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1
The Finsen Laboratory, Rigshospitalet, Copenhagen, Denmark.
a Address correspondence to this author at: The Finsen Laboratory, Strandboulevarden 49, DK-2100 Copenhagen, Denmark. Fax +45 31 38 54 50; e-mail p1000027{at}inet.uni-c.dk
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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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Measurement of the protein components of the urokinase system in tumor extracts reveals that high concentrations of uPA, uPAR, and plasminogen activator inhibitor type 1 (PAI-1) are associated with shorter survival of breast cancer patients (16)(17)(18)(19)(20)(21)(22)(23), and therefore may be used as prognostic markers. Clearly, determination of these indicators has value in the clinical setting, but suitable tissue extracts require careful tissue handling, compete with the needs of histopathology, and need optimized and calibrated extraction procedures. It would seem highly desirable to replace the requirement for a tissue sample with a sample of peripheral blood. This would also permit monitoring of the effect of treatment and the course of disease progression, which is not possible when only surgically removed tumor tissue samples are analyzed.
It is therefore important to note that soluble uPAR (suPAR) has been detected in plasma (24), and it seems plausible that this form has been released from the surface of cells. The cell-surface receptor consists of a glycolipid-anchored three-domain 60-kDa glycoprotein, the N-terminal domain 1 of which contains the binding site for the growth factor domain of uPA (25)(26). Cleavage of the glycolipid anchor by a phospholipase (25), and proteolytic cleavage between domains 1 and 2 (27), are two known ways in which release of soluble receptor forms may occur. Because the proteolytic activity of uPA can cleave its own receptor both in vitro and in vivo (28)(29), measurement of suPAR in blood may be a more accessible and reliable indicator of the activity of the uPA system in vivo. In this paper we extend our earlier findings (24) to better define optimal conditions for assay of suPAR in human blood and to establish values in healthy donors. Further, we report preliminary findings of increased plasma suPAR concentrations in patients with advanced cancers.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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blood collections and plasma and serum separation
Peripheral venous blood was drawn into prechilled citrate, EDTA,
or heparin collection tubes (Becton Dickinson) and quickly mixed by
inversion. The plasma was separated from blood cells within 1.5 h
by centrifugation at 4 °C at 1200g for 30 min, and stored
frozen at -80 °C before assay. Blood collected into dry tubes was
allowed to clot at room temperature and the serum was carefully
collected no later than 30 min from collection. Plasma and serum pools
were made with freshly collected samples from at least 10 donors,
aliquoted, and stored frozen at -80 °C.
modified supar elisa
Immunoassay plates (Maxisorp, Nunc) were coated for 16 h at
4 °C with 100 µL/well of purified rabbit anti-human uPAR IgG (0.5
mg/L) in 0.1 mol/L carbonate buffer, pH 9.5. This catching antibody was
previously absorbed on a column of mouse IgG to reduce the ELISA
background. Before use, the assay wells were rinsed twice with 200
µL/well of SuperBlockTM solution (Pierce Chemicals)
diluted 1:1 with PBS, followed by three washes with PBS containing 1
g/L Tween 20. Wells were then treated for 1 h at 37 °C with 100
µL/well of triplicate or duplicate 1:10 dilutions of plasma or serum
made in a sample dilution buffer of 50 mol/L phosphate, pH 7.2, 0.1
mol/L NaCl, 10 g/L bovine serum albumin (Fraction V, Boehringer
Mannheim), and 1 g/L Tween 20. On every assay plate a series of
calibrators was included that consisted of seven serial dilutions in
triplicate of purified recombinant suPAR (i.e., uPAR lacking the
glycolipid anchor; see ref. 30), starting from 1 µg/L,
then 0.5, 0.25, 0.125, 0.0625, 0.0313, and 0.0156 µg/L. Also included
on each plate were triplicate blank wells containing only sample
dilution buffer, and triplicate wells of a 1:10 dilution of a control
citrate plasma pool.
