[CANCER RESEARCH 43, 5517-5525,
November 1983]
Plasminogen Activator Secretion of Human Tumors in Short-Term Organ
Culture, Including a Comparison of Primary and Metastatic Colon
Tumors1
Gabor Markus,2 Sarah M. Camiolo, Shin Kohga,3 Judith M. Madeja, and Arnold Mittelman
Departments of Experimental Biology [G. M., S. M. C., S. K., J. M. M.] and of Surgical Oncology ¡A.M.], Roswell Park Memorial Institute, New York State Department ot
Health, Buffalo. New York 14263
ABSTRACT
The secretion of plasminogen activator by expiants of 92
human malignant tumors was studied in short-term organ cul
ture. Where possible, adjacent normal tissue of the presumed
origin of the tumors was also studied. The study included adenocarcinomas of the lung, colon, prostate, breast, and stomach
and different types of sarcomas. In addition to the measurement
of secretion rates, all tissues were quantitatively extracted to
determine the amount of cell-bound enzyme. Both culture fluids
and extracts were analyzed with respect to the type of plasmin
ogen activator they contained by immunoinhibition with goat
immunoglobulin G formed against purified human urinary urokinase sodium dodecyl sulfate:gel electrophoresis followed by
zymography. The study yielded the following conclusions: (a) the
measurement of plasminogen activator secretion rates gives a
much sharper differentiation between malignant and normal tis
sues than does the amount of extractable enzyme; (b) the
enzymes secreted in short-term organ culture are, in the great
majority of the cases studied, of the urokinase type, even when
a large fraction of the activator contained in the tissue is of the
vascular type; (c) the secretion rates of metastatic tumors of the
colon are much lower than those of the primary ones; (d) immunoperoxidase staining of tissue sections reveals that urokinase
is localized predominantly in the tumor cells. The low secretion
rates of metastatic tumors, probably a reflection of this property
in the original cell that gave rise to the metastatic focus, could
be of advantage to circulating cancer cells. Such cells would not
dissolve the microthrombus thought to be essential for the arrest
of cancer cells in the capillaries of target organs.
INTRODUCTION
The role plasminogen activators may be playing in the malig
nant behavior of cancer cells has been the subject of many
studies in recent years. While there is no unanimity concerning
their role(s) in this process, there are compelling reasons to
believe that they are of significance in more than one aspect of
neoplastic growth. There are several reviews dealing with these
enzymes (1, 20), including a recent one which is devoted entirely
to the relevance of the plasminogen activator-plasmin system to
malignant growth (12), as well as an organized collection of
relevant abstracts (27). Earlier studies from this laboratory dealt
1Supported in part by Grant BC-235 from the American Cancer Society and by
Grant 1 PO1 CA28853 from the National Cancer Institute.
2To whom requests for reprints should be addressed.
3 Eleanor Roosevelt Fellow of the International League against Cancer, on leave
from Miyazaki Medical College, Japan.
Received March 31,1983; accepted August 10,1983.
NOVEMBER
1983
with the analysis of plasminogen activators extracted from sur
gical specimens of human lung (14), colon (5), prostate (3), and
breast tumors (6), and from their normal tissue counterparts. In
these studies, the activator content was determined after quan
titative extraction, and the immunological identity of the activa
tors was established by inhibition studies with rabbit or goat IgG
directed against purified human urinary urokinase. In all these
studies, the amount of enzyme extractable from tumor tissue
was significantly higher than that from normal tissue. The pre
dominant type of activator, in most cases except prostatic tu
mors, was found to be of the urokinase type. In contrast, normal
tissues contained approximately equal proportions of urokinase
and vascular-type activator. Recently, several studies were pub
lished in which the plasminogen activator secretion of mammary
tumors, of both human (19) and rodent (17) origin, was measured
in short-term organ cultures. The advantage of this technique
over the measurement of extractable enzyme is that it yields a
functional parameter which is probably more relevant to the in
vivo behavior of a tumor than is the enzyme content, particularly
if one considers that the latter is likely to be simply the steadystate value resulting from the 2 opposing processes of enzyme
production and enzyme secretion, as was discussed earlier (6).
Its advantage over cell culture, aside from the greater simplicity
of the operation, is that here one measures the products of all
tumor cell variants present, rather than just those of a single,
and not necessarily typical, cell line selected for growth by a
particular set of culture conditions. The shortcoming of the
method, shared by all methods except cell culture, is that one
does not know the degree to which host cells present in the
tumors contribute to the enzyme activities measured. In many
cases, circumstantial evidence furnished by the pathology report
and analysis of adjacent normal tissues is sufficient to establish
the contribution of the tumor cells. In this paper, we present
immunohistochemical evidence of abundant presence of uroki
nase in cancerous colon tissue but not in the adjacent mucosa.
