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[Cancer Research 66, 1191-1198, January 15, 2006]
© 2006 American Association for Cancer Research

Clinical Research

Expression of a Fibroblast Growth Factor–Binding Protein during the Development of Adenocarcinoma of the Pancreas and Colon

Elena Tassi1, Ralf T. Henke1, Emma T. Bowden1, Matthew R. Swift1, David P. Kodack1, Angera H. Kuo1, Anirban Maitra2 and Anton Wellstein1

1 Lombardi Cancer Center, Georgetown University, Washington, District of Columbia and 2 Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland

Requests for reprints: Anton Wellstein, Lombardi Cancer Center, Georgetown University, Room E311, Research Building, 3970 Reservoir Road, Washington, DC 20057. Phone: 202-687-3672; Fax: 202-687-4821; E-mail: wellstea{at}georgetown.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The activity of growth factors is crucial for tumor progression. We previously characterized a secreted fibroblast growth factor–binding protein (FGF-BP1) as a chaperone molecule, which enhances the biological functions of FGFs by releasing FGFs from the extracellular matrix. Here, we characterize the frequency and pattern of FGF-BP1 expression during the malignant progression of pancreas and colorectal carcinoma. For this, we generated monoclonal antibodies that detect FGF-BP1 protein in formalin-fixed, paraffin-embedded tissues and applied in situ hybridization to detect FGF-BP1 mRNA in adjacent tissue sections. FGF-BP1 protein and mRNA were found up-regulated (>70% positive) in parallel (r = 0.70, P < 0.0001) in colon adenoma (n = 9) as well as primary (n = 46) and metastatic (n = 71) colorectal cancers relative to normal colon epithelia (all P < 0.0001, versus normal). Similarly, pancreatitis (n = 17), pancreatic intraepithelial neoplasia (n = 80), and pancreatic adenocarcinoma (n = 67) showed a significant up-regulation of FGF-BP1 compared with normal pancreas (n = 42; all P < 0.0001, relative to normal). Furthermore, the biological activity of FGF-BP1 is neutralized by one of the antibodies, suggesting the potential for antibody-based therapeutic targeting. We propose that the up-regulation of the secreted FGF-BP1 protein during initiation of pancreas and colon neoplasia could make this protein a possible serum marker indicating the presence of high-risk premalignant lesions. (Cancer Res 2006; 66(2): 1191-8)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Colorectal and pancreatic neoplasia, two main types of gastrointestinal carcinoma, are together responsible for 14% of cancer related mortality, ranking them third and fourth after lung cancer and breast or prostate carcinoma (1). As in other solid tumors, neoangiogenesis and stroma remodeling are crucial for the progression of a small lesion to extensive disease (2–4). To gain and sustain these activities, tumors rely on growth factor signaling. Among the protein growth factors and receptors involved in the malignant process, fibroblast growth factors (FGF), a family of more than 20 proteins, have been described to enhance tumor growth, angiogenesis, and progression (reviewed in refs. 5–8). Overexpression of FGF proteins was reported in tissue samples and cell lines, from lung (9, 10), prostate (11), pancreas (12), and colon cancer (13–17). FGFs are typically bound to heparan sulfate proteoglycans in the extracellular matrix and are released by heparanases or proteases from that storage to reach their cognate receptor (7).

Here we analyze the expression of FGF binding protein (FGF-BP1), a secreted protein that acts as an extracellular chaperone molecule for FGF-1 and FGF-2 and enhances their activity by release from the extracellular matrix storage (18, 19). Studies with FGF-BP1-negative cell lines showed that expression of FGF-BP1 increased tumor growth and angiogenesis in a nude mouse model (20). Complementary to that, depletion of endogenous FGF-BP1 from human squamous cell carcinoma and colorectal cancer cell lines reduced their tumor growth and angiogenesis (21). These findings support a potential role of FGF-BP1 as an angiogenic switch in human neoplasia (21). We and others previously found that FGF-BP1 mRNA is indeed elevated in primary breast cancer and squamous cell carcinoma samples (20, 22, 23). In addition, induction of FGF-BP1 expression was observed in human colorectal adenoma samples (24) and in adenoma in the ApcMin/+ mouse model where FGF-BP1 is induced due to the activation of the Wnt/ß-catenin pathway (24). In breast cancer, we found already an up-regulation of FGF-BP1 in in situ carcinoma with a further increase in invasive cancers (23). Due to the potential significance of this protein in different human cancers, we generated monoclonal antibodies (mAb) to identify the FGF-BP1 protein in various bioassays that are described here. In a series of tissues representing the progression of human pancreatic and colorectal adenocarcinoma, we then compared mRNA and protein expression and report high levels of FGF-BP1 expression in early neoplastic lesions that are maintained in all stages of colorectal carcinoma progression, including metastatic disease. In addition, we found that FGF-BP1 was overexpressed in pancreatic intraepithelial neoplasia (PanIN) and pancreatic adenocarcinoma compared with normal pancreatic ducts. One of the mAbs used for detection of FGF-BP1 also inhibited its biological activity, suggesting that antibodies could be used to therapeutically target FGF-BP1.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines, DNA constructs, and transfection. Human adrenal carcinoma SW-13 and human squamous cervical carcinoma ME180 cell lines (American Type Culture Collection, Rockville, MD) were maintained in modified IMEM (Invitrogen Corp., Carlsbad, CA) supplemented with 10% FCS.

