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Surgery Branch [A. L. F., J. C. Y., E. M. T., H. R. A., S. K. L.] and Urologic Oncology Branch [W. M. L.], National Cancer Institute, Bethesda, Maryland 20892; CytImmune Sciences, Inc., College Park, Maryland [L. T., G. F. P., B. W. S.]; and EntreMed, Inc., Rockville, Maryland [W. E. F.]
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ABSTRACT |
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 Top ABSTRACT IntroductionPatients and MethodsResultsDiscussionREFERENCES |
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Introduction |
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TopABSTRACTIntroductionPatients and MethodsResultsDiscussionREFERENCES |
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2% of all cancers, and its
incidence is increasing in North America and northern Europe
(1)
. Five-year survival rates, although gradually
improving, remain around 50–60% (1)
because of the high
resistance of metastatic disease to systemic therapy (2)
.
Renal cancers generally are highly vascular tumors known to secrete the
proangiogenic cytokine
VEGF2
in vitro and in vivo (3, 4, 5, 6, 7)
. Tumor
VEGF expression is correlated with the severity of disease in patients
with renal cell carcinoma (3
, 4
, 8)
, and some authors have
suggested using circulating VEGF as a prognostic factor or tumor marker
(3
, 5 , 9)
. In addition to producing proangiogenic cytokines, recent data demonstrate that tumors can produce antiangiogenic cytokines as well (10 , 11) . It has been suggested that, in humans, the generation of antiangiogenic compounds in the presence of a primary tumor suppresses the growth of distant metastases (12) . This phenomenon has been demonstrated in mice (11 , 13 , 14) . However, the presence of endogenous antiangiogenic cytokines in patients with renal cell carcinoma has not been reported. In this study, we sought to determine whether circulating levels of endostatin, an antiangiogenic cleavage product of C18 (10) , were elevated in patients with stage IV CCRC.
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Patients and Methods |
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TopABSTRACTIntroductionPatients and MethodsResultsDiscussionREFERENCES |
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/6. In addition, we obtained multiple aliquots of human serum from randomly selected volunteer blood donors (controls) from the Department of Transfusion Medicine, NIH. All serum samples were negative by ELISA for HIV as well as hepatitis B and C. We were provided with the age and gender of each control but received no other identifying information.
Methods.
Archival serum samples collected between 1988 and 1995 from patients
with CCRC who participated in an institutional review board-approved
NCI study were stored in liquid nitrogen. After retrieval from liquid
nitrogen, they were stored at -80°C until ready for use. Serum
samples from more recent CCRC patients and from volunteer blood
donors were stored at -80°C until ready for use. Sera were thawed at
room temperature, and VEGF and endostatin levels were measured using a
competitive EIA (ACCUCYTE; CytImmune Sciences, Inc., College Park, MD).
Each assay was developed with its respective recombinant protein.
Recombinant VEGF was obtained from PeproTech, Inc. (Princeton, NJ),
whereas recombinant endostatin was obtained from EntreMed, Inc.
(Rockville, MD). These recombinant proteins were the antigens for the
generation of the rabbit polyclonal antiserum and were biotinylated to
serve as the competitive ligands. After obtaining sufficient titers
of antibody, sera were fractionated by high-performance liquid
chromatography to obtain enriched immunoglobulin preparations.
Briefly, the assay was run by making 4- or 5-fold dilutions of samples,
which were added to a 96-well plate coated with goat antirabbit
polyclonal IgG antibody. After addition of the respective competitive
ligands, rabbit antihuman VEGF or endostatin polyclonal IgG antibody
was added, and the plates were incubated at room temperature for 3 h. After thorough washing, streptavidin-conjugated alkaline phosphatase
was added and incubated for 30 min at room temperature to
dephosphorylate NADPH to NADH. After further washing, color reagents
containing alcohol dehydrogenase and diaphorase were added. These
reagents use NADH as a cofactor to generate formazan.
Absorbances were measured at 492 nm when the
A492 for the negative control well was
between 1.5 and 2.0. Each sample was analyzed in triplicate, and
concentrations were calculated with reference to a standard curve using
Microplate Manager III (Bio-Rad, Hercules, CA). Each EIA was run
blinded to the origin of the serum samples.
