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Tumor Biology |
The Finsen Laboratory, Copenhagen University Hospital, DK-2100 Copenhagen, Denmark [T. L. F., C. H-H., B. S. N., I. J. C., N. B.]; Stereological Research Laboratory, Bartholin Bygningen, Aarhus University, 8000 Århus C, Denmark [J. R. N.]; and Center for Transgene Technology and Gene Therapy, Flanders Interuniversity Institute for Biotechnology, University of Leuven, B-3000 Leuven, Belgium [P. C.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In situ hybridization and immunohistochemistry studies of human breast cancer tissue have shown that uPA mRNA and protein is mainly expressed by myofibroblasts surrounding nests of cancer cells (1) , whereas uPAR is mainly expressed by infiltrating macrophages (2) , suggesting that the stromal-cell components of the tumor tissue are actively involved in the expression of proteolytic activity. Whether these stromal cells represent a particular subpopulation of stromal cells that are recruited into the tumor tissue, or whether they are preexisting but induced by the tumor cells to produce proteinases, has not yet been not clarified. We have shown previously that in human breast tumors grown in immunodeficient mice, murine uPA is expressed by the mouse stromal cells with a pattern similar to that described in human breast cancer (3) .
Experimental in vitro and in vivo studies have shown that inhibition of uPA activity or uPA-binding to uPAR results in tumor growth inhibition and reduced or abolished formation of metastasis (4, 5, 6, 7, 8) . uPA is most probably facilitating cancer invasion by stimulating extracellular matrix degradation, but the exact role of uPA in regulating tumor growth is not fully understood. uPA may also be involved in activation of latent growth factors, e.g., latent transforming growth factor ß (9) , and release of growth factors that are bound to the extracellular matrix, e.g., basic fibroblast growth factor (10) , and thereby regulate the growth of tumors by increasing the liberation and availability of these growth factors. In addition, uPA has been directly associated with cell proliferation, migration, and chemotaxis (11, 12, 13, 14, 15, 16) , all events that could have a significant impact on tumor cell dissemination.
Mice made genetically deficient for uPA show only minor phenotypic changes, such as occasional fibrin deposition in the intestines and in the sinusoids of the liver (17) . uPA-deficient mice have, in addition, been shown to have impaired neointima formation after electric or mechanical injury of arteries, possibly attributable to an impaired cellular migration of smooth muscle cells (18, 19, 20) , and a recent study showed that during the first 3 days of life, the epidermis from uPA gene-deficient mice have a significantly lower proliferative rate than the epidermis from wild-type mice, suggesting a role of uPA in epidermal cell proliferation (15) . In accordance, wound-healing experiments have shown delayed healing in uPA gene-deficient mice (21) . In another study, chemical induction of melanocytic neoplasms in mice revealed that wild-type and uPA gene-deficient mice developed blue nevi with the same frequency, whereas only wild-type mice had the ability to develop melanomas, suggesting a critical role of uPA in the final progression toward the fully developed malignant phenotype (22) .
In the present study, we have established combined uPA gene-disrupted and immunodeficient mice. By transplanting a human cell line expressing only small amounts of human uPA into these mice and their wild-type littermates, we have tested the hypothesis that stromal uPA plays an important role in the growth and dissemination of this xenograft tumor model. The results support a direct role of stromal uPA in the growth regulation of the MDA-MB-435 BAG xenograft, whereas only a trend toward fewer metastasis in uPA gene-disrupted mice was found.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Genotyping was performed by standard procedure PCR analysis of tail DNA specimens. Genotyping of the animals was performed before and after experiments. The following primers were used: (a) Neo, 5' ATG ATT GAA CAA GAT GGA TTG CAC G 3'; 5' TTC GTC CAG ATC ATC CTG ATC GAC 3'; and (b) uPA, 5' CTG GAA TGC GCC TGC TGT CCT TCA 3', and TGT CAC GAG CTG CCC TGG GAA TCA 3'.
Cell Lines.
Human breast cancer cell lines MDA-MB-435 BAG, MDA-MB-231 BAG, and
MCF-7 BAG, which have been transduced with a replication-defective
Moloney murine leukemia retroviral vector (M-MuLV) containing both
neoR (neomycin resistance) and lacZ genes
(23)
, were routinely propagated in Eagle’s MEM with
Glutamax-1 and supplemented with 5% FCS.