pRcCMV/FGF-BP1 was described earlier (20). Stable transfection of SW-13 with pRcCMV and pRcCMV/FGF-BP1 was done using Lipofectamine Plus reagent as recommended by the manufacturer (Invitrogen). Forty-eight hours after transfection, 750 µg/mL of G418 (Invitrogen) was added to the medium to select stable transfectants that were then expanded in the presence of G418. Transient transfection was done using FuGENE 6 Transfection Reagent as recommended by the manufacturer (Roche Diagnostics Corp., Indianapolis, IN). Twenty-four hours after transfection, cells were plated on glass coverslips in 12-well plates at 70% confluency for immunofluorescence.

To obtain agarose-embedded cell pellets, confluent SW-13 and ME180 cells were fixed in 10% buffered formaldehyde for 15 minutes, scraped off the plate, and washed once in cold PBS. To the cell pellets, an equal volume of 2% low melting agarose in PBS was added. Agarose-immobilized cell pellets were subsequently embedded in paraffin and sectioned for immunohistochemical analysis.

Xenografts and animal models. ME180 cells (2.5 million in 0.1 mL of PBS) were injected s.c. into female athymic nude mice (four sites per mouse). Tumors were allowed to grow for 1 month and then harvested. On sacrifice, the excised tumors were bisected; one half was snap-frozen in liquid nitrogen and the other half was fixed in 10% buffered formalin.

Production and characterization of mAbs to FGF-BP1. The production and purification of the recombinant human histidine-tagged FGF-BP1 (his-FGF-BP1) and glutathione S-transferase (GST)-FGF-BP1 fusion proteins were carried out as previously described (18). For the generation of antibodies, four female BALB/c mice were immunized by i.p. injection of 2 µg of purified full-length GST-FGF-BP1 mixed with complete Freund's adjuvant (Sigma, St. Louis, MO). Animals were then injected thrice at 3-week intervals with the same amount of antigen in incomplete Freund's adjuvant. Test bleeds were taken 7 days after each injection and tested by ELISA (see below) with purified his-FGF-BP1. Preimmune sera were used as negative control. Splenocyte/myeloma fusions were done with a modified protocol originally developed by Köhler and Milstein (25). Briefly, 3 days after the last immunization, a 4:1 ratio of mouse splenic cells and NSO/1 myeloma cells (5 x 105) was suspended in 1 mL of 50% polyethylene glycol/5% DMSO in RPMI medium (Invitrogen) and incubated at 37°C for 10 minutes. After centrifugation, cells were resuspended in 10 mL of RPMI medium (Invitrogen) with 20% fetal bovine serum, supplemented with hypoxanthine, aminopterin, and thymidine. Fused cells were equally distributed into twenty 96-well tissue culture plates and incubated at 37°C, 5% CO2 for several days. FGF-BP1-reactive clones were selected by ELISA (see below). Eighty-five positive clones were subjected to single-cell cloning by limiting dilution method. Of these, nine [eight immunoglobulin G1 (IgG1) and one IgG2b] that exhibited the highest affinity towards his-FGF-BP1 were expanded for further characterization. Isotyping of the hybridomas was done using the Sigma Immunotype Mouse Monoclonal Antibody Isotyping Kit (ISO-1, Sigma) according to the protocol of the manufacturer. Production of highly concentrated mAbs was carried out in the "CeLLine" device (BD Biosciences, San Jose, CA) as described by the manufacturer. mAbs were purified from hybridoma supernatants by protein A/G affinity purification [ImmunoPure (G) IgG Purification Kit, Pierce Biotechnology, Rockford, IL] according to the protocol of the manufacturer. 4E7, 3E11, 5G9, and 16H5 hybridoma were subcloned twice by limited dilution and their reactivity against FGF-BP1 was confirmed by ELISA after each cloning step. Antibodies were affinity purified from conditioned media of the hybridoma 4E7, 3E11, 16H5, and 6A6, with the latter serving as a negative control. The quality of the purification was ensured by Coomassie blue staining (not shown).