Rabbit antibodies against recombinant human endostatin for use in the EIA and Western blotting were generated by the method described by Shiosaka et al. (16) . Briefly, 0.5 mg of recombinant human Endostatin (EntreMed, Inc., Rockville, MD) was bound to 2 ml of 32-nm colloidal gold (CytImmune Sciences, Inc., College Park, MD) at pH 8. The solution was emulsified in Freund’s Complete Adjuvant and administered s.c. into a New Zealand White rabbit. Two weeks later, the rabbit was boosted with endostatin/colloidal gold emulsified in Freund’s Incomplete Adjuvant. Six weeks after the initial immunization, a 30-ml blood sample was collected. The serum was fractionated on a Bakerbond AbX mixed ion exchange HPLC column (Baker) according to manufacturer’s specifications. Fractions corresponding to the rabbit antibody peak were pooled and dialyzed against TBS.
The ACCUCYTE endostatin EIA was validated by Western blot analysis, parallelism, quantitative recovery, and cross-reactivity studies. Additionally, intra- and interassay variability was assessed.
For Western blotting, 800-µl serum samples from CCRC patient 15
(Table 2
; patient selected because of sample availability) and a
healthy, 45-year-old male control were diluted 5-fold in 0.5% SDS and
heated at 56°C for 5 min. The diluted samples were centrifuged in
Microcon 100,000 MWCO columns (Millipore, Bedford, MA), and the
filtrates were concentrated 20-fold in Microcon 10,000 MWCO columns.
Thus, serum proteins between Mr
10,000 and Mr 100,000 were
concentrated 4-fold compared with the original serum. Ten µl of
each sample were run under reducing conditions on a SDS-polyacrylamide
gel (NuPAGE; Novex) and transferred to a nitrocellulose membrane
(Hybond; Amersham Pharmacia Biotech, Buckinghamshire, England). The
membrane was incubated with 510 ng/ml purified rabbit antihuman
endostatin polyclonal IgG antibody (Cytimmune Sciences), followed by
horseradish peroxidase-conjugated goat antirabbit IgG antibody (Santa
Cruz Biotechnology, Santa Cruz, CA). Images were visualized using a
chemiluminescent detection kit (ECL and Hyperfilm ECL; Amersham).
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Quantitative recovery was assessed by adding a known amount of recombinant endostatin protein to a normal serum or plasma sample. A 0.5-ml aliquot of each sample received a 25 µl "spike" of a 2000 ng/ml stock endostatin solution, increasing the endogenous immunoreactivity by 100 ng/ml. A second and a third aliquot of the spiked and unspiked samples underwent one or two acute (i.e., within the same day) freeze (-80°C)/thaw (25°C) cycles. All samples were then analyzed by the ACCUCYTE endostatin EIA.
Cross-reactivity of the assay was tested against collagen I, collagen IV, vitronectin, fibronectin, bFGF, and Angiostatin and compared with the immunoreactivity of an equivalent amount of recombinant endostatin and heat-inactivated endostatin. To heat-inactivate endostatin, a 5 mg/ml solution of endostatin was heated to 90°C for 5 min. Each cross-reactive test molecule was run in the assay at a maximum concentration of 400 ng/ml. Two-fold serial dilutions of each test molecule to 50 ng/ml were also run to determine the specificity of any potential cross-reactivity. The potency estimates were determined based on the recombinant endostatin standard curve.
Intraassay variance was measured by analyzing three replicates of each of 26 serum samples. The mean of the individual potency estimates for each replicate was calculated, and the CV for the sample was determined. To determine interassay variation, aliquots of a single sample were analyzed over 5 days using 24 different plates.
The range of detection for the VEGF EIA was 0.195 to 50.0 ng/ml, whereas the range for the endostatin EIA was 1.95 to 500 ng/ml. Calculated concentrations exceeding the upper limit of detection for each EIA were reassayed using appropriate dilutions. Concentrations below the lower limit of detection were set at the midpoint between 0 and this lower limit (i.e., 0.098 and 0.98 ng/ml for VEGF and endostatin, respectively).
Two or three serum aliquots from the same venipuncture were analyzed for each subject. Subjects for whom only one aliquot was available were excluded from the study. The CV among the samples from each subject was calculated. For subjects from whom only two samples were available, the subject was excluded if the CV exceeded 40%. For subjects with three samples and a CV exceeding 40%, the outlying value was discarded, and the CV was recalculated using the two remaining values. If the CV still exceeded 40%, the subject was excluded. VEGF and endostatin levels were represented as the mean of sample values considered concordant using this method.
