchen.de Back

3 The abbreviations used are: MMP, matrix metalloproteinase; MMPI, matrix metalloproteinase inhibitor; X-gal, 5-bromo-4-chloro-3-indoyl-ß-D-galactopyranoside; TBST, Tris-buffered saline, 0.05% Tween 20; PCNA, proliferating cell nuclear antigen; TIMP, tissue inhibitor of metalloproteinase; VEGF, vascular endothelial growth factor; RT-PCR, reverse transcription-PCR. Back

Received 12/18/01. Accepted 7/26/02.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Yu A. E., Hewitt R. E., Connor E. W., Stetler-Stevenson W. G. Matrix metalloproteinases. Novel targets for directed cancer therapy. Drugs Aging, 11: 229-244, 1997.[Medline]
  2. Chambers A. F., Matrisian L. M. Changing views of the role of matrix metalloproteinases in metastasis. J. Natl. Cancer Inst., 89: 1260-1270, 1997.[Abstract/Free Full Text]
  3. Brown P. D. Ongoing trials with matrix metalloproteinase inhibitors. Expert Opin. Investig. Drugs, 9: 2167-2177, 2000.[Medline]
  4. Zucker S., Cao J., Chen W. T. Critical appraisal of the use of matrix metalloproteinase inhibitors in cancer treatment. Oncogene, 19: 6642-6650, 2000.[Medline]
  5. Nelson A. R., Fingleton B., Rothenberg M. L., Matrisian L. M. Matrix metalloproteinases: biologic activity and clinical implications. J. Clin. Oncol., 18: 1135-1149, 2000.[Abstract/Free Full Text]
  6. Patterson B. C., Sang Q. A. Angiostatin-converting enzyme activities of human matrilysin (MMP-7) and gelatinase B/type IV collagenase (MMP-9). J. Biol. Chem., 272: 28823-28825, 1997.[Abstract/Free Full Text]
  7. Sang Q. X. Complex role of matrix metalloproteinases in angiogenesis. Cell Res., 8: 171-177, 1998.[Medline]
  8. Della Porta P., Soeltl R., Krell H. W., Collins K., O’Donoghue M., Schmitt M., Krüger A. Combined treatment with serine protease inhibitor aprotinin and matrix metalloproteinase inhibitor Batimastat (BB-94) does not prevent invasion of human esophageal and ovarian carcinoma cells in vivo. Anticancer Res., 19: 3809-3816, 1999.[Medline]
  9. Krüger A., Soeltl R., Sopov I., Kopitz C., Arlt M., Magdolen V., Harbeck N., Gansbacher B., Schmitt M. Hydroxamate-type matrix metalloproteinase inhibitor batimastat promotes liver metastasis. Cancer Res., 61: 1272-1275, 2001.[Abstract/Free Full Text]
  10. Maquoi E., Munaut C., Colige A., Lambert C., Frankenne F., Noel A., Grams F., Krell H. W., Foidart J. M. Stimulation of matrix metalloproteinase-9 expression in human fibrosarcoma cells by synthetic matrix metalloproteinase inhibitors. Exp. Cell Res., 275: 110-121, 2002.[Medline]
  11. Lovejoy B., Cleasby A., Hassell A. M., Longley K., Luther M. A., Weigl D., McGeehan G., McElroy A. B., Drewry D., Lambert M. H., Jordan S. R. Structure of the catalytic domain of fibroblast collagenase complexed with an inhibitor. Science (Wash. DC), 263: 375-377, 1994.[Abstract/Free Full Text]
  12. Bode W., Maskos K. Structural studies on MMPs and TIMPs. Methods Mol. Biol., 151: 45-77, 2001.[Medline]
  13. Grams F., Brandstetter H., D’Alo S., Geppert D., Krell H. W., Leinert H., Livi V., Menta E., Oliva A., Zimmermann G. Pyrimidine-2, 4, 6-triones: a new effective and selective class of matrix metalloproteinase inhibitors. Biol. Chem., 382: 1277-1285, 2001.[Medline]
  14. Krüger A., Schirrmacher V., Khokha R. The bacterial lacZ gene: an important tool for metastasis research and evaluation of new cancer therapies. Cancer Metastasis Rev., 17: 285-294, 1998.[Medline]
  15. Kossakowska A. E., Urbanski S. J., Janowska-Wieczorek A. Matrix metalloproteinases and their tissue inhibitors-expression, role and regulation in human malignant non-Hodgkin’s lymphomas. Leuk. Lymphoma, 39: 485-493, 2000.[Medline]
  16. Krüger A., Umansky V., Rocha M., Hacker H. J., Schirrmacher V., von Hoegen P. Pattern and load of spontaneous liver metastasis dependent on host immune status studied with a lacZ transduced lymphoma. Blood, 84: 3166-3174, 1994.[Abstract/Free Full Text]