Direct ELISA. MaxiSorp microtiter plates (Nunc, Rochester, NJ) were coated with human his-FGF-BP1 (50 ng/well for mAbs screening; 25-0.39 ng for the dose-response assay) or 25 ng/well of recombinant rat FGF-BP1 (ref. 26; R&D Systems, Minneapolis, MN) and incubated overnight at 4°C. Plates were washed thrice between each incubation step with washing buffer [10 mmol PBS with 0.2% Tween 20, pH 7.4 (PBS-T)]. Blocking was carried out with 100 µL per well of 3% bovine serum albumin (BSA; Sigma) diluted in PBS-T for 1 hour at room temperature. Subsequently, plates were incubated for 1 hour at room temperature with 50 µL per well of (a) serial dilutions of mouse serum samples, (b) supernatant from different hybridomas, or (c) 0.2 µg/mL or serial dilutions (from 0.5 to 0.00016 µg/mL) of affinity-purified 3E11, 4E7, and 16H5 mAbs diluted in blocking solution. Next, 50 µL per well of an affinity-purified goat anti-mouse horseradish peroxidase (HRP)–conjugated antibody (1:1,000 dilution in blocking solution; Amersham Biosciences Corp., Piscataway, NJ) were used for detection and visualized by incubation for 5 to 30 minutes with 50 µL of developing solution [0.01 mg/mL of tetramethyl-benzidine diluted in 0.1 mol/L sodium acetate (pH 6.0) and 0.01% H2O2]. The enzymatic reaction was stopped by the addition of 50 µL of 1 mol/L sulfuric acid. The plate was analyzed by an Ultramark Microplate Imaging System (Bio-Rad Laboratories, Hercules, CA) at 450-nm absorbance.

Western blot analysis and immunoprecipitation. Frozen surgical samples of colorectal and head and neck cancers, as well as ME180 xenografts, were homogenized in 1 mL of 1x Laemmli's buffer with a Dounce tissue homogenizer. Confluent mock and FGF-BP1-transfected SW-13 cells were lysed with 1 mL of 1x Laemmli's buffer for Western blot analysis or with 0.5 mL of a lysis buffer [50 mmol/L Tris-HCl (pH 8.0), 150 mmol/L NaCl, 40 mmol/L ß-glycerophosphate, 1 mmol/L EGTA, 0.25% Na-deoxycholate, 1% NP40, 50 mmol/L Na-fluoride, 20 mmol/L Na-pyrophosphate, 1 mmol/L Na-orthovanadate, 2 µg/mL leupeptin, 2 µg/mL aprotinin, 1 µg/mL pepstatin, and 100 µg/mL pefabloc] for immunoprecipitation. FGF-BP1 was immunoprecipitated with 10 µg of purified 3E11 mAb for 2 hours at 4°C. Immunoprecipitation was carried out as described (18). For SDS-PAGE, all cell lysates and immunoprecipitants were heated for 5 minutes at 95°C and resolved on 4% to 20% gradient polyacrylamide gels. Proteins were transferred to polyvinylidene difluoride membranes, then blocked in 4% BSA (Sigma) in TBS-T buffer [20 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl, and 0.05% Tween 20] for 1 hour. FGF-BP1 was detected by incubating for 1 hour with 0.5 and 1 µg/mL of the 4E7 and 3E11 mAbs, respectively, diluted in TBS-T buffer. Visualization was done by enhanced chemiluminescence using HRP-linked donkey anti-mouse immunoglobulin G as secondary antibody (Amersham).

Immunofluorescence. Cells grown on coverslips were fixed and permeabilized twice with 3.7% formaldehyde, 0.1% Triton X-100 in PBS for 10 minutes at room temperature. Cells were washed thrice for 5 minutes with PBS at room temperature. Coverslips were incubated with affinity-purified 3E11 mAb (10 µg/mL in PBS) for 20 minutes at room temperature. After three washings, coverslips were incubated for 20 minutes with (a) Cy5 Affinipure Fab Frag Donkey Anti-Mouse IgG (H+L) [Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA); 2.5 ng/mL in PBS] and (b) Alexa Fluor 568 phalloidin [Molecular Probes, Inc. (Eugene, OR); 1:200 in PBS] for F-actin detection. After three washings with PBS, coverslips were mounted and fluorescence examined on an Olympus 1X70 inverted microscope equipped with fluorescence optics (Olympus, Hamburg, Germany).

Soft agar growth assay. Anchorage-independent growth assay of FGF-BP1 stably and mock transfected SW-13 cells was carried out as described (27). Briefly, 15,000 cells in 0.35% agar (Difco, Detroit, MI) were seeded on top of 1 mL of a solidified 0.6% agar layer in a 35-mm dish. mAbs (4E7 or 16H5) were added with or without FGF-2 (basic FGF; 2 ng/mL; Invitrogen) to the top layer, as indicated. Three independent experiments were conducted for 0 and 40 µg/mL mAb concentrations; in one of these, 10 and 20 µg/mL mAb were also tested. Each treatment was run in triplicates. After 8 days of incubation at 37°C, 5% CO2, colonies of >40 µm diameter were counted by an OMNICON TCA stage automated counter (IPI, Chantilly, VA).