After suPAR binding, the wells were washed six times, then treated for
1 h at 37 °C with 100 µL/well of a mixture of three murine
monoclonal anti-human uPAR antibodies [R2 (23 µg/L), R3 (281
µg/L), and R5 (70 µg/L); see refs. 24 and
31] in sample dilution buffer. After six washes the wells
were then incubated for 1 h at 37° with 100 µL/well of rabbit
anti-mouse immunoglobulins–alkaline phosphatase conjugate (Dako)
diluted 1:1000 in sample dilution buffer. After six washes with washing
solution and three washes with pure water, 100 µL of freshly made
p-nitrophenyl phosphate (Sigma) substrate solution (1.7 g/L
in 0.1 mol/L Tris-HCl, pH 9.5; 0.1 mol/L NaCl; 5 mmol/L
MgCl2) was added to each well and the plate was placed in a
Ceres 900TM plate reader (Bio-Tek Instruments). The yellow
color development at 23 °C was monitored automatically, with
readings taken at 405 nm against an air blank every 10 min for 60 min.
KinetiCalc II software was used to manage the data, calculate the rate
of color change for each well (linear regression analysis), and compute
from the rates for the suPAR calibrators a four-parameter fitted
calibration curve from which the suPAR concentration of each plasma or
serum sample was calculated. Development of color in each well was a
linear function of time for all concentrations of suPAR measured in
these experiments (see Fig. 1
A), with correlation coefficients for the automatically fitted
lines typically better than 0.97. The calibration curve of the rates
plotted against the suPAR concentration was slightly sigmoidal from 0
to 1.0 µg/L and the correlation coefficient for the four-parameter
fit was typically better than 0.999 (see Fig. 1B
). The rate with no
suPAR (read against air) was 0.167 ± 0.039 (mean ± SD)
milliabsorbance units/min (n = 46), whereas the rate with 1.0
µg/L calibrator suPAR was 12.16 ± 1.48 milliabsorbance
units/min (n = 46). The limit of detection for the assay, defined
as the concentration of suPAR corresponding to a signal 3 SD above the
mean for the suPAR blank, was 16 ng/L or 1.3% of the mean
concentration found in healthy plasma. The intraassay CV for 10
replicates of a control citrate plasma pool measured on the same plate
was 6.5%, and the interassay CV for 24 successive assays of the plasma
pool (on different days) was 14%. This plasma pool had a suPAR content
of 1.07 µg/L. The interassay CV of another plasma pool with suPAR
content of 1.83 µg/L was 11% (n = 27). Thus the precision did
not change markedly across a suPAR concentration range representing a
large proportion of the healthy blood samples assayed (median suPAR
concentration 1.2 µg/L).
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immunoabsorption of supar in citrate plasma pool
Citrate plasma pool (1.0 mL) was diluted 1:10 with sample dilution
buffer (9 mL), then preabsorbed to remove human IgG by repeated passage
through a 0.6-mL column of protein A–Sepharose (Pharmacia). The
preabsorbed plasma (2 mL) was applied to a 0.4-mL column of protein
A–Sepharose cross-linked through dimethylpimelimidate
(32) with 48 µg of monoclonal antibody against
trinitrophenyl hapten (anti-TNP; irrelevant antibody control
(24)). Another aliquot of preabsorbed plasma (2 mL) was
applied to a 0.4-mL column of protein A–Sepharose cross-linked with a
mixture of two monoclonals against uPAR, R4 and R9 (each 48 µg), and
a further aliquot (2 mL) was applied to a 0.4-mL column of protein
A–Sepharose cross-linked with 48 µg of rabbit anti-uPAR (suPAR ELISA
catching antibody). Samples of plasma pool before preabsorption, after
preabsorption, after anti-TNP absorption, after R4/R9 absorption, and
after anti-uPAR absorption were assayed for suPAR by ELISA as above.
recovery of calibrator supar signal in plasma and serum pools
The recovery of signal from calibrator suPAR was measured after
addition to 1:10 dilutions of citrate and EDTA plasma pools, and a 1:10
dilution of serum pool. Calibrator suPAR was added to these solutions
to final concentrations from 0 to 1.0 µg/L. The recoveries in each
case were calculated from the slopes of the lines representing suPAR
signal as a function of concentration, where 100% recovery was defined
as the slope obtained when suPAR was diluted in the sample dilution
buffer.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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).