For the animal experiments, cells were harvested from subconfluent monolayer cultures by scraping with a rubber policeman. Cells were inoculated (2 x 106 cells/site) s.c. bilaterally into the abdominal flanks of the animals. Tumors were measured twice weekly in two dimensions with a caliper. Mice inoculated with MCF-7 BAG cells received a 0.72-mg Estradiol pellet (Innovative Research), at the time of cell inoculation. At the end of the experiments, the tumor-bearing mice, which were used for in situ hybridization and immunohistochemistry, were sacrificed by perfusion-fixation with PBS and 4% paraformaldehyde. The tumors were removed, bisected, fixed in 4% paraformaldehyde overnight and then paraffin-embedded. Tumors being used for ELISA and Northern blotting were snap-frozen in liquid nitrogen and stored at -80°C until analyzed. Lungs for metastasis evaluation were removed from mice that had been perfused with PBS.
In Situ Hybridization.
The probe to murine uPA, designated pMUPA07, is a 608-1642-bp cDNA
fragment in pGEM5z, whereas the probe to human uPA, designated pHUPA13,
is a 791-1303-bp cDNA fragment in pBluescript KS(+). Plasmids were
linearized using the following restriction endonucleases:
(a) pMUPA07, EcoRI or PstI;
(b) pHUPA13, HindIII. The probes obtained from
these plasmids are specific for murine or human uPA mRNAs
and do not cross-react (3)
. Generation of
35S-UTP-labeled antisense and sense probes was
performed by in vitro transcription of the plasmids using
the relevant RNA polymerases (3)
. All probe preparations,
including both sense and antisense probes, were adjusted to
1x106 cpm/µl (3)
. In
situ hybridization was performed by the method of Kristensen
et al. (24)
. Briefly, 5-µm tumor tissue
sections cut from formalin-fixed, paraffin-embedded tissues were
treated with proteinase K. After incubation overnight at 47°C with a
hybridization solution containing radiolabeled RNA probe, the sections
were washed twice in a 50% formamide solution for 1 h. The
sections were then treated with RNase A, dehydrated, and air-dried.
Autoradiographic emulsion was applied and sections developed 1 week
after exposure.
ELISA for Human uPA.
Human xenograft tumors were pulverized with a precooled powder pistol.
The tissue powders were suspended at a ratio of 1:4 in extraction
buffer [75 mM potassium acetate, 0.3 M NaCl,
0.1 M L-arginine, 10 mM EDTA, and
0.25% Triton X-100 (pH 4.2); Ref. 25
] at 4°C. The
suspensions were centrifuged at 105,000 x g
for 1 h at 4°C, and the resulting supernatants were stored at
-80°C.
Total human uPA concentrations in tumor extracts were measured using an ELISA kit from Oncogene Science (Cambridge, MA; Ref. 26 ). This assay uses two murine monoclonal antibodies to human uPA for catching and a rabbit polyclonal antibody to human uPA for detection. To test for species specificity, serial dilutions in duplicates of purified murine pro-uPA (a generous gift from Karin List, The Finsen Laboratory, Copenhagen, Denmark) starting from 2.0 ng/ml, then 1.0, 0.5, 0.25, 0.125, 0.0625 ng/ml, were assayed. Protein concentrations of the tissue extracts were determined using the Bio-Rad protein assay (Bio-Rad, Richmond, CA) with BSA as standard.
Northern Blotting.
For Northern blots, 15 µg of total RNA derived from each tumor sample
was size-fractionated by electrophoresis in 1.0% denaturating agarose
gels containing formamide and transferred to reinforced nitrocellulose
membranes. The cDNA probes used were pHUK8, carrying a 1.6kb
PstI fragment of a human uPA cDNA (27)
and
pHFßA-3'UT, coding for the 3'-untranslated region of human ß-actin
cDNA (28)
. The probes were labeled by the random priming
method (29)
using dc tp
5'-
-32P triphosphate. Hybridization of blots
was carried out at 42°C for 16 h in 50% formamide, 10 x Denhardt’s solution, 0.5% SDS-5x SSC, 200 µg/ml salmon
sperm DNA, 10% dextransulfate, 200 µg/ml yeast-tRNA, and 2 x 109 dpm/µg of
32P-labeled probe. The filters were washed at
65°C for 1 h in 2x SSC-0.1% SDS, at 65°C for 1 h in
0.2x SSC-0.5% SDS, and at 65°C for one-half h in 0.1x SSC-0.5%
SDS. Autoradiography was performed at -80°C using Kodak T-MAT-G
films and Kodak Lanex x-omatic intensifying screens.