In the following, the results of short-term organ culture of
human tumor specimens comprising tumors of the lung, colon,
stomach, breast, and prostate, as well as a number of softtissue sarcomas, are presented. All specimens were also quan
titatively extracted, thus allowing a comparison between steadystate tissue enzyme levels and rates of enzyme secretion. The
main findings reported here are: (a) maximum secretion rates
give a sharper differentiation between tumor and normal tissues
than do determinations of extractable plasminogen activator; (b)
the secretion rates of metastatic colon tumors are significantly
lower than those of primary ones; and (c) the enzymes secreted
in short-term organ cultures are, in the great majority of the
cases, of the urokinase type, even when the tissue contains a
large proportion of vascular activator.
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G. Markus et al.
MATERIALS AND METHODS
Handling of Tumor Specimens. All specimens in this study were
obtained by surgery with none by autopsy. The surgical samples were
sent to the pathology department immediately after removal, where the
collaborating pathologist removed pieces of tumor and adjacent normal
tissue by a sterile technique and sent the specimen to our laboratory.
Short-Term Organ Culture. The technique described here was based
on the work of Recklies ef al. (19) and Ossowski ef al. (17). In most
cases, the surgical samples were immediately set up for organ culture.
When this was not possible, the tumor was kept in an ice-packed jar
until it could be worked up, usually not longer than a few hr. We found,
however, that not even overnight storage on ice impaired the secretory
ability of the specimens. The entire preparation was done under sterile
conditions. Small pieces of tissue were rinsed with Roswell Park Me
morial Institute Tissue Culture Medium 1640 (Grand Island Biological
Co., Grand Island, N. Y.) to remove blood from the cut surfaces, blotted,
and then cut up with a scalpel into 1- to 2-cu mm pieces, 10 to 15 of
which were placed on weighed rafts made of polypropylene monofilament
screen cloth with 1000-//m mesh openings (Small Parts, Inc., Miami,
Fla.), cut into 19-mm diameter discs. The rafts were weighed again,
placed in polyethylene screw caps (inner diameter, 22 mm), and floated
on 1 ml of Roswell Park Memorial Institute Tissue Culture Medium 1640
with L-glutamine, containing penicillin (100 units/ml) and streptomycin
(100 Mg/ml). No fetal calf serum supplement was used in this work in
order to avoid the introduction of inhibitors and of plasminogen into the
media, all of which would have interfered with the subsequent analysis
of activator and protease activity. The cultures in their screw caps were
placed into individual plastic Petri dishes and kept in a 37° incubator,
flushed with a water-saturated
gas mixture of 95% air:5% CO2. Media
were replaced daily, and the conditioned media were stored frozen for
subsequent analysis. In all cases, at least 2, but often 3 or 4, replicate
cultures were set up from the same tissue specimen. Bacterial contam
ination of cultures occurred only 3 times during this study and was easily
detected by rapid discoloration of the indicator in the culture medium
and by high protease activity, i.e.. high caseinolytic activity in the absence
of added plasminogen. Microscopic examination of smears obtained by
touching a glass slide to the fragments during culture fluid replacement
showed that, in the first 48 hr of culture, most cells so obtained looked
healthy and were not stained by trypan blue, but after this period, rapid
deterioration could be observed. We have also sectioned tissue frag
ments removed at daily intervals in 2 cultures and examined them after
staining with hematoxylinieosin. In one of these cases, namely, the breast
tumor shown in Chart 2, the tissue appeared healthy during the first 48
hr but showed signs of rapid decay beyond this period, which appeared
to parallel the decreasing slope of the plasminogen activator secretion
curve.
Extraction of Plasminogen Activator. This was carried out as de
scribed earlier with tissues that had been kept frozen (14), except that
this time, homogenization in the Tekmar Tissumizer was done in a
different extraction medium; instead of the one recommended by Nagy
ef al. (15), the tissues were suspended in the modified medium of Camiolo
ef al. (4) found recently to be 1.5 to 3 times more efficient for the
extraction of soft tissue. This medium has the following composition:
0.07 M potassium acetate:0.3 M NaCMO mM EDTA:0.1 M u-arginine:0.25% (v/v) Triton X-100, pH 4.2.
Activator Assay. The azocaseinolytic method used was described
earlier (14). Results were expressed in CTA4 units (11) and were calcu
lated with reference to a standard curve made with purified human
urinary urokinase (Winthrop Laboratories). Each sample was analyzed
for: (a) total activator activity by using azocasein to which human plas
minogen was added: (b) activator activity due to activators other than
urokinase, by including into the plasminogen-containing azocasein also
goat antibody formed against human urinary urokinase; and (c) protease
activity due to caseinolysis by active proteases, by omitting plasminogen
from the digestion mixture. Typically, to 1 ml of azocasein (9 mg/ml)
containing 15 /¿gof human plasminogen were added 30 to 100 ¿ilof
tissue extract or culture fluid. For the determination of activator type, 10
n\ of solution of goat anti-human urokinase antibody (1.3 mg/ml) were
included. This amount of antibody was found sufficient to inhibit >98%
of the urokinase activities encountered in the samples. The protease
activities (determined in the absence of plasminogen) were usually not
more than 5% of the total activity and were subtracted in order to arrive
at activator activities.