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The contribution of each monoclonal detection antibody to ELISA signal
was determined by measurements with monoclonal mixtures lacking one of
the three monoclonal antibodies at a time. In the concentration range
of suPAR calibrator comparable with that found in a 1:10 dilution of
plasma or serum from a healthy donor (i.e., 0.1 to 0.2 µg/L), R2
contributed ~65–70% of the ELISA signal and R3 contributed 25–35%
(Fig. 2
), whereas R5 only contributed significantly at higher suPAR
concentrations (not shown). A closely similar epitope pattern was found
for the signal from 1:10 dilutions of citrate plasma pool, EDTA plasma
pool, and serum pool (see Fig. 2
). Thus the epitope contributions to
the signal for endogenous suPAR in plasma and serum were consistent
with the presence of full-length suPAR, comprising all three protein
domains.
immunoabsorption of supar elisa signal
When citrate plasma pool was absorbed on a protein A–Sepharose
column with two anti-uPAR monoclonal antibodies (R4 and R9) different
from those used in the ELISA, the suPAR signal in the subsequent ELISA
was reduced by ~80% (Fig. 3
). The epitope recognized by R4 is located in domain 3 of uPAR,
whereas R9 binds to an epitope in domain 1 (9). Absorption
with protein A–Sepharose alone removed only some 10% of the signal,
as did protein A–Sepharose with an irrelevant monoclonal antibody
(anti-TNP) of the same IgG subclass (Fig. 3
). If the plasma pool was
absorbed with the same rabbit polyclonal antibody as used for catching
in the ELISA, then the signal was totally abolished (Fig. 3
).
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recovery of supar calibrator after dilution in plasma and serum
Specific signal recovery was determined by addition of increasing
concentrations of purified suPAR calibrator to a fixed 1:10 dilution of
plasma or serum pool and subsequent measurement of the ELISA signal. In
diluted citrate plasma pool, 96% recovery of suPAR signal was
obtained, 91% in diluted EDTA plasma pool and 94% in diluted serum
pool (Fig. 4
A). Loss of specific suPAR signal after addition to samples was
therefore barely significant.
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dilution curves for plasma and serum supar signal
The suPAR ELISA signal produced by different dilutions of citrate
plasma pool, EDTA plasma pool, and serum pool are shown in Fig. 4B
.
Serum gave the best linearity of signal as a function of dilution,
while EDTA and citrate plasmas showed some curvature. However, the
error due to nonlinearity was not considered significant at 1:10
dilution and this dilution was therefore used in all subsequent
determinations.
supar in citrate plasma and serum from the same healthy donors
A collection of citrate plasma and serum samples taken
simultaneously from 93 healthy donors was available for this study. The
percentile plots for suPAR concentrations determined in these samples
are shown in Fig. 5
. The values in each set approximated a normal distribution; the
citrate plasma suPAR concentrations had a reference range (10th to 90th
percentile) of 0.82 to 1.7 µg/L, and the mean of 1.2 ± 0.34
µg/L was indistinguishable from the median (Table 1
). Similarly, the reference range for the serum suPAR
concentrations was 0.92–1.8 µg/L, and the mean of 1.3 ± 0.39
µg/L was close to the median of 1.2 µg/L (Table 1
). A paired means
comparison showed that the concentration in citrate plasma is
significantly lower by 0.12 µg/L (95% lower 0.077, 95% upper 0.16,
P <0.0001) than the concentration in serum from the same
individual. However, this likely reflected the small and variable
systematic error (x ~9/10) introduced by dilution of whole
blood with citrate buffer in the plasma preparation. The concentration
of suPAR in serum correlated with citrate plasma from the same
individuals: The linear regression plot in Fig. 6
has a regression coefficient of 0.84, and a nonparametric
Spearman's rank test for the data set gave 
= 0.84 and P
<0.0001.