Quantitation of Lung Metastasis.
Lung metastases were stained and counted essentially as described
previously (23)
. Briefly, anesthetized animals were
PBS-perfused, after which the lungs were dissected from the animals and
placed in a mixture of 2% (v/v) paraformaldehyde and 0.2% (v/v)
glutaraldehyde in PBS for 3–4 h at 4°C. After fixation, the tissue
blocks were rinsed three times with PBS and then incubated for 24 h at 4°C in 1 mg X-gal/ml; 35 mmol/l potassium ferricyanide; 35
mmol/l potassium ferrocyanide; 2 mmol/l MgCl2;
0.02% (v/v) NP40; and 0.01% (w/v) sodium deoxycholate in PBS. The
lungs were then rinsed, first with 3% (v/v) dimethyl sulfate in PBS
and then with PBS only. Until evaluation of surface metastases, the
lungs were stored at 4°C in 0.02% sodium azide in PBS. Metastases
were evaluated by counting surface metastases through an inverted
dissecting microscope.
ß-Gal Measurement in Plasma.
Blood was obtained from each mouse, and citrate plasma was separated.
Plasma ß-gal activity was measured by an immunocapture assay as
described previously (30)
. Briefly, white microwell plates
(Berthold, Bad Wildbad, Germany) were coated for 16 h at 4°C
with 200 µl/well of the murine monoclonal antibody to
Escherichia coli ß-gal clone 5B88 (2.5 µg IgG/ml; Life
Technologies, Inc., Gaithersburg, MD) in 0.1 M
carbonate buffer (pH 9.5). Before use, the assay wells were rinsed once
with 200 µl/well of SuperBlock solution (Pierce Chemicals, Rockford,
IL) diluted 1:1 with PBS, and then by three washes with PBS containing
1 mg/ml Tween 20. Wells were then incubated for 1 h at 37°C with
200 µl/well in triplicate of 1:20 diluted plasma samples. After
ß-gal binding, the wells were washed three times with PBS containing
1 mg/ml Tween 20 and then treated for 45 min at 37°C with 200
µl/well of Galacto-Light Reaction Buffer Diluent with Galacton-Plus
chemiluminescent substrate (Tropix, Bedford, MA). After the enzymatic
cleavage of the substrate, chemiluminescent light emission was
triggered by adding automatically 100 µl/well of the alkaline
polymeric enhancer (Accelerator-II; Tropix) using the pump incorporated
in the luminometer (Berthold LB 96 P-2 MicroLumat). Chemiluminescence
intensity (relative light units) was measured over a 5-s
interval with a delay time of 2 s. The results are presented as
the mean ± SE.
CD34 Immunohistochemistry.
Five-µm paraffin sections of the tumors were deparaffinized in xylene
and hydrated through ethanol/water dilutions. Proteolytic digestion was
performed with 0.025% trypsin (Sigma T8128) in 50 mM Tris
(pH 7.6) containing 0.1% CaCl2 for 7 min at
37°C. Sections were blocked for endogenous peroxidase activity by 1%
hydrogen peroxide for 15 min. Then, sections were washed in TBS [50
mM Tris, 150 mM NaCl (pH 7.6)] containing
0.5% Triton X-100. Slides were mounted into Shandon sequenza slide
racks with immunostaining cover plates (Shandon Inc. Pittsburgh, PA,
USA), for additional incubations. The sections were incubated overnight
at 4°C with rat antimouse CD34 (clone MEC 14.7, HyCult Biotechnology,
Uden, Netherlands) at 1:30 (3 µg/ml). The rat antibody was detected
with affinity purified biotinylated rabbit antirat antibodies (E468,
Dako, Glostrup, Denmark) followed by streptavidin horseradish
peroxidase complexes (Dako, Glostrup, Denmark). Each antibody
incubation was followed by washes with 6 ml of Triton X-100.