Human tissues. Permission to conduct this study was obtained from the appropriate Institutional Review Boards of Johns Hopkins University and Georgetown University, respectively. FGF-BP1 mRNA and protein expression were examined in sections of paraffin-embedded archival tissues and tissue microarrays, as well as in frozen tissues (Western blot). Briefly, formalin-fixed, paraffin-embedded blocks of surgically resected tissues were retrieved from the surgical pathology files of both institutions. The diagnosis was confirmed for each case by a pathologist (A.M.). In situ hybridization and immunohistochemistry were done each on a set of 17 tissue microarrays: 5 colorectal cancer, 6 colorectal cancer metastasis, 1 pancreatitis, 1 PanIN, 4 pancreatic adenocarcinoma, and 2 pancreatic nonadenocarcinoma. All tissue microarrays contained 1.4-mm cores of reference tissues and multiple cores of cancer samples. The total number of tumor-cases (t) and tumor-cores (c) was as follows: primary colorectal adenocarcinoma, 51(t), 332(c); colorectal adenocarcinoma metastases, 75(t), 430(c); pancreatic adenocarcinoma, 69(t), 277(c); and pancreatic nonadenocarcinoma, 36(t), 144(c). The grades of the PanIN lesions (n = 98) had been assigned using a previously described classification scheme: PanIN-1A and PanIN-1B were classified as "low grade" (n = 76) and PanIN-2 and PanIN-3 as "high grade" (n = 22; refs. 28–30). In addition to the series of tissue microarrays, 34 slides with full-sized sections of normal colon and colorectal adenoma were processed for immunohistochemistry only. Control samples were evaluated from the tissue microarrays and slides: normal colon (immunohistochemistry: 110, in situ hybridization: 75); colorectal adenoma (immunohistochemistry: 29, in situ hybridization: 9); normal pancreas ducts (89); liver, stomach, bile, and kidney (115 total); and brain, breast, lymph node, skin, lung, prostate hyperplasia, and uterus (362 total).

In situ hybridization. To assess mRNA expression in archival, formalin-fixed, paraffin-embedded tissue, in situ hybridization was done as previously described (23, 28). Digoxigenin-labeled sense and antisense riboprobes were transcribed from a 668-bp DNA template using the DIG RNA labeling kit (Roche) according to the protocol of the manufacturer. Tissue slides were stained by in situ hybridization using these riboprobes as previously described (23, 31). In brief, slides were deparaffinized and then tissue proteins were digested with proteinase K and then acetylated. The RNA probe was mixed with hybridization solution (Sigma; 1.5 ng probe/1.0 µL solution) and incubated with the tissue for 16 to 18 hours at 42°C. Slides were washed and digested with RNase A (Roche). After refixation, cross-linking, and blocking, a 1:250 solution of alkaline phosphatase–tagged antidigoxigenin antibody fragments (Roche) in buffer I (31) was applied and incubated with the tissue for 16 to 18 hours at 4°C. Staining solution was applied (0.375 mg/mL nitroblue tetrazolium, 0.175 mg/mL 5-bromo-4-chloro-3-indolyl phosphate; Roche) in buffer II (31). Once sufficient staining was observed, the reaction was terminated with buffer III (31). Slides were washed in 0.5% Tween-20 and water for 5 minutes each, then allowed to dry completely before mounting with sealing solution and coverslipped. (For further discussion and review, see ref. 32).

Immunohistochemistry. Four-micrometer sections were deparaffinized by xylene and rehydrated with decreasing concentrations of ethanol and finally H2O. Antigen retrieval was done with 1x citrate buffer (pH 6.0; Zymed Laboratories, South San Francisco, CA) at 100°C for 10 minutes with subsequent cool-down in the solution for 20 minutes. Endogenous peroxidases were inactivated with 3% H2O2 (Ventana Medical Systems, Tucson, AZ) for 10 minutes. Tissues were incubated with anti-FGF-BP1 monoclonal mouse IgGs (45 minutes at room temperature) diluted in Common Antibody Diluent (BioGenex, San Ramon, CA). Detection of mAb was done using the StrAviGen Multilink Kit (BioGenex) and 3,3'-diaminobenzidine tetra-hydrochloride (Liquid DAB; BioGenex) according to the protocols provided by the manufacturer. Counterstaining of nuclei was done by 15-second immersion in Meyer's hematoxylin (BioGenex). Excess of hematoxylin was removed by rinsing the slides several times in distilled H2O and once in 1x OptiMax Wash Buffer (BioGenex). Slides were dehydrated with increasing concentrations of ethanol and a final immersion in 100% xylene, then mounted with Cytoseal 60 low-viscosity mounting medium (Richard-Allan Scientific, Kalamazoo, MI) and coverslipped.