Polyacrylamide Gel Electrophoresis. This was described in detail
recently (6). The subsequent zymographic visualization of the activator
bands, using the substrate copolymerization technique of Heussen and
Dowdle (10) but utilizing casein instead of gelatin, was also described
recently.5
Immunoperoxidase Staining. Immunoperoxidase staining was per
formed according to published procedures (25). Surgically resected colon
cancer tissues were fixed in 10% formalin solution in 0.9% NaCI solution
and embedded in paraffin. These blocks were cut into 4-^m-thick sec
tions, mounted on glass slides, and dried at 60°for 2 hr. After deparaffinization, endogenous peroxidase activity was blocked with pure methanol containing 0.3% hydrogen peroxide for 30 min (22). The specimens
were then rinsed in distilled water and placed in 0.05 M Tris:0.1 M NaCI,
pH 7.6. The sections were first incubated with 1:40 dilution of a 25-mg/
ml stock solution of goat IgG directed against human urokinase for 60
min at 37°.Normal goat IgG, adjusted to the same protein concentration,
served as control. The sections were then incubated for 30 min with
rabbit IgG conjugated with horseradish peroxidase and directed against
goat IgG. The stock solution (Dako, Denmark, as supplied by Accurate
Chemicals, Westbury, N. Y.; Lot 102) was diluted 1:20 for use. All
dilutions of antisera were made in 0.05 M Tris:0.1 M NaCI. Antibody
localization was determined by detection of peroxidase activity after
reaction with a freshly prepared solution of 0.05% 3',3'-diaminobenzidine tetrahydrochloride (Sigma Chemical Co.) containing 0.01 % hydrogen
peroxide for 8 min at room temperature. The sections were washed in
distilled water, counterstained
with hematoxylin,
dehydrated, and
mounted.
RESULTS
The information collected in this study is summarized in Table
1, which shows the means of the extractable plasminogen acti
vator content from 6 tumor categories, as well as the means of
the maximum plasminogen activator secretion rates observed in
short-term organ culture of the same tumors. The type of plas
minogen activator in extracts and culture fluids was determined
by inhibition with goat antibody against human urokinase, and
the distribution between the 2 types was expressed as percent
age of total activator present as urokinase.
Tumor versus Normal Tissue. Table 2 presents the data
obtained with tumor-normal pairs from 5 lung cancer cases,
using both extraction and the measurement of secretion rates.
Chart 1 shows the time course of activator secretion from one
such pair. It can be seen that by extraction the tumors contain,
on the average, 2.33 times more activator than do their normal
counterparts, in fair agreement with the value of 3.3 obtained
earlier on a series of 37 paired lung samples (14). The maximum
secretion rates obtained with replicate samples from the same
tumors can be quite variable, probably reflecting degrees of
histológica! inhomogeneity in the cultures. Variable as these rates
may be, the difference between the secretion rates of the tumors
5 S. M. Camiolo, L. S. Englander, G. Markus. M. R. Siuta, J. E. Pontes, G. H.
4 The abbreviations used are: CTA, Committee on Thrombolytic Activity; EGF,
epidermal growth factor.
5518
Hobika, and S. Kohga. Plasminogen activator content in explants of benign and
neoplastic human prostate tissue, submitted for publication.
CANCER
RESEARCH
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VOL. 43
Plasminogen Activator Secretion of Human Tumors
Table 1
Summary of data on plasminogen activators in human tumors
se
cretion rate
CTA
of
(CTA units/
of
urokinase895
TissueLungTumorNormalColonTumor
units/g38.019.312.92±29°±13±
cases5521146211451111Maximum
9/hr)4.58
urokinase9766989793£+•+-+-+•97
cases5522176201351112
±3.60.17
±0.186.37
±51
±88
(primary)MetastasisNormalProstateTumorBenign
6.49.96
±93
±0.29
8.915.110.77.018.320.723.47±21±14±
±
±61
±0.63
±48
±1.19±0.71
±38
±94
hypertrophyStomach
3.5±
cancerBreast
5.1±20±28%of
±89
cancerSarcomasExtracted
±98+96±99±724231593242±36No.
±1.58±7.00.270.581.10.91.90.84
±
±728151540242561458 0.80.72
±41No.
±1.04%of
" Percentage of total activator activity inhibitable by goat antibody against human urinary urokinase (see
"Materials and Methods").
6 Corresponds to the steepest slope observed in plots of CTA units/g tissue versus hr in culture.
c Mean ±S.D.
