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National Jewish Medical and Research Center, Denver, Colorado 80206 [F. Z., L. N., K. T., E. W. G., L. Z., R. J. M.]; Denver Health and Hospitals, Denver, Colorado 80204 [J. H. F.]; University of Colorado Health Sciences Center, Denver, Colorado 80262 [A. M. Ma., A. M. Me., L. D. D-N.]; Memorial Sloan Kettering Cancer Center, New York, New York 20021 [W. P.]; and National Cancer Institute, Rockville, Maryland 20850 [S. M. U., S. B. J.]
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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 a recent study of urethane-induced adenomas in mice, we showed that these tumors expressed relatively high levels of SPs3 on the basis of immunostaining and in situ hybridization (6) . These tumors all expressed SP-C, a marker restricted to alveolar type II epithelial cells, but not CC-10, a marker of nonciliated bronchiolar epithelial cells, commonly referred to as Clara cells. Hence, SPs could potentially serve as biomarkers of lung cancer in mice. Of the SPs, only SP-A and SP-D are hydrophilic and readily measured in serum. Serum levels of SP-A and SP-D are increased in acute lung injury and several interstitial lung diseases in humans (7 , 8) . In addition, serum SP-D is a biomarker of acute lung injury and inflammation in rats (9) . SP-D also has the advantage that it should theoretically be useful in tumors derived from either type II cells or Clara cells. Wikenheiser and Whitsett (10) , who studied murine tumors induced by SV40 early region genes under the control of the SP-C promoter, demonstrated that these murine tumors could express either CC-10, a marker of bronchiolar epithelial cells, or SP-C. SP-D was not measured in those mice.
SP-D is a calcium-binding lectin that is important in host defense (11 , 12) . Although SP-D can be detected at very low levels in a variety of tissues, it is most highly expressed in type II cells and Clara cells (12 , 13) . SP-D is a collagenous glycoprotein whose monomeric structure is composed of an NH2-terminal unit, a long collagenous portion, a neck region, and a highly conserved globular carbohydrate recognition domain that is the functional unit for attachment to microorganisms (11) . SP-D binds to viruses, bacteria, yeast, and mycobacteria, and it can also alter inflammatory cell responses (11 , 12) . Mature SP-D is most commonly composed of four trimeric units with a molecular mass of about 516 kDa. SP-D is very similar to bovine conglutinin, a circulating protein (14) . Although SP-D is conventionally referred to as a SP, it binds surfactant poorly and is not found in lamellar bodies. SP-D is probably not a true SP but part of the innate immune system.
In a preliminary survey of SP-D serum levels in inflammatory lung disease, we found an extremely high level in a BALB/c mouse that had a spontaneous adenocarcinoma. We therefore sought to determine whether serum SP-D could be used to detect lung epithelial tumors in mice.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Ovalbumin Sensitization and Challenge.
BALB/c mice were sensitized and exposed to aerosolized ovalbumin to produce airway inflammation and hyperresponsiveness, as described previously (17
, 18)
. In this protocol there are two control groups [i.e., one immunized by i.p. injection (IP only), and one that was not sensitized but received nebulized ovalbumin on three successive days (3 Neb only)]. The experimental group is sensitized by i.p. injection and challenged by nebulization (IPN), and this is the group that manifests airway hyperresponsiveness to methacholine, airway inflammation, and epithelial metaplasia (17)
. Serum samples were collected after the third day of nebulized ovalbumin or 24 h after the third nebulization. This experimental protocol was approved by the Institutional Animal Care and Use Committee of National Jewish Medical and Research Center.
Urethane-induced Adenoma.
Male A/J mice at 6 weeks of age were purchased from Jackson Laboratory (Bar Harbor, ME). Breeder pairs from B6.A chromosome substitution strains (CSS or consomic strains; C57BL/6J mice bred to contain one A/J chromosome) were a gift from Dr. Joseph Nadeau (Case Western Reserve University, Cleveland, OH) and were bred to provide mice for experimental use at the University of Colorado Health Sciences Center Center for Laboratory Animal Care. Five A/J mice, 21 male B6.AY mice, and 7 male and 6 female B6.A14 CSS mice (each at 8 weeks old) all received i.p. injection with 1 mg/g urethane in saline as described previously (19)
. Twenty weeks after urethane treatment, mice were euthanized with a lethal dose of pentobarbital, and plasma was collected by cardiac puncture. Lung tumors were dissected and counted under a dissecting microscope (Wild Inc., Heerbrugg, Switzerland). Plasma was also collected from four age-matched A/J control mice (26 weeks of age).