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tests for correlations to gender and age of donor
The percentiles, divided according to gender, for suPAR in 93
serum samples are shown in Fig. 7
. The median value for 51 men was 1.1 µg/L and for the 42
women in this set it was 1.4 µg/L. A Mann–Whitney U-test
indicated that a difference may exist between the two populations
(P = 0.04). There was no significant correlation
between serum suPAR concentration and age of the donors (Spearman's
= 0.12, P = 0.24).
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plasma sets prepared with edta and heparin
Two other sets of plasmas from healthy donors, made with EDTA and
heparin anticoagulants, were analyzed for suPAR. For EDTA plasmas
(Table 1
), the reference range for 44 donors was 1.2–1.9 µg/L with a
mean of 1.5 ± 0.34 µg/L and a median of 1.5 µg/L. For heparin
plasmas, the reference range for 46 donors was 0.83–1.7 µg/L with a
mean of 1.2 ± 0.41 µg/L and a median of 1.1 µg/L (Table 1
).
Unpaired t-tests showed that there was no significant
difference between the means for serum and heparin plasma
(P = 0.11), and between citrate and heparin plasma
(P = 0.93). There were significant differences between
the means for serum and EDTA plasma (P = 0.003),
citrate plasma and EDTA plasma (P < 0.0001), and
heparin plasma and EDTA plasma (P = 0.0001). The mean
concentration of suPAR in EDTA plasma was 0.33 µg/L higher than the
mean for citrate plasma (95% lower 0.21, 95% upper 0.45). Thus it is
important that the same blood preparation is used consistently for
comparisons of different donor groups, or for comparison of healthy
donor sets with patient material.
preliminary studies of plasma supar concentrations in patients with
advanced malignancies
A pilot study was carried out in which the suPAR concentrations
were measured in 19 citrate plasma samples from advanced (stage IV)
breast cancer patients (see Fig. 8
). The 10th to 90th percentile range of suPAR in the breast
cancer plasmas was 1.9–7.1 µg/L, with a mean of 3.9 ± 4.0
µg/L and a median of 2.9 µg/L, compared with the reference range of
0.82–1.7 µg/L with a mean of 1.2 ± 0.35 µg/L and a median of
1.2 µg/L for 42 citrate plasma samples from healthy female donors. A
Mann–Whitney U-test for this data indicated a highly
significant difference with P <0.0001, but the data set
reported is small and all the patients had advanced disease.
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We also measured suPAR concentrations in citrate plasma from 10 patients with Duke's stage D colon cancer, and found a 10th to 90th percentile range of 1.4–4.7 µg/L, with a mean of 2.3 ± 1.3 µg/L and a median of 1.9 µg/L. This was also significantly different (Mann–Whitney U-test, P <0.0001) from the suPAR concentration in citrate plasmas from healthy individuals.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The epitope contributions to total suPAR signal that we have found in ELISAs of healthy donor plasma and serum samples suggest that suPAR is present in plasma as the full-length, three-domain receptor protein. Many cell-surface proteins have been found to have soluble counterparts (35), and in vivo this gives rise to soluble receptors circulating in the blood. In some cases these have already proven to be useful diagnostic indicators (e.g., 36–38). The occurrence of suPAR in healthy human plasma has been conclusively demonstrated previously by immunoaffinity purification and cross-linking to 125I-labeled amino-terminal fragment of human uPA (24). Thus in these earlier studies the soluble receptor present in plasma was able to efficiently bind its ligand, and therefore most probably consisted of more than domain 1. If the suPAR had been proteolytically cleaved to the known products (i.e., domains 2 + 3, and domain 1), then the affinity for uPA would have been reduced by ~1500-fold (39). Moreover, full-length suPAR was also indicated by the molecular mass of the labeled cross-linked uPA/suPAR complex (24). Our epitope studies provide additional evidence for full-length suPAR protein in the peripheral blood of healthy people. This soluble form has been found in the culture supernatant of human HT-1080 fibrosarcoma cells, from which it may be released by the action of a phospholipase (40). In patients with the hereditary disease of paroxysmal nocturnal hemoglobinuria, a defect in the C-terminal processing of the receptor prevents its anchorage in the plasma membrane of blood neutrophils, and increased concentrations of full-length suPAR protein are therefore found in blood from these patients (24). However, the tissue origin of the suPAR found in human blood from healthy donors has not been established, nor is the mechanism by which it is released in vivo, or the functional significance it may have.