Sections were developed with NovaRED substrate as specified by the
manufacturer (Vector Laboratories, Burlingname, CA) for 10 min.
Finally, sections were counterstained in diluted Mayers Hematoxylin for
30 s, dehydrated in ethanol, and mounted.
Quantitation of Angiogenesis.
The computer-assisted stereology setup consisted of a Leica Dialux 22
microscope equipped with a CCD Camera (JAI 2040) and a motorized X-Y
stage directed by a multicontroller unit (Olympus, Albertslund,
Denmark). The computer (IBM, PC 300 GL) had a 24-bit framegrabber board
(Screen Machine, FAST SMII) and CAST-grid software (Olympus, Denmark)
installed as described
elsewhere.3
The vascular length density was estimated with an unbiased counting
frame as described by Gundersen et al. (31)
,
with the assumption that the vessels were isotropic in the tumor.
Counting was performed on CD34 immunohistochemically stained sections
(two from each tumor), which were slightly counterstained with
hematoxylin. The overall magnification used on the computer monitor was
x318. The vascular length density
[LV(vasc/tumor)] estimates were obtained by
counting vascular profiles [QA(vasc)] within
the counting frame. The upper right corner was used to estimate the
tumor tissue by point-counting [
P(tumor)]. The area associated to
this test point was denoted (a/p). Only vital tumor areas
and vascular profiles located within living tumor areas were counted.
The vascular length density was estimated as:
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(tumor/vasc), was obtained
by:
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Statistical Methods.
For the comparison of primary tumor growth curves of the two groups,
repeated measures of ANOVA were used. The statistical significance of
differences in plasma ß-gal activity, vascular length density,
maximal diffusion distance, and total length of vessels were analyzed
by Student’s t test. The number of metastases formed in the
two groups was compared by Fischer’s exact test and the nonparametric
Mann-Whitney test. In all statistical analyses, Ps <
0.05 were considered statistically significant.
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RESULTS |
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. Extracts from MDA-MB-231 BAG and MDA-MB-435 BAG tumors were
investigated in a human-specific uPA ELISA, and in accordance with the
Northern blot analysis, the ELISA measurement showed that whereas
MDA-MB-231 BAG tumors express high amounts of human uPA (84 ng/mg
protein ± SD), MDA-MB-435 BAG cells contained 400-fold
less uPA/mg protein (0.22 ng/mg protein ±SD). By applying the ELISA to
a standard curve of murine uPA, it was confirmed that this assay does
not detect mouse pro-uPA (not shown). Thus, the uPA protein measured is
exclusively produced by the human cancer cells.
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In Situ Hybridization of Human and Murine uPA.
In situ hybridization of MDA-MB-435 BAG xenografts for human
uPA using a species-specific RNA probe (3)
, showed a weak
but homogenous signal over the tumor cells, with no signal observed in
the host stromal tissue (data not shown). No specific signal was
obtained by the corresponding sense probe (data not shown). In
contrast, when using the mouse-specific probe, distinct regions of
murine uPA expression were apparent within the stromal cells of the
tumors. Expression was predominantly seen at the stromal regions near
the tumor periphery, usually in the areas of invasion (Fig. 2, A and C)
. Murine uPA mRNA was also strongly
expressed in regions bordering the necrotic areas (Fig. 2, B and D)
. At higher magnification, murine uPA was found to be
expressed by stromal cells near the tumor-stroma border, including some
endothelial-like cells. Although some endothelial cells (or cells in
the vicinity of endothelial cells) did appear to express murine uPA
mRNA, the signal was generally not associated with vascular-appearing
structures, suggesting nonendothelial stromal cells as the major
expressors of murine uPA. No murine uPA mRNA signal was detected in
tumors obtained from uPA-/- mice or when using
the sense probe on tumors from wild-type mice (data not shown).