  17. Eccles S. A., Box G. M., Court W. J., Bone E. A., Thomas W., Brown P. D. Control of lymphatic and hematogenous metastasis of a rat mammary carcinoma by the matrix metalloproteinase inhibitor batimastat (BB-94). Cancer Res., 56: 2815-2822, 1996.[Abstract/Free Full Text]
  18. Edwards D. R., Leco K. J., Beaudry P. P., Atadja P. W., Veillette C., Riabowol K. T. Differential effects of transforming growth factor-beta 1 on the expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases in young and old human fibroblasts. Exp. Gerontol., 31: 207-223, 1996.[Medline]
  19. Krüger A., Fata J. E., Khokha R. Altered tumor growth and metastasis of a T-cell lymphoma in Timp-1 transgenic mice. Blood, 90: 1993-2000, 1997.[Abstract/Free Full Text]
  20. Balbin M., Fueyo A., Knauper V., Lopez J. M., Alvarez J., Sanchez L. M., Quesada V., Bordallo J., Murphy G., Lopez-Otin C. Identification and enzymatic characterization of two diverging murine counterparts of human interstitial collagenase (MMP-1) expressed at sites of embryo implantation. J. Biol. Chem., 276: 10253-10262, 2001.[Abstract/Free Full Text]
  21. Krüger A., Schirrmacher V., von Hoegen P. Scattered micrometastases visualized at the single-cell level: detection and re-isolation of lacZ-labeled metastasized lymphoma cells. Int. J. Cancer, 58: 275-284, 1994.[Medline]
  22. St-Pierre Y., Aoudjit F., Lalancette M., Potworowski E. F. Dissemination of T cell lymphoma to target organs: a post-homing event implicating ICAM-1 and matrix metalloproteinases. Leuk. Lymphoma., 34: 53-61, 1999.[Medline]
  23. Kossakowska A. E., Urbanski S. J., Watson A., Hayden L. J., Edwards D. R. Patterns of expression of metalloproteinases and their inhibitors in human malignant lymphomas. Oncol. Res., 5: 19-28, 1993.[Medline]
  24. Koop S., MacDonald I. C., Luzzi K., Schmidt E. E., Morris V. L., Grattan M., Khokha R., Chambers A. F., Groom A. C. Fate of melanoma cells entering the microcirculation: over 80% survive and extravasate. Cancer Res., 55: 2520-2523, 1995.[Abstract/Free Full Text]
  25. Sounni N. E., Devy L., Hajitou A., Frankenne F., Munaut C., Gilles C., Deroanne C., Thompson E. W., Foidart J. M., Noel A. MT1-MMP expression promotes tumor growth and angiogenesis through an up-regulation of vascular endothelial growth factor expression. FASEB J., 16: 555-564, 2002.[Abstract/Free Full Text]
  26. Chambers A. F., MacDonald I. C., Schmidt E. E., Morris V. L., Groom A. C. Preclinical assessment of anti-cancer therapeutic strategies using in vivo videomicroscopy. Cancer Metastasis Rev., 17: 263-269, 1998.[Medline]
  27. Chambers A. F., Naumov G. N., Varghese H. J., Nadkarni K. V., MacDonald I. C., Groom A. C. Critical steps in hematogenous metastasis: an overview. Surg. Oncol. Clin. N. Am., 10: 243-255, vii 2001.[Medline]
  28. Harris A. C., Ben-Ezra J. M., Contos M. J., Kornstein M. J. Malignant lymphoma can present as hepatobiliary disease. Cancer, 78: 2011-2019, 1996.[Medline]
  29. Foda H. D., Zucker S. Matrix metalloproteinases in cancer invasion, metastasis and angiogenesis. Drug Discov. Today, 6: 478-482, 2001.[Medline]
  30. Egeblad M., Werb Z. New functions for the matrix metalloproteinases in cancer progression. Nature Rev. Cancer, 2: 161-174, 2002.[Medline]
  31. Wojtowicz-Praga S., Torri J., Johnson M., Steen V., Marshall J., Ness E., Dickson R., Sale M., Rasmussen H. S., Chiodo T. A., Hawkins M. J. Phase I trial of Marimastat, a novel matrix metalloproteinase inhibitor, administered orally to patients with advanced lung cancer. J. Clin. Oncol., 16: 2150-2156, 1998.[Abstract]
  32. Stetler-Stevenson W. G., Yu A. E. Proteases in invasion: matrix metalloproteinases. Semin. Cancer Biol., 11: 143-152, 2001.[Medline]



This article has been cited by other articles:


Home page
 Mol Cancer ResHome page
M. Gerg, C. Kopitz, S. Schaten, A. Tschukes, C. Kahlert, M. Stangl, C. W. Hann von Weyhern, B. L.D.M. Brucher, D. R. Edwards, K. Brand, et al.