Our quantitative studies of soluble receptor in blood from healthy donors show that all forms of blood preparations appear to be suitable for ELISA determination of suPAR concentrations—serum as well as plasma made with citrate, EDTA, or heparin anticoagulant. Delay in processing blood did not seem to be a significant source of error, at least in the first hour after collection, implying that neutrophils in healthy blood are not rapidly shedding suPAR. On the contrary, inflammatory neutrophil exudates have previously been shown to contain considerable amounts of suPAR (41). Similar median values and ranges were found for healthy serum, citrate plasma, and heparin plasma, but the concentration of suPAR in EDTA plasma was significantly higher. If comparisons of donor groups or healthy donors with patient material are to be valid, the same blood preparation (e.g., serum) should be used consistently. If the receptor is considered as the full-length protein of Mr ~60 000, the median of 1.2 µg/L for citrate plasma and serum corresponds to a molar concentration of ~20 pmol/L (cf. 28 pmol/L found in ref. 24). The receptor ligand uPA also occurs in plasma at only ~20 pmol/L (42), so given that the binding affinity of the receptor is ~100 pmol/L, it would seem that the majority of the receptor in vivo is unoccupied by uPA. It is probably for this reason that Pedersen et al. (43) were able to affinity-purify suPAR from plasma of ovarian cancer patients with a column of immobilized uPA. At least some of the suPAR present in sepsis plasma was also able to bind uPA (41).
The remodeling of tissues that characterizes several physiological and pathological events, such as wound healing, trophoblast invasion, and tumor invasion, is well known to involve the proteolytic activity of the urokinase system in degrading tissue matrix. Increases in uPAR expression occur in such events and a corresponding increase in suPAR in blood may be a consequence of this. In our studies, an indication of this possibility is found in the slightly higher suPAR serum concentrations in women compared with men. One possible explanation of this is the increased activity of the uPA system in ovulation, and menstrual degradation of uterine endometrium (44). To study this possibility, a considerably larger number of blood samples from postmenopausal women would be required than were obtainable from our collections of voluntary blood donors that included only four women age 50 years or more.
However, in cancer the uPA system is clearly very active in many types of tumors, and uPAR is often overexpressed. Furthermore, the concentration of suPAR released by cells within a tumor appears to be more closely related to prognosis than the total membrane-bound fraction of uPAR (17), suggesting that measurement of suPAR in blood in malignancy may have important prognostic value. Appreciable amounts of suPAR have been previously identified and characterized in plasma and ascites fluid from ovarian cancer patients (43), and more recently increased concentrations of suPAR have been found in plasma from non-small-cell lung cancer patients (45). With the quantitative ELISA described above, our preliminary findings suggest that increases in the concentration of plasma suPAR are also found in patients with advanced cancers of breast and colon.
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Acknowledgments |
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Footnotes |
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1 Nonstandard abbreviations: uPA, urokinase plasminogen
activator; pro-uPA, proenzyme form of uPA; uPAR, cell-surface uPA
receptor; suPAR, soluble uPAR; and TNP, trinitrophenyl hapten. 
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-
), a mixture of R3 and R5 monoclonal anti-human uPAR
antibodies (
), R2 and R5 (
), R2 and R3 (
), and R2, R3, and R5
together (
-