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In the uPA+/+ mice, 34 of 34 animals (100%) developed tumors in the experimental period. MDA-MB-435 BAG cells formed tumors in uPA-/- mice with the same frequency as in uPA+/+ mice, suggesting that host (murine) uPA is not essential for MDA-MB-435 BAG tumor formation. No differences in length of lag phase (time to palpable tumors) was observed between tumors growing in the two mouse genotypes.
MDA-MB-435 BAG tumors in uPA+/+ mice grew rapidly
and reached a mean size of 2000 mm3
50 days after
cell inoculation (Fig. 3A)
. In contrast, tumors grown in
uPA-/- mice had a significantly slower growth
rate reaching a mean size of 1200 mm3
50 days
after cell inoculation (P = 0.007). The
growth curves shown in Fig. 3A
included 20
uPA+/+ and 19 uPA-/-
mice, and presents one experiment (experiment 3) representative
of the three separate experiments performed.
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Local Invasion and Metastasis Formation of MDA-MB-435 BAG
Xenografts.
Histological inspection of the tumors grown in
uPA+/+ or uPA-/- mice
showed that the tumors grew locally invasive, infiltrating the
peritoneal wall of the animals in both groups. Fig. 2,
E and F, show a representative area with invasion
of MDA-MB-435 BAG cells into the peritoneal wall of a
uPA-/- mouse.
Lungs from tumor-bearing animals in experiment 3 were removed at autopsy (day 52) and processed for X-gal staining. Eighteen of 20 lungs from wild-type mice were evaluable. Fourteen of 18 (78%) of uPA+/+ mice had stereomicroscopically visible metastases (blue dots) on the surface of the lungs. In contrast, only 10 out 19 uPA-/- mice (53%) showed surface lung metastases. Fishers exact test showed that uPA-/- mice had a trend toward a reduced number of animals with lung metastases (P = 0.2). By counting the number of surface metastasis (blue dots) on individual lungs, the mean number of metastases in uPA+/+ mice was 12.7 (range, 0–36) whereas the mean number of surface lung metastases in uPA-/- was 4.7 (range, 0–80). By applying the Mann-Whitney nonparametric test, a trend appeared toward fewer lung surface metastases in uPA-/- mice (P = 0.15).
Thus, the difference between the groups regarding lung metastases did not reach statistical significance, but a tendency toward fewer metastases in the uPA-/- mice was evident.
CD34 Immunostaining of MDA-MB-435 BAG Tumors.
Microscopic inspection of CD34-stained MDA-MB-435 BAG tumors from both
uPA+/+ and uPA-/- mice
revealed positively stained vessels and capillaries throughout the
tumor tissue (Fig. 2, G and H)
and in the
connective tissue surrounding the tumors.
No obvious differences appeared between the two groups, including no apparent differences in size or morphology.
Quantitation of Tumor Angiogenesis.
In experiment 2, stereological evaluation of the number of
CD34-immunostained capillaries (vascular profiles) showed a
nonsignificant difference (P = 0.15) in
vascular length density between uPA-/- mice
(n = 11; 19 ± 4
profiles/mm2
) and uPA+/+
mice (n = 8; one mouse was lost before
termination of the experiment; 22 ± 4
profiles/mm2
). In addition to vascular length
density, we examined at average maximal diffusion distance from
capillary to tumor (0.13 mm ± 0.01 (SE) in
uPA-/- mice and 0.12 mm ± 0.004
(SE) in uPA+/+ mice) as well as total length of
vessels in vital tumor tissue (8.5 m ± 1.7 (SE) in
uPA-/- mice and 14.0 m ± 4.6
(SE) in uPA+/+mice). In both cases we found the
differences to be nonsignificant (P = 0.16
and P = 0.23, respectively), but with a trend
toward reduced angiogenesis in tumors grown in the uPA
gene-deficient mice. Thus, for all 3 types of measurements for tumor
angiogenesis, we found a nonsignificant trend toward lower tumor
angiogensis in the uPA-/- mice as compared with
uPA+/+ mice.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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For the mRNA expression studies, the applied human- and mouse-specific uPA probes were clearly species-specific, and by including both murine and human uPA standard curves in the ELISA, we unequivocally demonstrated that this uPA ELISA does react with human, but not with murine, uPA. By the use of littermate animals as controls, we have attempted to minimize other host factors than uPA, influencing tumor growth and dissemination. Genotypes of the animals included in the experiments were in addition to PCR analyses, confirmed by subsequent murine uPA in situ hybridizations of the tumors.