Tables
Extractable plasminogen activator and activator secretion rates in lung tumors and
normal lung
Extracted
CTA units/
g tissue
Case
Tumor
78.3
Normal
38.3
Tumor:
normal
ratio
15.0
4.4
2.04
5
Tumor
Maximum secretion
rate8 (CTA units/g/hr)
51.9
15.4
3.7
o
0.13
12.8
2
o
14.2
22
Tumor
47.5
0.1
(7.65)
38.7
6.14
Normal
49
Tumor
6.3
0.06
8.1
0.43
0.16
10.6
75Hours 50
in Culturesomedato
)25
127.5
50
«>*>
Normal
22.8
0.76
Normal
500
<4-75'
0.90
Normal
^, 1.000
13.5
4.5
8
Tumor
Tumor
to
(A
(7.7)"
S
28.4
1,500
Tumor:
normal
ratio
1.83
Normal
Lung Tumor
0.018
25
0.007
Tumor
Normal
38.0 ±29e 2.33 ±2.2
19.6 ±13
4.43 ±3.4
0.16 ±0.19
45.3 ±48
50
75
Chart 1. Cumulativeplots of plasminogenactivator secretion in short-term organ
culture by a human lung adenocarcinomaand by adjacent normal lung tissue. The
time course of activator secretion by the normal lung tissue is enlarged at the
bottom of the chart to show continued enzyme output.
* Corresponds to the steepest slope observed in plots of CTA units secreted
per g of tissue versus hr in culture.
6 Numbers in parentheses, maximum secretion rate value per tissue.
c Mean ±S.D.
and those of the normal tissues is usually much greater than the
individual differences within replicate samples of either tumor or
normal tissue. What is important here is that ratios of the
secretion rates in the tumor-normal pairs are much larger than
can be obtained by extraction (mean ratios, 45.3 versus 2.33).
NOVEMBER
1983
As can be seen in Table 1, the same conclusions can be drawn
from a comparison of the secretion and the extraction results
from primary colon tumors and normal colon mucosa (10.0
versus 0.85). Normal breast tissues were not available in this
series; thus, the appropriate comparisons cannot be made for
secretion. Our earlier study has shown, however, that, as far as
extractable enzyme is concerned, tumors contain significantly
larger amounts than does normal breast tissue (6). Dexametha-
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G. Markus et al.
sonéinhibited plasminogen activator secretion in the case illus
trated by Chart 2, as it did in all other cases where it was tried.
This shows, among other things, that secretion from organ
culture is an active process and not merely leaking of tissuebound activator.
In the case of prostatic tumors, no significant difference was
found between the adenocarcinomas and prostates with benign
hypertrophy whether extractable amounts or secretion rates
were measured (surgical samples of normal prostates were not
available). As with breast tumors, earlier work on a much larger
case load did show a moderate but statistically significant ele
vation of extractable enzyme in the prostatic adenocarcinoma
specimens (3). The small stomach cancer series is distinguished
so far only by its remarkably uniform enzyme content, as shown
by the small S.D. Some of the sarcomas were distinct in that
they both contained and secreted large amounts of vasculartype activator. The character of the secretion curves varied from
case to case. In 60% of the tumors studied, the initial secretion
rate (slope of curve from 0 to 24 hr) was the highest in a run,
while in 40%, the rate gradually increased in the first 48 hr. Most
of the colon tumors belonged to the first type, while many of the
prostatic tumors showed varying degrees of initial acceleration.
Types of Plasminogen Activators Found in Extracts and
Secretions. Earlier studies from this laboratory, quoted above,
indicated that the predominant activator in extracts of tumors of
Dexamethasone
50
10
M
100
150
Time in Culture (hours!
Chart 2. Cumulative plots of plasminogen activator (PA) secretion in short-term
organ culture by a human adenocarcinoma of the breast in 4 replicate cultures.
The 2 bottom curves show suppression of secretion by 1CT5 M dexamethasone
present in the Roswell Park Memorial Institute Tissue Culture Medium 1640.
5520
the lung (14), colon (5), and breast (6) is of the urokinase type.
In prostatic tumors, however, urokinase and vascular-type acti
vators were about equally represented (3). This was also true
for normal tissues with the exception of normal breast tissue,
which was mostly of the urokinase type. These conclusions hold
also for the series of tissues presented here. The measurement
of plasminogen activators in conditioned media of short-term
organ cultures now shows that the predominant activator se
creted into the culture media by tumors is urokinase. This is the
case even when the tissue extracts contain appreciable amounts
of vascular activator, as in the case of one of the tumors
illustrated in Fig. 1. This figure shows the zymographic visualiz
ation of electrophoretically distinct plasminogen activator com
ponents derived from extracts and culture fluids of a primary
gastric and a primary colon tumor. A comparison of the visual
evidence gained by inspection of zymogram pairs with the ana
lytical results obtained by azocaseinolysis in the presence and
absence of antiurokinase antibody shows good overall agree
ment in the activator composition of these samples. The zymograms, in addition, show that urokinase occurs mostly as the M,
55,000 species, with smaller amounts having mobilities corre
sponding to smaller molecules. Vascular activator in these spec
imens occurs only as the M, 70,000 species, but other zymograms often contain some M, 100,000 vascular activator, as well
as a M, 95,000 urokinase.