TGF-ß Deletion and K-ras Mutation.
C57BL/6NCr mice heterozygous for deletion of one TGF-ß1 allele (TGF-ß1+/-; Ref. 20
) were mated with C57BL/6/129/SV mice heterozygous for latent mutation of one K-ras allele (K-ras+/-; Ref. 21
). This mating generated four genotypes (TGF-ß1+/+/K-ras+/+, TGF-ß1+/-/K-ras+/+, TGF-ß1+/+/K-ras+/-, and TGF-B1+/-/K-ras+/-). Confirmation of the genotypes was performed on DNA isolated from the tails of each offspring by using PCR amplification. Specific TGF-ß1 and K-ras oligonucleotide primers were designed to distinguish heterozygous from wild-type littermates (20
, 21)
. All mice were maintained in a pathogen-free barrier facility under conditions of constant room temperature (22 ± 1°C) and on a 12-h light/12-h dark cycle. Water and rodent food were available ad libitum. Wild-type littermates and heterozygous mice were humanely sacrificed, serum was collected by cardiac puncture, and the lungs were removed quickly. Total tumor number was determined, and the sizes of individual tumors were measured with a metric caliper. At all times, mice were treated in accordance with the guidelines for animal care and use established by the NIH.
Inducible Activated K-ras Mouse Model of Lung Cancer.
Mice that form lung tumors due to expression of a tetracycline-regulated K-ras oncogene were generated as described previously (22)
. Bitransgenic (CCSP-rtTA/tet-op-K-ras4bG12D) animals and littermate controls were bred on wild-type (FVB), p53-deficient (FVB), or INK4a-Arf-deficient (C57BL/6) backgrounds. Genotyping was performed as published. The expression of activated K-ras was induced in lung epithelia by feeding adult animals (4–6 weeks of age) doxycycline-impregnated food pellets (Harlan-Teklad, Madison, WI) for varying amounts of time, ranging from 3 weeks to 11 months. Serum was collected from mice by retroorbital eye bleed in anesthetized mice before sacrifice. Half of the left lung was prepared for H&E staining. Multiple sections were examined by light microscopy to determine the presence or absence of tumor. This experimental protocol was approved by the animal care committee at Memorial Sloan-Kettering Cancer Center.
ELISA for Mouse SP-D.
Serum SP-D levels were measured by ELISA. Initially, the samples were coded, and the ELISA was performed without knowledge of the tumor status of the mice. When it was clear that some of the values for tumor-bearing mice were very high, appropriate dilutions of the serum were made based on the presence or absence of a tumor so as to avoid wasting serum. Recombinant mouse SP-D produced in Chinese hamster ovary cells was used as the SP-D standard. Polyclonal antirecombinant mouse SP-D rabbit IgG (10 µg/ml in 0.1 M sodium bicarbonate) was bound to wells in microtiter plates (Immulon 1 plates; Dynatech Laboratories, Alexandria, VA) overnight at room temperature. The wells were then incubated for at least 30 min at room temperature with a 4% (w/v) solution of nonfat dry milk in PBS containing 1% Triton X-100 (blocking buffer) to block nonspecific binding. The wells were washed with blocking buffer, and 100 µl of purified mouse SP-D (0–20 ng/ml) for standards or appropriately diluted mouse serum samples were added to each well. Plates were incubated for 90 min at 37°C and then washed with blocking buffer. One hundred µl of horseradish peroxidase-conjugated anti-SP-D antibody (10 µg/ml) were added to each well, and the plates were incubated for 90 min at 37°C. After washing with 1% Triton X-100 in PBS, 100 µl of the color-developing agent [0.1% O-phenylenediamine and 0.015% hydrogen peroxide in 0.1 M citrate buffer (pH 4.6)] were added. The reaction was carried out for 5 min at room temperature in a darkened room and was stopped by the addition of 100 µl of 2N sulfuric acid. Absorbance was measured with Microplate Autoreader EL-311s (Bio-Tek Instruments, Inc., Winooski, VT).
SP-D Protein Identification.