We have described previously that mouse uPA is expressed in the human MDA-MB-231 breast cancer when grown in nude mice (3) . Although using another human breast cancer cell line in the present study, the data confirm that the murine compartment of the human breast cancer xenografts express uPA. The murine uPA mRNA expression in MDA-MB-435 BAG tumors was confined to fibroblast-like cells, however, not all stromal cells with fibroblast-like appearance expressed murine uPA. Thus, the expression pattern of murine uPA in the xenografted human tumors resembles that described for human breast cancer (1) .
The binding of uPA to its receptor strongly enhances cell-surface plasminogen activation (33) . However, uPA binding to uPAR shows strict species specificity, e.g., murine uPA does not bind human uPAR and vice versa (34) . Thus, to fully exert its proteolytic potential, murine uPA must bind murine uPAR. In our previous study of MDA-MB-231 xenografts (3) , murine uPA and uPAR were shown to be expressed by cells that were either the same or located very close to each other. Although we did not include analysis of murine uPAR in the present study, we assume that murine uPAR is also expressed in MDA-MB-435 BAG xenografts. Thus, coexpression of murine uPA and uPAR in the same or adjacent cells in the xenografted tumors makes it likely that the murine uPA system is involved in plasmin generation in the tumor tissue. The exact mechanisms regulating stromal cell uPA expression in tumors is unknown. In vitro studies have shown that a number of cytokines regulate uPA expression (35) . In the xenografted tumors, the human cancer cells may be the source of cytokines, which, by a paracrine mechanism, induces uPA (and uPAR) expression in the surrounding stromal cells.
There is ample experimental evidence that uPA has importance for tumor cell growth, e.g., it has been shown by different groups that uPA has a direct stimulative effect on tumor cell proliferation in vitro (11 , 12) . Inhibition of uPA activity (6 , 7) or inhibition of uPA-uPAR interaction (36 , 37) , results in impaired growth of experimental tumors. However, of particular interest is that in a recent experimental in vivo study (36) using the MDA-MB-231 human xenograft implanted into immunodeficient mice, both human and murine uPA-uPAR antagonists showed significant inhibition of primary tumor growth, suggesting that both tumor- (human) and stromal- (murine) cell uPAR dependent plasminogen activation can modulate tumor growth in vivo.
Our experimental data are in agreement with the study of Tressler et al. (36) . By genetic disruption of the uPA gene, we have shown that tumors grown in the gene-deleted animals have a significantly slower growth rate than tumors grown in littermate control animals. One explanation to the observed decreased growth rate of tumors in uPA-/- mice is that the local activation/release of growth factors is decreased attributable to decreased proteolytic activity in the tumor tissue (9 , 10) . Another explanation relates to the putative role of proteinases in the angiogenic process. Although the bioavailability of growth factors such as basic fibroblast growth factor, which is a highly efficient stimulator of angiogenesis, may be reduced in the tumor tissue of the uPA-/- mice, proteinases have also been ascribed a direct role in the migration of endothelial cells (review by Pepper et al. in Ref. 38 ). Endothelial cells express both uPA and uPAR (38 , 39) , and the interaction between these two molecules may govern endothelial cell migration in the extracellular matrix. In support of this notion is the demonstration of decreased angiogenesis in murine tumors exposed to a murine uPA-uPAR-inhibitor (37) . In our study we found a nonsignificant trend toward a lower angiogenesis as measured by vascular density, total vascular length, as well as the maximal diffusion distance, in tumors in the uPA-/- mice.
Dissemination of cancer cells with subsequent formation of secondary tumors (metastasis), require proteinase activity and the ability to migrate and attach of not only the cancer cells but also of cells of the supporting stroma. A number of experimental tumor studies have shown that inhibition of uPA activity (4 , 5) or uPA-uPAR interaction (40) results in a reduced number of metastatic foci. In our model, a difference although not significant was seen between uPA+/+ and uPA-/- mice with regard to the number of lung surface metastases formed from the primary tumors, suggesting that stromal uPA is also involved in the mechanisms of metastasis formation. However, because of the significant effect of uPA-deficiency on the growth of the primary tumors, it cannot be excluded that the effect seen on the number of lung surface metastases reflects the effect on growth, i.e., some of the metastases in the uPA-/- mice are under the detection limit of our evaluation system. In support of this notion is that tumors showed an invasive growth pattern in both wild-type and uPA-/- mice.