Comparison of Primary and Metastatic Colon Tumors. This
study yielded the unexpected result that metastatic colon tumors
secrete plasminogen activator at a much lower rate than do
primary ones. Table 3 shows both extraction and secretion data
of these colon tumors. While the extractable amounts are not
significantly different (at least not at the present number of cases
collected), the difference between the mean secretion rates, 6.31
and 0.29, is highly significant (p < 0.001). The ratio of the means
in this group is 22. Confidence in this result is greatly increased
when we consider Cases 64, 68, 72, and 214, where primary
and metastasis were removed together from the same patients.
In all 4 cases, the maximum secretion rate is at least 10 times
higher in the primary than in the corresponding metastasis, and
in Case 72, this ratio is 500. Chart 3 illustrates 2 of these cases.
While the slopes or the general height of the curves for the
primary tumors is variable, in the replicate runs, some character
istic features of each tumor are well conserved. Thus, the 50-hr
initial lag period in Colon Tumor 68 or the absence of lag and
the presence of a change in the slope between 50 and 75 hr in
Colon Tumor 72 are characteristic individual features of each
tumor. While the curves for the metastatic tumors look flat on
these plots, they do possess a definite slope; i.e., they still
secrete enzyme. It should also be added that most primary colon
tumors behave like Tumor 72, rather than Tumor 68 (Chart 3);
i.e., secretory activity is already very high, or is highest, in the
first 24 hr of culture. Metastatic tumors, on the other hand,
exhibit low activities already in the first time period, indicating
that the property of low secretory activity in this group of tumors
is not due to progressive tissue death but is present at the time
the cultures are set up.
It was of importance to establish that the large difference in
the secretion rates of primary and metastatic tumors was not
due to trivial causes, such as a greater degree of necrosis or a
larger relative amount of connective, or liver, tissue in the métas
tases. In order to evaluate the effect of these variables, the slides
of the 4 cases (Cases 64, 68, 72, and 214) where primary and
CANCER
RESEARCH
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VOL. 43
Plasminogen Activator Secretion of Human Tumors
Tabte3
Plasminogen activator content and secretion in primary and metastatic adenocarcinomas
of the colon
Primary tumors
activatorStage
Plasminogen
(Duke'sclassifica-Case575964e206882223321968e214e91932182272247072e8223221723442Age7771556865565260696277606667656773686166651863
originLeft
units/gtissue20.216.623.714.58.810.18.913.43.09.38.822.421.38.714.64.89.918.014.28.055.01
rate(CTA
urokinase0859696949885839493100959747868997999796878690.5
of
units/g/hr)c4.81.62.820.01.023.14.80.834.30.89.42.411.
of
colonSigmoidCecumRectumRectosigmoidSigmoidSigmoidSigmoidSigmoidSigmoidSigmoidRectumSigmoidSigmoidSigmoidCecum
WD-MDINFMDMDMDMD-PDMD-PDINFMDINFMDINFMDINFMDINF
MD-PDINFPDMucinousMucinousMucinousINF
colonCecumRight
and right
colonRectumIleocecumSigmoidLeft
mucinousINF
mucinousSignet
colonPathologyWDdWDWDWD-MDINFringtion)8DC2DC1B282DDDC2B1DCIB2C1B2DC1B2DB2C2ExtractionCTA
7.04922%ofurokinase"98971001009895979710098989
±
1.422Métastases44465064e207297190213221233689214e20820922572e7357685569645768616051696265311973MFMMFFMFMFMMFFFFFLiverLiverMesenteric
±6.0221%
321SecretionMaximum
±11.
±
±22"SexFMMMMFMMMFMFFMMFFMFFMF12'Site
nodeLiverAbdominal
lymph
wallLiverOmentumLiverLiverOmentumLiverLiverMesenteric
nodeLiverOmentumAbdominal
lymph
MD-PDPDPDPDMucinousDDDDDDDDDDDDC2DDDD11.03.610.13.99.46.415.015.937.08.256.30.58.93.29795971009196951001
wallMesenteric
lymph nodeWDWDWDWDWD-MDMDMDMDMDMDMDMD-PDINF
59 ±14'
17"
9.96 ±8.9
14
92.8 ±15.5
13
0.29 ±0.279
17
97.2 ±5
17
a See Ref. 2.
" Percentage of total activator activity.
c Corresponds to the maximum slope obtained in plots of activity versus time.
a WD, well differentiated; MD, moderately differentiated; INF, infiltrating; PD, poorly differentiated.
e In these cases, primary and metastasis were removed from the same patients.
' Mean ±S.D.