The renal cancer cell lines 1581-RCC, 1764-RCC, UOK-125, and UOK-131 (developed in our laboratories), as well as the transformed human embryonic kidney cell 293 (American Type Culture Collection, Manassas, VA), were assayed for supernatant endostatin and VEGF concentrations. Cells were plated in 12-well plates at a density of 5 x 105 cells/well in 0.5 ml of DMEM containing 10% FCS, 100 units/ml penicillin, 100 µg/ml streptomycin, 50 µg/ml gentamicin, 0.5 µg/ml Fungizone, and 4 mM glutamine (Biofluids, Rockville, MD). After 24 h incubation at 37°C, supernatants were harvested and centrifuged at 2000 x g for 5 min. All experiments were performed in triplicate. Each sample was assayed in duplicate for endostatin and VEGF concentrations by EIA as described above.
The upper limit of normal for endostatin and VEGF levels was defined as 2 SD above the mean. Comparisons between groups were performed using the Mann-Whitney U test to compare groups according to their median values with no assumption about the scatter of the data. Correlations were performed using the Spearman rank correlation. All calculations were done using Instat 2.01 (GraphPad Software), and P < 0.05 was considered significant.
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Results |
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TopABSTRACTIntroductionPatients and MethodsResultsDiscussionREFERENCES |
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. Mean age was 48 years in both groups with similar age ranges. Of the
15 patients with CCRC, 12 (80%) were men, whereas 9 (50%) of the 18
controls were men.
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). Tumor thrombus in the renal vein was present in 3 (20%) of the 15
patients, and the inferior vena cava was uninvolved in all patients.
All but one patient had solid organ metastases, with the lung being the
most common site (12 of 14 patients). The remaining patient was
classified as stage IV because of N2 nodal
disease.
Serum Endostatin and VEGF Levels in Patients with CCRC and
Controls.
Median serum endostatin levels were 24.6 ng/ml (range, 15.1–54.0
ng/ml) in CCRC patients and 14.1 ng/ml (range, 1.0–19.3 ng/ml) in
controls (P < 0.0001; Fig. 1
). Median serum VEGF levels were 3.4 ng/ml (range, 0.1–11.2 ng/ml) in
CCRC patients and 2.5 ng/ml (range, 0.1–4.2 ng/ml) in controls
(P = 0.065). As defined in "Patients and Methods,"
the upper limits of normal for serum concentrations of endostatin and
VEGF were 22.1 and 4.3 ng/ml, respectively. By these criteria,
endostatin levels were abnormally elevated in 8 of 15 (53%) CCRC
patients, and VEGF levels were abnormal in 6 of 15 (40%).
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. In the control group, median endostatin levels were 13.8 ng/ml
(range, 11.1–15.4 ng/ml) and 15.5 ng/ml (range, 1.0–19.3 ng/ml) in
men and women, respectively (P = 0.67). Median VEGF
levels were 2.5 ng/ml (range, 0.3–4.2 ng/ml) and 2.4 ng/ml (range,
0.1–3.7 ng/ml) in men and women, respectively (P >
0.99). In the CCRC patients, median endostatin levels were 21.9 ng/ml
(range, 15.1–54.0 ng/ml) and 40.6 ng/ml (range, 19.2–44.5 ng/ml) in
men and women, respectively (P = 0.63). Median VEGF
levels were 3.2 ng/ml (range, 0.1–10.8 ng/ml) and 4.8 ng/ml (range,
3.4–11.2 ng/ml) in men and women, respectively (P =
0.18).
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). There was, however, a highly significant correlation between
endostatin and VEGF levels among the CCRC patients (r =
0.81, P = 0.0003; Fig. 3
). Neither endostatin levels
nor VEGF levels were correlated with the size of the primary tumor
(r = 0.05, P = 0.82; and
r = 0.26, P = 0.16, respectively;
n = 14). Similarly, there was no significant difference
in median levels of endostatin or VEGF in CCRC patients with or without
renal vein thrombus (endostatin, P = 0.36; VEGF,
P = 0.94).