Evaluation of mRNA and protein expression. The results obtained from immunohistochemistry and in situ hybridization were categorized using identical stratifying criteria for ease of analysis. In situ hybridization staining was evaluated in conjunction with the sense (control) probe and immunohistochemistry in conjunction with a nonspecific primary antibody. Staining results were assigned to four distinct groups: negative (–), staining comparable to sense probe (in situ hybridization) or <5% of cells (immunohistochemistry); low positive (+), staining exceeding sense probe intensity in 5% to 50% of cells (in situ hybridization) or staining in 5% to 50% of cells (immunohistochemistry); positive (++), moderate to high staining in >50% of the cells; and high positive (+++), intense staining in ~100% of cells. Subsequently, this four-tier scheme was converted to a numerical "score" [ranging from 1 (for –) to 4 (for +++)] for each section or tissue microarray core. To consider the heterogeneity of expression frequently observed in adenocarcinoma, we calculated mean values from the staining of different cores of each case. Mean values were then reassigned to the closest discrete value. Details are discussed and reviewed in ref. 32.

Statistical analysis. Binding affinity of mAbs to the immobilized antigen was calculated by nonlinear regression analysis. The correlation analysis of mRNA with protein expression was done using Spearman nonparametric analysis on paired observations; P value and 95% confidence interval (95% CI) are given. To compare the frequencies of expression levels between different tissues and between tumors with and without a metastasis at the time of diagnosis, a {chi}2 test for trend was used on appropriate contingency tables. Prism/GraphPad 4.0 software was used.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
FGF-BP1 detection by ELISA. From the hybridoma generated from spleens of BALB/c mice immunized with a GST-fusion protein of human FGF-BP1, we observed 687 aminopterin-resistant colonies from 1,920 wells. The supernatants of 78 of these colonies were screened by ELISA for the production of mAbs to FGF-BP1 and categorized as high reactivity (A450 ≥ 1.5; 13 of 78), low reactivity (1.5 > A450 > 0.2; 57 of 78), and nonreacting clones (A450 ≤ 0.2; 8 of 78). All positive hybridomas were further expanded and supernatants were retested. Figure 1A summarizes the results of this second screening and shows the respective isotypes of the antibodies. To determine the sensitivity of the mAbs, a direct ELISA with decreasing concentration of the antigen was done with affinity-purified 4E7, 3E11, and 16H5 and a nonspecific antibody as negative control. The limit of detection of FGF-BP1 was slightly below 1 ng/well (Fig. 1B). From titration curves with different antibody dilutions, we estimated that the dissociation constant of the antibodies for the immobilized antigen is between 0.1 nmol/L (15 ng/mL; 4E7) and 0.3 nmol/L (3E11; Fig. 1C). Recombinant rat FGF-BP1 protein, which is 57% identical at the amino acid sequence to its human homologue (26), was used in the same titration assay to assess cross-reactivity. We observed no signal for the rat homologue with the three antibodies used even at their highest concentration (Fig. 1C, diamond). This finding shows the specificity of the antibodies for human FGF-BP1.


Figure 1
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  		Figure 1. ELISA assays with mouse mAbs to human FGF-BP1. A, isotyping and ELISA. Immobilized his-FGF-BP1 (50 ng; ref. 18) was incubated with different hybridoma clones as primary antibodies as described in Materials and Methods. Black columns, hybridoma used in the present study. Representative of two independent experiments. B, sensitivity of the mAbs. Different concentrations of his-FGF-BP1 antigen were immobilized and incubated with 0.2 µg/mL of the mAbs indicated. The 6A6 mAb served as the background control (nsb). Representative experiment done in duplicate. C, specificity of the mAbs. Fifty nanograms of his-FGF-BP1 were immobilized in a microtiter plate and detected by direct ELISA with different concentrations of the mAbs indicated. Representative experiment done in triplicate. Recombinant rat FGF-BP1 protein (26) was used as a specificity control with all three antibodies and the result is indicated by a single symbol ({diamond}).

 
Western blot analysis of different samples showed that the mAbs 4E7 and 3E11, but not 16H5 (not shown), detect FGF-BP1 at an apparent molecular mass of 34 kDa (Fig. 2A) and also recognize a smaller FGF-BP1 fragment of 17 kDa in transfected cells, xenograft tissues, and tumor samples (Fig. 2B-D). The 17-kDa protein is a truncated product of FGF-BP1 that was first purified from A431 cells, sequenced, and named HBp17 by Wu et al. (19). Similar to our present findings with the mAbs, we reported earlier that a polyclonal rabbit antibody to FGF-BP1 also detects the 34- and 17-kDa proteins in the FGF-BP1-transfected SW-13 BP cells used in the experiments in Fig. 2B and C (33).