9 The difference between the 2 means in this column is highly significant (p < 0.001) by Student's i test.
h Number of cases.
metastatic tumors were both available from the same patients
were carefully examined. Two of the primaries and 3 of the
métastases showed some necrotic areas; in Case 72, neither
primary nor metastasis was necrotic. The métastasesshowed
relatively more connective tissue than did the primaries, and
islands of liver tissue were indeed present in the 2 liver métas
tases. However, the differences between primary and metastatic
tumors in these 2 respects were relatively small and could not
have accounted for the 10- to 500-fold differences in the secre
tion rates measured in these paired samples. The most convinc
ing argument against dominance of noncancerous tissue as the
reason for the large difference in the secretion rates is the
NOVEMBER
1983
following. Table 3 shows that the average amount of extractable
plasminogen activator in the 2 groups consists of about 90%
urokinase. Control measurements on the predominantly connec
tive tissue of the separated colon submucosa (verified histologically) showed that the activator extractable from this tissue
consisted of 90% vascular activator and only 10% urokinase.
Since the extractable activator in the métastasesis 93% uroki
nase (Table 3), connective tissue could not have represented a
sizable fraction of the tissue used for extraction and secretion
measurements. The presence of significant amounts of normal
liver tissue in the liver metastasis samples can also be excluded
by the finding that normal liver tissue contains only about 0.1
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G. Markus et al.
1000
tOOO-
ChartS. Cumulative plots of plasminogen
activator secretion in short-term organ culture
by the 2 primary colon adenocarcinomas and
their métastases (MefasÃ-.), to the liver in the
case of Colon Tumor 68, and to a mesenterio
lymph node in that of Colon Tumor 72. The
differences in the activities of the replicate cul
tures of the tumors probably reflect histological
as well as functional inhomogeneity in the tumor
fragments used for the experiments.
750-
750-
500-
500-
u
250-
250-
50
(00
(50
Hours in Culture
units of activator per g of tissue; a larger representation of
normal liver tissue in the metastatic samples would have de
creased the extractable activator by much more than 20%.
Finally, the difference in activator secretion rates could have
been due to the release into the medium of inhibitors of activation,
or of plasmin action, by the metastatic tissues. To examine this
possibility, 3 culture fluid specimens from metastatic and 3 from
highly active primary tumors were mixed with Sepharose-coupled antiurokinase antibody to absorb all endogenous activator
activity. After separation from the gel, a standard amount of
purified urokinase was added to the culture fluids, and 15 min
later, azocaseinolytic activator assays were carried out to see if
the added urokinase activity had been inhibited by the condi
tioned media. Controls indicated that absorption of endogenous
activator was complete in all cases. One of the culture fluids
from a metastasis showed a 25% inhibition of the added uroki
nase; none of the other samples was inhibitory. The inhibitory
power of that sample, however, could have only neutralized 1%
of the activator activity of the culture fluid from the average
primary tumor and thus could not have been the cause for the
low secretion rates of the metastatic tumors. These tests, how
ever, only proved that metastatic tumor did not release enough
inhibitor to be detected in the culture fluids. It was therefore
considered that the inhibitor levels in the fluid may have been
low due to their instantaneous removal by cells. To test this
possibility, fragments of a metastatic tumor of low secretory
activity were cocultivated with fragments of a highly active
primary tumor. The resultant secretory activity, however, was
indistinguishable from that of the primary tumor by itself. Thus,
it appears that the average of 22-fold-lower secretion rate in the
metastatic tumors cannot be due to any of these causes alone
and must represent a genuine difference in the ability of these
tumors to secrete plasminogen activator into the culture medium
under the prevailing experimental conditions. What cannot be
excluded at present is the possibility that the observed difference
in the rate of plasminogen activator secretion could be a reflection
of a differential reactivity of primary and metastatic tumors to an
environmental factor, e.g., gas composition, or the presence or
absence of certain nutrients in the culture medium. If this were
found to be true in the course of further studies, an equally
important, and perhaps more fundamental, difference could be
defined.
5522
50
(00
(50
Hours in Culture
Besides the difference just discussed, no statistically signifi
cant differences were as yet found between extractable plasmin
ogen activator or secretion rate, on one hand, and a number of
other parameters, on the other. These included degree of differ
entiation, stage of disease, infiltrative tendency, and segment of
the large bowel involved. It should be stated, however, that, in
some of the cases, a larger number of specimens would have
very likely yielded significant differences. Thus, the mean secre
tion rate of infiltrative tumors was about twice that of the noninfiltrative ones (8.76 versus 4.38), and rates were also higher in
tumors of the left colon than those of the right (7.1 versus 3.88).
Such marginal significance was also present in a comparison
between Stage B (8.27) and Stage D (4.4) cases. In all of these
comparisons, however, p was greater than 0.05. It seems very
probable that, as a greater number of cases will accumulate,
some of these differences will become significant.