SP-D was partially purified from serum, as described previously (9)
. SP-D binds Saccharomyces cerevisiae in a calcium-dependent manner. Aliquots of serum from normal, tumor-bearing, and SP-C/TNF-
transgenic mice were dialyzed against 5 mM TBS (pH 7.4) containing 1 mM EDTA to remove glucose, a competitive inhibitor. After dialysis, the sera were adjusted to 5 mM Ca2+ with CaCl2 and incubated with paraformaldehyde-fixed S. cerevisiae cells (strain SEY6210) for 2 h at room temperature by gentle shaking. After incubation, the cells were washed three times with TBS containing 5 mM CaCl2. The cells were then incubated with 50 mM inositol for 1 h at room temperature to remove bound SP-D. The cells were centrifuged, and the supernatant was removed, pooled, and analyzed for SP-D by immunoblotting. The samples were placed in Laemmli reducing sample buffer, boiled, and layered onto precast 8–16% Tris-glycine polyacrylamide slab gels (Novex), and proteins were separated by electrophoresis in a Novex X cell Mini-cell (Invitrogen, Carlsbad, CA). Proteins were transferred to nitrocellulose membranes using the Novex X cell blot module according to the manufacturer’s instructions. Nonspecific binding sites on the nitrocellulose membranes were blocked by incubating the blots in 5% nonfat dry milk in 20 mM Tris-HCl (pH 7.5), 137 mM NaCl, and 0.05% Tween 20 (TTBS; all from Sigma, St. Louis, MO) at 4°C overnight. The blots were incubated with rabbit antimouse SP-D IgG in 5% nonfat milk in TTBS (final concentration, 0.35 µg/ml) for 1 h at room temperature. Horseradish peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA) were added for 30 min at room temperature, and the antigens were detected by enhanced chemiluminescence (ECL-plus; Amersham Pharmacia Biotech, Piscataway, NJ) on Hyperfilm (Amersham Pharmacia Biotech).
Immunostaining.
This was done with polyclonal rabbit IgG antimurine SP-D as detailed previously (6
, 23)
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In Situ Hybridization.
Tissues were fixed with 4% paraformaldehyde, embedded in paraffin, and sectioned. The sections were dewaxed, hybridized with sense or antisense riboprobes to rat SP-D or SP-C, and processed as reported previously (6
, 24
, 25)
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Data Analyses.
All statistical tests were done with the SAS statistical analysis package (version 8.2). Significance level was set to 0.05 for all comparisons. For Table 1
and Fig. 1
, we compared multiple groups with a single control group. Because the groups had different variances, Dunnett’s multiple comparison test was used with the Proc Mixed procedure where each group has its own variance. The nonparametric Wilcoxon’s rank-sum test was used for the comparisons in Fig. 2, A and B
, and Fig. 5
. For Fig. 4
, both tumor volume and SP-D levels were first transformed to their logarithmic scale of base 10 because both variables were not normally distributed. The transformed data were then fitted to a linear regression equation using Proc GLM procedure. For the sensitivity and specificity analysis, tumor data were combined with normal control data. Because tumor volumes for normal group were all zero, logarithm (base 10) transformation was performed on (tumor volume + 1). A linear regression equation was derived to predict either tumor volume or SP-D if only one of them is known. We used whether or not the mouse had a tumor as the outcome variable and calculated the sensitivity and specificity for confidence limits of 95% or 99% for serum SP-D.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, SP-D was increased in transgenic mice that overexpress SP-D in type II cells driven by the SP-C promoter and was essentially zero in SP-D nulls.
Ovalbumin Sensitization and Challenge.
In a preliminary study, we measured SP-D in sera of mice sensitized and challenged with nebulized ovalbumin in a well-characterized model of airway inflammation and hyperresponsiveness (17)
. We knew from previous work that lavage levels of SP-D were elevated after Aspergillus fumigatus allergen challenge in sensitized mice (26)
and in mice challenged with ovalbumin.4
Serum SP-D levels increased after challenge (Fig. 1)
. However, one value was excluded as an outlier because the value was 1420 ng/ml. This animal had a large tumor, which stained positive for SP-D (data not shown), and provided the rationale for the subsequent experiments.
Urethane-induced Adenoma.
Because urethane-induced lung adenomas express SP-D (6)
, we evaluated serum from mice that had few [1–2 adenoma(s); CSS mice] or many (30–50 adenomas; A/J) adenomas. Serum SP-D was elevated in A/J mice bearing multiple tumors over their age-matched controls (Fig. 2A)
. All CSS mice received injection with urethane, and SP-D plasma levels were grouped according to whether the mouse did or did not develop lung tumors. Most CSS mice with 1 or 2 tumors had serum SP-D levels within the normal range, similar to that in animals bearing no tumors (Fig. 2B)
. Serum SP-D levels in mice without tumors were slightly higher than those in other cohorts. This could be because all of these animals were exposed to urethane, these CSS strains might exhibit higher basal SP-D concentrations (basal levels have not been measured in these strains), or they may contain microscopic adenomas undetectable under the dissecting microscope.