In conclusion, the experimental data obtained points to a direct role of stromal-derived uPA for the growth of the human MDA-MB-435 BAG breast cancer xenograft.
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FOOTNOTES |
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1 To whom requests for reprints should be
addressed, at The Finsen Laboratory, Copenhagen University
Hospital, Strandboulevarden 49, Boulevard 86.2, DK-2100 Copenhagen,
Denmark. Phone: 45-35455606; Fax: 45-35251117; E-mail: nils{at}finsenlab.dk 
2 The abbreviations used are: uPA, urokinase
plasminogen activator; uPAR, uPA-specific receptor; PAI-1 and
PAI-2, plasminogen activator inhibitors type 1 and 2; pro-uPA, uPA
pro-enzyme; ß-gal, ß-galactosidase.
3 Nielsen, B. S., Lund, L. R.,
Christensen, I. J., Johnsen, M., Autzen, P., Andersen, L. W.,
Frandsen, T. L., Danø, K., and Gundersen, H. J. A precise
and efficient method to determine murine lung metastasis volumes using
stereology, submitted for publication.
Received 6/26/00. Accepted 11/13/00.
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R. Das, G. H. Mahabeleshwar, and G. C. Kundu Osteopontin Stimulates Cell Motility and Nuclear Factor {kappa}B-mediated Secretion of Urokinase Type Plasminogen Activator through Phosphatidylinositol 3-Kinase/Akt Signaling Pathways in Breast Cancer Cells J. Biol. Chem., August 1, 2003; 278(31): 28593 - 28606. [Abstract] [Full Text] [PDF]
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Q. Han, J. Leng, D. Bian, C. Mahanivong, K. A. Carpenter, Z. K. Pan, J. Han, and S. Huang Rac1-MKK3-p38-MAPKAPK2 Pathway Promotes Urokinase Plasminogen Activator mRNA Stability in Invasive Breast Cancer Cells J. Biol. Chem., December 6, 2002; 277(50): 48379 - 48385. [Abstract] [Full Text] [PDF]
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Y. Guo, P. Pakneshan, J. Gladu, A. Slack, M. Szyf, and S. A. Rabbani Regulation of DNA Methylation in Human Breast Cancer. EFFECT ON THE UROKINASE-TYPE PLASMINOGEN ACTIVATOR GENE PRODUCTION AND TUMOR INVASION J. Biol. Chem., October 25, 2002; 277(44): 41571 - 41579. [Abstract] [Full Text] [PDF]
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S. A. Rabbani and J. Gladu Urokinase Receptor Antibody Can Reduce Tumor Volume and Detect the Presence of Occult Tumor Metastases in Vivo Cancer Res., April 1, 2002; 62(8): 2390 - 2397. [Abstract] [Full Text] [PDF]
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E. K. Rofstad, H. Rasmussen, K. Galappathi, B. Mathiesen, K. Nilsen, and B. A. Graff Hypoxia Promotes Lymph Node Metastasis in Human Melanoma Xenografts by Up-Regulating the Urokinase-Type Plasminogen Activator Receptor Cancer Res., March 1, 2002; 62(6): 1847 - 1853. [Abstract] [Full Text] [PDF]
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J. Chen, C. Baskerville, Q. Han, Z. K. Pan, and S. Huang alpha v Integrin, p38 Mitogen-activated Protein Kinase, and Urokinase Plasminogen Activator Are Functionally Linked in Invasive Breast Cancer Cells J. Biol. Chem., December 14, 2001; 276(51): 47901 - 47905. [Abstract] [Full Text] [PDF]
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, wild-type mice
(n = 20). The growth curves shown are
representative of three separate experiments. B,
plasma ß-gal activity in mice carrying MDA-MB-435 BAG tumors.
Bars, SE.