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. VEGF levels varied from below the limit of detection (0.195 ng/ml) to
7.3 ng/ml. Neither endostatin nor VEGF was detectable in the
supernatant of 293 cells.
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. This band was not visualized in the lane containing the normal human
serum sample (endostatin concentration, 13.4 ng/ml by EIA).
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. Dilution of both the serum and plasma sample led to diminished
endostatin immunoreactivity paralleling the dilution curve of the
recombinant endostatin standard. Quantitative recovery validation of
the ACCUCYTE endostatin EIA yielded recovery of the spike ranging from
83 to 96%, with an average recovery of 90%. Of the molecules tested
(collagen I, collagen IV, vitronectin, fibronectin, basic fibroblast
growth factor, and angiostatin), angiostatin had the highest
cross-reactivity, which was 0.04% as compared with an equivalent dose
of endostatin. Heat-inactivated endostatin retained 1%
immunoreactivity. No visible precipitates were observed in the boiled
preparation; therefore, the diminished immunoreactivity of
heat-inactivated protein did not appear related to a change in
solubility. Under less damaging conditions, such as preparing the
molecule for Western blot analysis under reducing conditions, the
endostatin retained its immunoreactivity (data not shown; the
endostatin was not heated for the Western blot in Fig. 4
). Intraassay
evaluation of three replicates of each of 26 serum samples yielded an
average CV of 6.4%. Evaluation of interassay variation yielded a SD of
0.83 ng/ml and a CV of 13%.
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Discussion |
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TopABSTRACTIntroductionPatients and MethodsResultsDiscussionREFERENCES |
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The nucleotide sequence encoding endostatin resides within the COOH-terminal, noncollagenous domain of C18, termed NC1 (10) . C18 has been shown to be a member of the unique collagen family, the multiplexins, that resides in basement membranes and particularly in the liver (17, 18, 19) . Most collagens undergo cleavage of their noncollagenous domains after secretion from the cell (19 , 20) . This phenomenon would explain the lack of endostatin secretion from most tumor cell lines in vitro, including those from renal cell carcinomas. More likely, endostatin is cleaved from C18 extracellularly. Recently, elastase and cathepsin activities have been shown to cleave endostatin from NC1 in vitro (21 , 22) . The mechanisms of endostatin generation in vivo remain unknown.
Our findings suggest that endostatin is detectable in healthy subjects. The validation data evaluating parallelism and quantitative recovery using the ACCUCYTE endostatin EIA show that the immunoreactivity detected in normal human serum and plasma is essentially identical to that of recombinant endostatin. The absence of a Mr 20,000 endostatin-immunoreactive band Western blotting of normal human serum was to be expected, because the 4-fold concentration of serum samples would yield an estimated endostatin concentration (53.6 ng/ml) below the sensitivity of the blot (125 ng/ml). Endostatin in the circulation of healthy subjects may play a role in the homeostatic regulatory network controlling angiogenesis, termed the "angiogenic switch" (23, 24, 25) . Alternatively, it may be generated as a by-product of physiological collagen turnover.
Western blotting of the concentrated serum sample from CCRC patient 15
(calculated endostatin level after concentration, 177.2 ng/ml),
however, revealed a band with mobility equal to that of recombinant
endostatin (Fig. 4)
. We believe that the larger, ill-defined areas of
intensity on the Western blot are most likely attributable to
nonspecific binding to ubiquitous serum proteins. John et
al. (26)
have reported the presence in human plasma
of a number of endostatin antibody-immunoreactive protein fragments;
however, these are relatively small molecules with molecular weights
ranging from Mr 16,000 to Mr 22,000.
In this study, patients with CCRC had higher serum VEGF levels than healthy controls, although this difference was not quite significant. Given previous data in the literature with larger numbers of patients (3) , we suspect that the high VEGF levels seen in our series represent a real phenomenon. Nonetheless, the difference in endostatin levels between CCRC patients and controls was highly significant. Elevations in circulating endostatin levels are not unique to patients with cancer. Perturbed regulation of angiogenesis is an important feature of rheumatoid, vascular, and other nonneoplastic diseases (25) ; recently, circulating endostatin levels were reported to be elevated in patients with systemic sclerosis (27) .