We had reported previously that urethane-induced tumors expressed SP-D based on immunostaining (6)
. Here, we used in situ hybridization to demonstrate the relative mRNA levels in the tumors. Although these tumors expressed SP-D as expected (Fig. 3)
, there was also enhanced expression of SP-D mRNA in the type II cells surrounding the tumor. This proximity or field effect was not observed for SP-C expression. The implication is that the tumors produce a substance(s) that increases the expression of SP-D in neighboring normal type II cells.
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. If one uses tumor volume as the response variable and SP-D as the predictor, the linear equation is Log10 (mm3 volume +1) = -0.656 + 1.003 x Log10 (ng/ml SP-D). Only animals with tumors were included in this analysis to prevent anchoring the curve at the low end due to the large number of normal samples. Mice whose serum values were greater than 11.4 ng/ml all had tumors in this data set. All samples from tumor-free mice had serum values of <11.4 ng/ml (specificity is 100%), and 80% of the tumor-bearing mice had serum values of >11.4 ng/ml (sensitivity is 80%). A serum level of 11.4 ng/ml corresponds to a total tumor burden of 1.84 mm3.
Conditional Induction of Lung Tumors with Activated K-ras.
To extend these observations to a third experimental system, we measured SP-D levels in serum from mice bearing lung adenomas and adenocarcinomas due to the induced expression of an activated K-ras (22)
. This protocol used bitransgenic animals (on wild-type or tumor suppressor-deficient backgrounds) in which the expression of murine K-ras4bG12D in lung epithelial cells is under the control of doxycycline. Tumors express SP-C, a type II cell marker, but not CC-10 (10-kDa Clara cell-associated protein), a Clara cell marker. This protocol was chosen because of the rapid induction of tumors and their propensity for malignant transformation. In our studies, mice on wild-type or tumor suppressor-deficient backgrounds were tested after the addition of doxycycline for 3 weeks to 11 months (22)
. Similar to the other inductive protocols, serum SP-D level differentiated the control mice from the tumor-bearing mice (Fig. 5)
.
Verification of SP-D in Serum.
SP-D has been isolated from human and rat serum, and its identity has been verified by immunoblotting (8
, 9)
. We isolated SP-D from serum by binding to yeast and eluting with inositol, as we reported previously (9)
. As shown in Fig. 6
, a 43-kDa protein was isolated from serum, which has the same molecular mass as recombinant SP-D, and was recognized by a rabbit polyclonal IgG specific for murine SP-D. We chose serum from TNF--overexpressing mice as a positive control because we knew that the serum level in these mice was 343 ± 28 ng/ml (n = 4). These mice have significant chronic lung inflammation (27)
. Under nonreducing conditions, both serum SP-D and recombinant SP-D showed higher-order oligomers characteristic of SP-D (data not shown).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, during respiratory syncytial virus infections, after ozone exposure, and after ovalbumin challenge in sensitized mice (some data not shown). Serum SP-D measurement is a relatively simple means of distinguishing normal mice from those with tumors or those with chronic inflammation. Serum SP-D will likely increase with tumor growth and decrease with regression, although this has not been tested in a prospective manner. Because measurement of tumor volume is difficult, and imaging techniques are not widely available, serum SP-D measurements should be useful to identify mice with tumors or to monitor response to therapy over time. The importance of this finding for human lung cancer remains to be defined. Some human adenocarcinomas and large cell carcinomas express SP-D or SP-A, whereas small cell tumors and squamous cell tumors do not (28, 29, 30, 31, 32) . However, about 75% of pulmonary adenocarcinomas express thyroid transcription factor 1, and about 50% of pulmonary adenocarcinomas express SP-A (32) . Expression of SP-D should be similar to SP-A in adenocarcinoma (28) . Although a large study of serum SP-D in human lung cancers has not been done, serum values of SP-D are elevated in some patients with adenocarcinoma and increase further if patients develop radiation pneumonitis (33 , 34) . To define the role of serum SP-D in human lung cancer, serum values of SP-D will need to be determined in a large cross-sectional study with multiple tumor histological types. In our murine tumor study, only mice with adenomas or adenocarcinomas were tested. Because normal human serum levels are higher and more variable than those in mice (7) , serum SP-D levels may not be as distinctive a biomarker for lung tumors in humans. Serum SP-D levels may only be useful in that subset of patients with adenocarcinomas, such as the bronchioloalveolar carcinoma subtype.