Figure 2
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  		Figure 2. mAbs recognize human FGF-BP1 in immunoblot and immunoprecipitation analyses. A, Western blot analysis of 25 ng of recBP1 by 4E7 and 3E11 mAbs. B and C, Western blot analysis (B) and immunoprecipitation (C) from mock and FGF-BP1 transfected SW-13 cells. D, Western blot analysis of FGF-BP1 from frozen surgical samples of colon and head and neck (H/N) cancer tissues and ME180 xenografts. Both mAbs recognize two specific bands of 34 and 17 kDa apparent molecular mass (arrows).

 
Detection of FGF-BP1 protein in cells and in tissue sections. To assess the utility of the mAbs described above for detection of FGF-BP1 in tissues, we used ME180 squamous cervical carcinoma cells that express endogenous FGF-BP1 and are tumorigenic in athymic nude mice, as well as FGF-BP1-negative SW-13 cells, as model systems. As illustrated in Fig. 3A, SW-13 cells transiently transfected with an FGF-BP1 expression vector show the protein in the cytoplasm as a punctuated stain (green). The subpopulation of nontransfected SW-13 cells visualized by staining for actin (red) lacked staining for FGF-BP1. ME180 cells showed cytoplasmic FGF-BP1 staining with a similar punctuated pattern in all of the cells. Furthermore, in agarose-embedded, formalin-fixed and sectioned ME180 and SW-13 cell pellets, we detected a strong staining signal (brown) for endogenous FGF-BP1 in ME180 cells and no staining in the FGF-BP1-negative SW-13 cells (Fig. 3B).


Figure 3
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  		Figure 3. Specificity of FGF-BP1 immunostainings. A, immunocytochemistry of FGF-BP1 transiently transfected SW-13 cells and wild-type ME180 cells that express endogenous FGF-BP1. Only some of the transiently transfected SW-13 cells expressed detectable FGF-BP1 protein (green) and the F-actin stain highlights all cells (red). Representative of three independent experiments. B, immunohistochemistry of agarose-immobilized cell pellets from ME180 (right, FGF-BP1 positive) and SW-13 cells (left, FGF-BP1 negative). Different hybridoma supernatants were used at 1:10 dilution. C, immunohistochemistry of mouse xenograft tumors from ME180 cells using FGF-BP1-reactive 3E11, 4E7, 5G9, and 16H5 hybridoma supernatants (1:10 dilution). Secondary antibody only and the nonreactive 6A6 hybridoma supernatant (1:10 dilution) were used as negative controls.

 
For the initial immunohistochemical analyses of formalin-fixed, paraffin-embedded tissue samples, we used human ME180 cell xenograft tumors grown s.c. in athymic nude mice. Sections from these xenografts were stained with a panel of mAbs (3E11, 4E7, 5G9, and 16H5) indicated in Fig. 3C. These mAbs detected abundant FGF-BP1 in the cytoplasm and in the extracellular matrix of the tumor cells. In contrast, no immunoreactivity was detected with a control mAb (6A6) that is nonreactive to FGF-BP1 or in the absence of a primary antibody (Fig. 3C, bottom). From these data, we conclude that the newly generated mAbs specifically recognize FGF-BP1 by immunofluorescence and immunohistochemistry and can be used to monitor protein expression in archival human tissues.

Detection of FGF-BP1 protein in pancreatic and colorectal tissues with different malignant progression. In previous studies, we found that FGF-BP1 was overexpressed in dysplastic colon lesions both in human and mouse samples (24). Here we studied the expression of FGF-BP1 in different stages of progression towards pancreatic adenocarcinoma and nonadenocarcinoma of the pancreas as well as in invasive primary human colorectal carcinomas and metastases. The 3E11 mAb (see above) was used to detect FGF-BP1 protein in tissue microarrays and in full sections of archival colorectal and pancreatic tissues. The protein was mostly detected in the cytoplasm of tumor cells and in the tumor stroma of some of the samples (Fig. 4). Relative to normal colon and normal pancreatic ducts, respectively, we found a significant increase in FGF-BP1 protein expression in colon adenoma, invasive adenocarcinomas, and metastases, as well as in pancreatitis, PanIN, and pancreatic adenocarcinomas (P < 0.0001). Representative examples of the staining in tissues are shown in Fig. 4 (left) and the evaluation is summarized in Fig. 5A.