Visualization of Urokinase in Colon Cancer by the Immunoperoxidase Technique. As pointed out above, there is always
some uncertainty in the interpretation of activator measurements
obtained from work with tissue fragments, since it is not possible
to be certain that the enzyme was contained in, or was secreted
by, the tumor cells alone. One way to solve this problem is to
visualize the enzyme histochemically. Fig. 2 shows that, by using
an indirect immunoperoxidase technique, the location of uroki
nase can be visualized in the tumor tissue. The reaction is almost
completely restricted to the tumor, with only an occasional goblet
cell showing it in the normal mucosa. The heaviest stain was
seen at the luminal surface of the glandular structures formed
by the cancer cells and also at the basal area of these cells.
Reactive material was also observed in many of the lumina,
corresponding to desquamated cells. Control slides (not shown)
prepared by the use of nonspecific goat IgG, instead of antiuro
kinase goat IgG, showed minimal staining of the cancer tissue.
It should be noted that Paul ef al. (18) were the first ones to
demonstrate the presence of plasminogen activator by an im
munoperoxidase technique in cultures of porcine kidney cells.
DISCUSSION
Nature of the Secretion of Plasminogen Activators by Can
cer Tissue. It is important to establish that the observed release
of plasminogen activator into the culture medium is an active
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Plasminogen Activator Secretion of Human Tumors
biological process and not leakage of enzyme from a disinte
grating tissue. The following observations indicate that we are
indeed dealing with a secretory process, (a) During the period of
culturing, the tissue releases much more enzyme than is con
tained in the specimen at the start of the culture period. For
example, Colon Tumor 72, illustrated in Chart 3, contained a
total of 0.087 CTA units in the 18 mg of tissue set up for culture,
as determined by extraction of the bulk of the tumor. During the
150 hr in culture, a total of 15 CTA units of activator was released
into the medium. Thus, the tumor produced 172 times more
enzyme in 150 hr than it contained at zero time. Recent results
from this laboratory,5 obtained by extraction of tissue particles
(in this case, prostate) that had been used for organ culture,
indicate that activator contained in the tissue, far from being
exhausted, is actually greatly enriched at the end of the life of
the tissue in culture, (b) Dexamethasone, a glucocorticoid shown
to inhibit plasminogen activator secretion in many systems and
found effective in the present study as well (Chart 2), exerts its
effect by an active process which requires de novo protein
synthesis (21). Work of others furnished other examples of
hormone effects on activator secretion in organ culture (e.g.,
Ref. 17).
Plasminogen Activator Secretion and Metastasis. The po
tentially most important observation made in this study was that
the secretion rates of metastatic tumors were significantly lower
than those of the primary ones. While this conclusion is based
mostly on data from colon cancer, by far the largest group in the
present study, similar results obtained with a smaller number of
other types of cancer suggest that it will hold for other metastasizing carcinomas as well. It is important to point out that the
difference is far more pronounced in the secretory activities;
extractable, i.e., tissue-bound, plasminogen activator is only
slightly less in the métastases.While at present it is not possible
to unequivocally interpret the phenomenon, attention may be
drawn to some areas of potential relevance. Gross ef al. (9) have
reported recently that the down-regulation of the EGF receptor
that follows addition of EGF to a culture of human epidermoid
cells is accompanied by the induction of plasminogen activator
synthesis, the 2 events occurring with identical kinetics. Further
more, blocking of plasminogen activator synthesis or activity or
inhibition of plasmin action blocks both EGF-induced receptor
down-regulation and the increase of plasminogen activator activ
ity itself. If these observations are relevant to our results, they
may imply that primary and metastatic tumors could differ in their
responsiveness to growth factors. This important question is
now under investigation. The results are also in harmony with
current views on the role of hemostatic and fibrinolytic factors in
the growth and spreading of tumors. It is accepted by many that
the successful lodgement of circulating tumor cells in small blood
vessels of target organs requires the development of a small
thrombus consisting of platelets and fibrin. [This theory and the
extensive experimental evidence relevant to it were reviewed
recently by one of us (13).] It would not be unexpected to find
that cells that give rise to metastatic foci should not be secretors
of plasminogen activator, as this property, if present, would
result in activation of blood plasminogen to plasmin, with sub
sequent destruction of the microthrombus deemed essential for
arrest and continued proliferation. This theory appears reason
able, since experimental studies indicate that: (a) primary tumors
exhibit gross phenotypic heterogeneity, particularly with respect
to metastatic potential (7) and, therefore, could well contain
NOVEMBER
1983
subsets of cells with greatly diminished ability to release plas
minogen activator; (b) the metastatic process is very inefficient
in that only a minute fraction of circulating cancer cells actually
give rise to métastases(26), implying that the cells that do so
do not have to exhibit the average properties of the cells of the
primary tumor; and (c) métastasesappear to be of monoclonal
origin, or at least to develop from a few cells only (24), and are
thus likely to exhibit phenotypic properties of the parent cell.