Most importantly, serum endostatin levels correlated significantly with serum VEGF levels in patients with CCRC but not in controls. There was no correlation of either endostatin or VEGF levels with primary tumor size, which represented the majority of disease burden in these patients. The association between endostatin and VEGF demonstrated in this study does not necessarily indicate a causal link between the elevated levels of these cytokines in CCRC patients. It is possible that elevated serum levels of VEGF and endostatin in advanced CCRC are unrelated and attributable to some clinical factor not examined in the present study. To demonstrate the role of various clinical factors will require multivariate analysis of a larger group of patients with more detailed prospective data collection. We are now conducting such a study. Alternatively, elevated VEGF and endostatin levels may be correlated because of the homeostatic interrelationship between pro- and antiangiogenic substances, which has been hypothesized but not fully elucidated. The interaction between VEGF and endostatin might be a direct or an indirect one.
We do not believe that VEGF expression increases in response to
circulating endostatin levels in CCRC, because of the angiogenic
phenotype of the tumor and the relatively low levels of endostatin
present. In vitro, the threshold concentration of murine
endostatin for inhibition of endothelial cell proliferation is 100
ng/ml (10
, 28) and may be much higher for human endostatin
(29)
. Furthermore, our data indicate that VEGF is secreted
by at least some renal cancer cell lines in vitro in the
absence of endostatin. Finally, in a murine model (28)
,
adenoviral delivery of the endostatin gene leads to high plasma
concentrations of endostatin (mean, 1770 ng/ml) without a concomitant
elevation in VEGF levels (data not shown).
We therefore have proposed two hypotheses to explain the correlation of
endostatin and VEGF in this study, summarized in Fig. 6
. In addition to VEGF, invasive tumors secrete multiple collagenases,
including matrix metalloproteinases, which facilitate digestion of the
extracellular matrix and basement membrane, allowing the tumor access
to the circulation. This is true of renal carcinomas as well (30
, 31) . One of these collagenases may cleave endostatin from C18
(Fig. 6
A). Evolution may have favored the development of
cleavage products that are opposite in activity to the process which
created them to keep such pathological processes in check or as part of
the "off" switch that controls physiological angiogenesis. The
relationships between PEX and matrix metalloproteinase-2 and between
angiostatin and macrophage-derived metalloelastase may be other
examples of this phenomenon (32
, 33)
.
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B). We believe that both of these processes may occur
simultaneously, because tumor neoangiogenesis is known to involve
degradation of extracellular matrix components by both endothelial
cells and tumor cells. The expanding list of endogenous pro- and
antiangiogenic factors reveals the complexity of the homeostatic system
controlling angiogenesis. In conclusion, serum endostatin levels can be reliably determined using a competitive enzyme immunoassay, are detectable in normal subjects, and are elevated in patients with CCRC. Furthermore, endostatin levels are significantly correlated with VEGF levels in CCRC patients but not in healthy controls. We hypothesize that elevated endostatin levels represent an attempt at a compensatory response to the angiogenic phenotype of CCRC. We are currently investigating the interrelationship of endostatin, VEGF, and other related cytokines in vitro and in in vivo animal models, as well as in patients with other tumor histologies. We believe that elucidating the nature of the homeostatic relationship between pro- and antiangiogenic, tumor-derived cytokines will be critical in developing and refining treatment strategies to inhibit tumor angiogenesis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 To whom requests for reprints should be
addressed, at Surgery Branch, National Cancer Institute, NIH, 9000
Rockville Pike, Bethesda, MD 20892. Phone: (301) 496-6457; Fax:
(301) 402-1788. 
2 The abbreviations used are: VEGF, vascular
endothelial growth factor; CCRC, clear cell renal carcinoma; C18,
collagen XVIII; NCI, National Cancer Institute; EIA, enzyme
immunoassay; CV, coefficient of variation; EC, endothelial cell.
Received 4/18/00; revised 8/22/00; accepted 10/10/00.
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REFERENCES |
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1(XVIII), a collagen chain with frequent interruptions in the collagenous sequence, a distinct tissue distribution, and a homology with type XV collagen. Proc. Natl. Acad. Sci. USA, 91: 4234-4238, 1994.This article has been cited by other articles:
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,
individual subjects; ----, median values. Endostatin levels were
significantly elevated in the CCRC patients (P <
0.0001, Mann-Whitney U test). VEGF levels were higher in
CCRC patients than in controls, but this difference was not significant
(P = 0.065).