Figure 4
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  		Figure 4. Staining for FGF-BP1 protein and mRNA. Tissue microarrays and full-size sections from tissues were stained for FGF-BP1 protein using the 3E11 mouse mAb (immunohistochemistry) and for mRNA using digoxigenin-labeled riboprobes (in situ hybridization). Different tissues at low (10x objective; left) and high magnification (40x objective; right). Rectangles, magnified areas depicted to the right. For the different tissue types, adjacent paraffin sections were selected to compare protein and mRNA stainings side by side. Normal colon showed mostly only background signals in the stainings. In colorectal adenoma, carcinoma, and organ metastases, high expression levels of protein (brown stain) and mRNA were typically observed. In some samples, extracellular FGF-BP1 protein was found in the tumor stroma (red arrows). In normal pancreatic ducts, protein or mRNA stainings were below detection whereas PanIN and pancreatic adenocarcinoma showed distinct expression of protein and mRNA. Staining with and without the 3E11 primary mAb, as well as antisense and sense mRNA probes, was used as control.

 

Figure 5
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  		Figure 5. FGF-BP1 protein and mRNA expression analysis. A and B, the distribution of protein (A) and mRNA (B) expression levels [– to +++] are shown for different tissues as indicated. Control tissues are from different organs described in the text. n, number of cases. P values from the {chi}2 analysis for trend comparison of the results are indicated. ns, P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.0001. Each case was represented by an average of five to six cores.

 
FGF-BP1 expression during pancreatic malignant transformation showed the highest rate in early stages of malignant transformation [i.e. the high-grade PanIN (68% positive) and somewhat less frequently in low-grade PanIN and adenocarcinoma (43% and 49%, respectively)]. FGF-BP1 protein was also detected in 47% of the pancreatitis samples. All of these groups had significantly higher expression than normal pancreas ducts (P < 0.0001). Much in contrast, only 23% of nonadenocarcinoma of the pancreas showed any expression of FGF-BP1 although this was still an increase relative to normal pancreas ducts (P = 0.0044) but significantly lower than high-grade PanIN and adenocarcinoma (P < 0.003; Fig. 5A). The highest intracellular levels and frequencies of FGF-BP1 protein expression were observed in the colorectal adenoma (90% positive) and this was slightly higher than adenocarcinoma and metastases (72% and 80%, respectively).

FGF-BP1 protein expression in reference tissues, including small intestine, brain, skin, lymph nodes, prostate hyperplasia, lung, and normal breast samples, was low and similar to normal colon (P > 0.1), with pancreas ducts showing the lowest levels of staining. It is noteworthy that nonspecific stainings were observed in the absence of the primary antibody in several specimens from liver (hepatocytes), stomach crypts, bile ducts, and kidney tubules. These tissues also lacked FGF-BP1 mRNA by in situ hybridization and we thus concluded that these nonspecific stainings did not mask endogenous FGF-BP1 protein expression. From these data, we conclude that FGF-BP1 protein overexpression initiates early in the development of pancreatic and colorectal neoplasia before the progression to invasive carcinoma and remains overexpressed in metastasis. This early initiation of expression of this secreted protein may also provide a serum marker to discover subjects at risk who developed early pancreatic or colorectal lesions.

Detection of FGF-BP1 mRNA in tissues. To measure the expression of FGF-BP1 in human tissues at the mRNA level, in situ hybridization with digoxigenin-labeled RNA probes was done in the same series of tissues as the protein detection. Cytoplasmic mRNA staining was evaluated according to the grading system used for protein and average expression scores were calculated for tumors with multiple cores as described (28, 32).

Similar to the protein expression, a significant increase in FGF-BP1 mRNA expression was observed in pancreatitis, PanIN, and pancreatic adenocarcinomas, as well as in colorectal adenoma, adenocarcinoma, and metastases, when compared with reference tissues (normal pancreatic duct or colon, respectively; all P < 0.001). Representative examples are shown in Fig. 4 (right) and the evaluation of the samples is summarized in Fig. 5B. FGF-BP1 mRNA expression was found in all adenoma, in 80% of primary colorectal adenocarcinoma, and in 91% of metastases. It is noteworthy that metastases showed higher levels of expression than primary tumors (P = 0.036). In pancreatic adenocarcinoma, the level and frequency of mRNA expression were lower than those in the colorectal cancers (P < 0.003) and followed the pattern observed in the protein analysis: The highest expression frequency was observed in high-grade PanIN (79% positive) with somewhat lower rates in pancreatic adenocarcinoma (60%), low-grade PanIN (52%), and pancreatitis (40%), all being significantly above normal pancreas ducts (P < 0.0001). Nonadenocarcinoma of the pancreas showed increased expression frequency (26%) relative to normal pancreas ducts (P = 0.002) and was indistinguishable from pancreatitis (P > 0.2) but significantly lower than low-grade or high-grade PanIN and adenocarcinoma (all P < 0.015). The mRNA expression in other reference tissues was low and similar to normal colon (P > 0.1) with pancreatic ducts showing the lowest staining levels. From these data, we conclude that FGF-BP1 mRNA overexpression is an early event in the development of colorectal and pancreatic adenocarcinoma and remains high during carcinoma progression. A comparison between protein and mRNA expression in this series of parallel samples showed a significant correlation between protein and mRNA expression: all samples, r = 0.67 (95% CI, 0.62-0.71), P < 0.0001, n = 718; colon, r = 0.62 (95% CI, 0.53-0.69), P < 0.0001, n = 264; pancreas, r = 0.55 (95% CI, 0.50-0.63), P < 0.0001, n = 285.