Distinct Functionality of Tumor Cell-Derived Gelatinases during Formation of Liver Metastases
Mol. Cancer Res., March 1, 2008; 6(3): 341 - 351.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
C. Kopitz, M. Gerg, O. R. Bandapalli, D. Ister, C. J. Pennington, S. Hauser, C. Flechsig, H.-W. Krell, D. Antolovic, K. Brew, et al.
Tissue Inhibitor of Metalloproteinases-1 Promotes Liver Metastasis by Induction of Hepatocyte Growth Factor Signaling
Cancer Res., September 15, 2007; 67(18): 8615 - 8623.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
Y. Miyata, T. Iwata, K. Ohba, S. Kanda, M. Nishikido, and H. Kanetake
Expression of Matrix Metalloproteinase-7 on Cancer Cells and Tissue Endothelial Cells in Renal Cell Carcinoma: Prognostic Implications and Clinical Significance for Invasion and Metastasis
Clin. Cancer Res., December 1, 2006; 12(23): 6998 - 7003.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
M. J.E. Arlt, I. Novak-Hofer, D. Gast, V. Gschwend, G. Moldenhauer, J. Grunberg, M. Honer, P. A. Schubiger, P. Altevogt, and A. Kruger
Efficient Inhibition of Intra-Peritoneal Tumor Growth and Dissemination of Human Ovarian Carcinoma Cells in Nude Mice by Anti-L1-Cell Adhesion Molecule Monoclonal Antibody Treatment
Cancer Res., January 15, 2006; 66(2): 936 - 943.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
C. Kopitz, M. Anton, B. Gansbacher, and A. Kruger
Reduction of Experimental Human Fibrosarcoma Lung Metastasis in Mice by Adenovirus-Mediated Cystatin C Overexpression in the Host
Cancer Res., October 1, 2005; 65(19): 8608 - 8612.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
A. Kruger, M. J.E. Arlt, M. Gerg, C. Kopitz, M. M. Bernardo, M. Chang, S. Mobashery, and R. Fridman
Antimetastatic Activity of a Novel Mechanism-Based Gelatinase Inhibitor
Cancer Res., May 1, 2005; 65(9): 3523 - 3526.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
C. Van Themsche, T. Alain, A. E. Kossakowska, S. Urbanski, E. F. Potworowski, and Y. St-Pierre
Stromelysin-2 (Matrix Metalloproteinase 10) Is Inducible in Lymphoma Cells and Accelerates the Growth of Lymphoid Tumors In Vivo
J. Immunol., September 15, 2004; 173(6): 3605 - 3611.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Pathol.Home page
E. Van Valckenborgh, P. I. Croucher, H. De Raeve, C. Carron, E. De Leenheer, S. Blacher, L. Devy, A. Noel, E. De Bruyne, K. Asosingh, et al.
Multifunctional Role of Matrix Metalloproteinases in Multiple Myeloma: A Study in the 5T2MM Mouse Model
Am. J. Pathol., September 1, 2004; 165(3): 869 - 878.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
N. E. Sounni, C. Roghi, V. Chabottaux, M. Janssen, C. Munaut, E. Maquoi, B. G. Galvez, C. Gilles, F. Frankenne, G. Murphy, et al.
Up-regulation of Vascular Endothelial Growth Factor-A by Active Membrane-type 1 Matrix Metalloproteinase through Activation of Src-Tyrosine Kinases
J. Biol. Chem., April 2, 2004; 279(14): 13564 - 13574.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
S. Roelle, R. Grosse, A. Aigner, H. W. Krell, F. Czubayko, and T. Gudermann
Matrix Metalloproteinases 2 and 9 Mediate Epidermal Growth Factor Receptor Transactivation by Gonadotropin-releasing Hormone
J. Biol. Chem., November 21, 2003; 278(47): 47307 - 47318.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
Y.-G. Zhao, A.-Z. Xiao, R. G. Newcomer, H. I. Park, T. Kang, L. W. K. Chung, M. G. Swanson, H. E. Zhau, J. Kurhanewicz, and Q.-X. A. Sang
Activation of Pro-gelatinase B by Endometase/Matrilysin-2 Promotes Invasion of Human Prostate Cancer Cells
J. Biol. Chem., April 18, 2003; 278(17): 15056 - 15064.
[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 Email this article to a friend
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
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Arlt, M.
Right arrow Articles by Krüger, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Arlt, M.
Right arrow Articles by Krüger, A.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online