The mechanism responsible for the increased serum level of SP-D is not known. The most likely reason for this elevation is the production of SP-D by epithelial cells within the tumors. Tumor cells express SP-D mRNA and have detectable SP-D by immunostaining (6) . Presumably, SP-D is secreted, enters the extracellular space, and reaches the systemic circulation via local lymphatic drainage. It is unlikely that the bronchoalveolar lavage fluid levels of SP-D would vary greatly depending on the presence or absence of tumors, although these values have not been measured to date. However, we cannot eliminate a contribution from the neighboring type II cells that overexpress SP-D. Although SP-D can be expressed on many mucosal surfaces, the highest concentration is found in the lung and bronchoalveolar lavage fluid (12 , 13 , 35) . It is highly unlikely that the increased serum SP-D came from an extrapulmonary source. It is also unlikely that the volume of distribution or clearance of SP-D from the vascular compartment is altered in animals with tumors, although decreases in either parameter would theoretically increase serum concentrations.
The cause of the increased expression of SP-D in the type II cells surrounding the tumor is unknown. In vitro, SP-D expression is altered by factors that affect the state of differentiation of type II cells but, in general, is less responsive to alterations in culture conditions than expression of other SPs (36 , 37) .5 In vivo, pulmonary SP-D expression is increased by acute inflammation (endotoxin, antigen challenge, and so forth) and by overexpression of interleukin 4 (38 , 39) . We presume that some factor is expressed in the tumor that diffuses to the neighboring type II cells and increases SP-D expression. The factor could be produced by inflammatory cells within the tumors (40) . However, this factor(s) has not been identified and will be the subject of future studies.
In summary, murine lung tumors express SP-D, and the tumor burden is reflected in the serum level of SP-D. However, serum SP-D is also elevated with lung inflammation, and SP-D is not specific for lung tumors.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by grants from the Environmental Protection Agency (Grant R825702) and the NIH (Grants HL-67671, HL-36577, HL-61005, CA33497, CA96133, and CA09712). W. P. was supported by NIH Grant K12-CA09712. 
2 To whom requests for reprints should be addressed, at National Jewish Medical and Research Center, 1400 Jackson Street, Denver, CO 80206. Fax: (303) 398-1806; E-mail: masonb{at}njc.org
3 The important abbreviations used are: SP, surfactant protein; TGF, transforming growth factor; TBS, Tris-buffered saline.
4 K. Takeda, unpublished observations.
5 R. J. Mason, unpublished observations.
Received 3/31/03. Revised 6/11/03. Accepted 6/25/03.
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REFERENCES |
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produces an increase in lung volumes and pulmonary hypertension. Am. J. Physiol. Lung Cell Mol. Physiol., 280: L39-L49, 2001.This article has been cited by other articles:
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  D. D. Sin, S. F. P. Man, A. McWilliams, and S. Lam Surfactant Protein D and Bronchial Dysplasia in Smokers at High Risk of Lung Cancer Chest, September 1, 2008; 134(3): 582 - 588. [Abstract] [Full Text] [PDF]
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 D. D. Sin, P. S. Pahlavan, and S.F. P. Man Review: Surfactant protein D: A lung specific biomarker in COPD? Therapeutic Advances in Respiratory Disease, April 1, 2008; 2(2): 65 - 74. [Abstract] [PDF]
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 F. Kassie, L. B. Anderson, R. Scherber, N. Yu, D. Lahti, P. Upadhyaya, and S. S. Hecht Indole-3-carbinol Inhibits 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone Plus Benzo(a)pyrene-Induced Lung Tumorigenesis in A/J Mice and Modulates Carcinogen-Induced Alterations in Protein Levels Cancer Res., July 1, 2007; 67(13): 6502 - 6511. [Abstract] [Full Text] [PDF]
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Y. Lu, D. Bellgrau, L. D. Dwyer-Nield, A. M. Malkinson, R. C. Duke, T. C. Rodell, and A. Franzusoff Mutation-Selective Tumor Remission with Ras-Targeted, Whole Yeast-Based Immunotherapy Cancer Res., August 1, 2004; 64(15): 5084 - 5088. [Abstract] [Full Text] [PDF]
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A. M. Malkinson Evidence That Inflammation Encourages Pulmonary Adenocarcinoma Formation in Mice: Clinical Implications Chest, May 1, 2004; 125(5_suppl): 154S - 155S. [Full Text] [PDF]
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