Inhibition of FGF-BP1-dependent growth by mAb 4E7. We have previously shown that FGF-induced SW-13 cells form colonies in soft agar and that this can be significantly enhanced by the expression or exogenous addition of FGF-BP1 (20). We applied this cell model here to assess whether the FGF-BP1 effect would be neutralized by the mAbs. Indeed, the highest-affinity mAb 4E7 (see Fig. 1C) inhibited FGF-induced colony formation of FGF-BP1-transfected SW-13 cells (Fig. 6). In contrast, colony formation in mock-transfected cells was not affected by mAb 4E7. Another mAb, 16H5, with a lower affinity than 4E7 (see Fig. 1C), did not inhibit the FGF-BP1 effect (not shown). These data suggest that at least one of the newly developed mAbs not only specifically recognizes FGF-BP1 in tissues but also functionally interacts with FGF-BP1 and blocks its biological effects, suggesting that this protein could also be targeted therapeutically using antibodies.


Figure 6
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  		Figure 6. Growth inhibition by the anti-FGF-BP1 mAb 4E7. Soft agar colony formation of pRcCMV/FGF-BP (SW-13/BP) or mock transfected SW-13 cells treated with the mAb 4E7 (40 µg/mL). The number of colonies is given in the absence and presence of FGF-2 ± mAb 4E7. ns, not significant; **, P < 0.01 (ANOVA).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
FGF-BP1 is a secreted protein that is thought to enhance FGF activity and, among other functions, to drive tumor angiogenesis (20, 21) as well as to modulate vascular permeability in cooperation with FGF (34). In more recent studies, we found that the FGF-BP1 protein was up-regulated early during wound healing of mouse and human skin as well as during the initiation of skin neoplasia by chemical carcinogens (35). Furthermore, in a series of studies using rabbit polyclonal antibodies, we had found differential regulation of the FGF-BP1 protein during rodent embryonic development (26, 36, 37) and in diseased children (38) and adult tissues (24). Based on the striking differential protein expression observed in those studies, we sought to generate mAbs to FGF-BP1 as more sustainable detection reagents to detect this protein in human archival tissues.

With these newly created antibodies on hand, we now report increased FGF-BP1 protein during the early phases of malignant progression in both pancreatic and colorectal tissues (i.e., in PanIN and adenoma). This distinct increase in protein expression during the tumor initiation process was paralleled at the mRNA level. For the early stages of colorectal tumorigenesis, the present study corroborates our previous report that showed FGF-BP1 up-regulation as a direct target gene of ß-catenin in early stages of colorectal neoplasia in ApcMin/+ mouse and human adenomatous lesions (24). Beyond this early induction, overexpression of FGF-BP1 is maintained throughout colorectal and pancreatic tumor progression to invasive and metastatic cancers. Furthermore, overexpression of FGF-BP1 was also found at high levels in organ metastases of colorectal carcinoma. It is noteworthy to point to the significantly lower expression of FGF-BP1 in nonadenocarcinoma versus adenocarcinoma of the pancreas, supporting the notion of a potentially distinct role of FGF-BP1 in the progression of PanIN to adenocarcinoma (39). In addition, it is conceivable that the secreted FGF-BP1 may be shed at sufficient amounts from PanIN lesions to be detectable in the circulation as an early warning sign of a high risk of patients with PanIN for the onset of invasive adenocarcinoma.

As previously published (21), the growth of xenograft tumors arising from colorectal carcinoma cells can be significantly reduced by ribozyme depletion of FGF-BP1, suggesting that inhibition of this protein could be a viable addition to the available cancer therapeutic approaches. Although the primary purpose of generating mAbs to FGF-BP1 was to generate better detection tools, we sought to also assess the potential of any of these mAbs to inhibit the function of FGF-BP1 in a bioassay. For this, we used a previously established in vitro model (20) and identified the 4E7 mAb as a blocking antibody that reduces anchorage-independent growth dependent on FGF-BP1 (see Fig. 6). This suggests that it is conceivable to tackle FGF-BP1 therapeutically with mAbs.


   Acknowledgments
 
Grant support: NIH/National Cancer Institute grant R01 CA71508 and the Gordon Family Foundation (A. Wellstein), the German department of education and research (R.T. Henke), a Johns Hopkins Clinical Scientist Award (A. Maitra), and a generous gift from the family of Margaret Lee.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   Footnotes
 
Note: E. Tassi and R.T. Henke contributed equally to this work.

Received 8/16/05. Revised 9/23/05. Accepted 11/10/05.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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