We emphasize that, while this discussion had dealt with the
relevance of only one property, fibrinolytic potential for metas
tasis, there must be many others of equal or greater significance.
The experiments of Nicolson (16) have established the essential
role of cell surface glycoproteins in determining both metastatic
ability and organ preference for metastasis. It seems evident that
the ability of a cancer cell to successfully initiate a metastasis
will always be determined by a combination of specific properties,
each of which will contribute to its ability to escape the defense
mechanisms of the host.
Fibrinolytic Activity of Colon Tumors. Szczepanski ef al. (23)
have investigated recently the fibrinolytic activity of human colorectal tumors in surgical specimens, using a modification of the
histochemical method of Todd for the visualization of lysed areas
in a fibrin overlay, and by fibrin plate analysis of KSCN extracts
of tissues. They found that the fibrinolytic activity of normal colon
mucosa was higher than that of colorectal tumors and that
fibrinolytic activity in tumors was restricted to perivascular zones.
In interpreting their results and comparing them with our earlier
study (5), they assumed correctly that the differences could be
explained by the fact that vascular activator, in the presence of
fibrin, is far more efficient than urokinase in activating plasmino
gen (3). This explanation indeed fully accounts for the differences;
while the total amount of activator (by azocaseinolysis) is much
greater in colon cancer tissue than in normal mucosa, about half
of the activator content of the normal mucosa is of the vascular
type, with the tumor containing less than 10% of that type of
enzyme (5). In any assay, therefore, which greatly favors the
vascular-type enzyme, the activity of the mucosa will appear to
be greater than that of cancer. This interpretation is also sup
ported by the finding in the study quoted that activator activity
is localized to the perivascular areas. It should be noted that
similar observations to those of Szczepanski ef al. have been
published earlier by Franklin ef al. (8). Since they also used fibrin
overlay techniques, their data can be interpreted similarly. The
above comments point out the necessity, when discussing plas
minogen activator results, to be aware of the type of assay that
was used; clearly, different assays emphasize different functional
aspects of plasminogen activation. The immunohistochemical
evidence presented in this paper established that urokinase is
indeed present in the neoplastic portions of human colon tumors.
ACKNOWLEDGMENTS
The authors wish to express their gratitude to Dr. John L. Evers and Dr. Grant
H. Hobika for carrying out the electrophoresis runs and to Professor Désiré
Collen
of the University of Luvain, Belgium, for his kind gift of the melanoma-derived
activator.
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Vase
Act
Colon
Extract
%UK:
95
97
100
Ca
Cult. Med
0
Fig. 1. Sodium dodecyl sulfate:polyacrylamide gel electrophoresis of tissue extracts and culture fluids, followed by zymographic visualization of plasminogen activatorcontaining bands in the presence and absence of goat antibody against human urinary urokinase. Gastric Ca, primary adenocarcinoma of the stomach; Colon Ca. primary
adenocarcinoma of the colon; UK, purified M, 55,000 human urinary urokinase; Vase. Act., vascular-type M, 70,000 plasminogen activator, purified from the culture fluid
of a human melanoma cell line (kind gift of Dr. D. Collen); Ab, goat antiurokinase antibody; %UK (anal.), percentage of activator activity inhibitable by antiurokinase
antibody, as determined by azocaseinolysis in the presence of plasminogen; 70K, 55K, and 33K, molecular weight in thousands. Note: none of the samples shown here
exhibited proteolytic activity when examined in the absence of added plasminogen. Experiment was carried out using the substrate copolymerization technique of
Heussen and Dowdle (10): the 8.5% acrylamide gel contained 1 mg of casein and 12.7 ng of human plasminogen per ml. and where indicated, also 25.7 ^g of the
antiurokinase antibody per ml. Activator applied. 0.001 to 0.0025 CTA units/lane.
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VOL. 43
Plasminogen Activator Secretion of Human Tumors
Fig. 2. Immunoperoxidase staining for urokinase in a primary colon adenocarcinoma (Table 3, Case 227), counterstained with hematoxylin. A, left side of picture
showing the normal mucosa with only goblet cells stained. Short arrow points to a normal gland. Right side is occupied by moderately differentiated adenocarcinoma
which shows many areas of dark staining. Long arrow points to one of the glands, x 80. B, higher magnification (x 200) of an area from the same field. Strong
accumulation of dark reaction product can be seen at the luminal (long arrow) and the basal area of the tumor cells which form the glandular structures. Short arrow,
strongly stained desquamated cells which occupy the lumina of the glands.
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5525
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Plasminogen Activator Secretion of Human Tumors in
Short-Term Organ Culture, Including a Comparison of Primary
and Metastatic Colon Tumors
Gabor Markus, Sarah M. Camiolo, Shin Kohga, et al.
Cancer Res 1983;43:5517-5525.
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