SYMPOSIUM PAPER |
Correspondence to: Lawrence F. Brown, Dept. of Pathology, Beth Israel Deaconess Medical Center, East Campus, 330 Brookline Ave., Boston, MA 02215.
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Summary |
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The formation of vascular stroma plays an important role in the pathophysiology of malignancy. We describe the use of in situ hybridization in our laboratory as a tool to study the role of vascular permeability factor/vascular endothelial growth factor in the angiogenesis associated with malignancy. (J Histochem Cytochem 46:569575, 1998)
Key Words: VPF, VEGF, angiogenesis, KDR, flt-1, carcinoma
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Introduction |
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The formation of vascular stroma (angiogenesis) is essential for tumor growth beyond a minimal size (
Vascular permeability factor (VPF), also known as vascular endothelial growth factor (VEGF), is a multifunctional angiogenic cytokine expressed by many tumors (reviewed in
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Materials and Methods |
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In Situ Hybridization
In situ hybridization (ISH) was performed on 6-µm frozen sections of tissues that had been fixed in 4% paraformaldehyde in PBS, pH 7.4, for 24 hr at 4C and then transferred to 30% sucrose in PBS overnight at 4C and frozen in OCT compound (Miles Diagnostics; Elkhart, IN) and stored at -70C or, alternatively, on 4-µm-thick sections of archival formalin-fixed, paraffin-embedded tissue.
Briefly, slides with paraffin sections were passed through xylene and graded alcohols; 0.2 M HCl; 10 mM Tris/1 mM EDTA with 3 µg/ml proteinase K; 0.2% glycine; 4% paraformaldehyde in PBS, pH 7.4; 0.1 M triethanolamine containing 1/200 (v/v) acetic anhydride; and 2 x SSC. Slides were hybridized overnight at 50C with 500,000 counts of gel-purified 35S-labeled riboprobe in the following mixture: 0.3 M NaCl, 0.01 M Tris, pH 7.6, 5 mM EDTA, 50% formamide, 10% dextran sulfate, 0.1 mg/ml yeast tRNA, and 0.01 M dithiothreitol. Posthybridization washes included 2 x SSC/50% formamide/10 mM dithiothreitol at 50C; 4 x SSC/10 mM Tris/1 mM EDTA with 20 µg/ml ribonuclease at 37C; and 2 x SSC/50% formamide/10 mM dithiothreitol at 65C and 2 x SSC. Slides were then dehydrated through graded alcohols containing 0.3 M ammonium acetate, dried, coated with Kodak NTB 2 emulsion, and stored in the dark at 4C for 2 weeks. The emulsion was developed with Kodak D19 developer and the slides were counterstained with hematoxylin. Slides with frozen sections were not passed through xylene and alcohol initially but placed directly into 0.2 M HCl, and less proteinase K was used (1 µg/ml). Otherwise, processing was the same.
Anti-sense single-stranded 35S-labeled VPF/VEGF RNA probe and its sense control were designed by Brygida Berse and have been described previously (
Immunohistochemistry
Preparation of an affinity-purified rabbit antibody to the N-terminal 26 amino-acid peptide of human VPF/VEGF has been previously described (
Staining for factor VIII-related antigen utilized a rabbit polyclonal antibody (Dako; Santa Barbara, CA) with an avidinbiotinperoxidase conjugate protocol.
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Results and Discussion |
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VPF/VEGF Expression in Invasive Carcinomas
We first used in situ hybridization studies to study expression of VPF/VEGF in an animal carcinoma model (
The first human tumor we studied for the expression of VPF/VEGF, flt-1, and KDR mRNAs was the highly vascular renal cell carcinoma (
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In an effort to extend these findings to other common human malignancies, we studied adenocarcinomas of the colon, stomach, small intestine, and pancreas (
In situ hybridization with radioactive probes can be evaluated in a semiquantitative manner by counting the number of grains in the photographic emulsion overlying the cells. For example, if large numbers of grains are counted over malignant cells in invasive carcinoma but few grains are counted over normal epithelium, then expression is strong in the tumor compared to the normal tissue. Many blocks in our studies were chosen to contain both tumor and normal tissue, thereby allowing direct comparison of mRNA expression in the same section. In almost all cases we have studied, expression of VPF/VEGF and its receptors has been far stronger in carcinomas than in adjacent normal tissue.
In summary, VPF/VEGF and its receptors are strongly expressed in many common invasive human carcinomas, and probably play an important role in the formation of vascular stroma in these malignancies. This makes VPF/VEGF an especially attractive target for antiangiogenic therapy, because such therapy could be effective across a broad range of common human carcinomas.
VPF/VEGF Expression in Dysplasia and Carcinoma In Situ
In situ hybridization is an even more critical tool in the study of the early steps in neoplastic progression. When large invasive carcinomas are resected, a pathologist can often select a piece of tumor that is not important for diagnostic purposes to be optimally prepared for molecular studies such as Northern analysis. However, small biopsy specimens must be routinely processed through paraffin in their entirety for diagnosis. It is in these biopsies that the precursor lesions of invasive carcinoma, i.e., dysplasia and in situ carcinoma, can be found.
In situ hybridization can be performed on archival paraffin-embedded material, although in our experience with less sensitivity than when used on fixed frozen sections. In our hands, in situ hybridization is optimally performed on tissue immersed in freshly made 4% paraformaldehyde immediately after surgical removal, fixed for 24 hr at 4C, cryoprotected with sucrose, and embedded for frozen sectioning, with all steps using solutions that are ribonuclease-free. In contrast, in specimens undergoing routine histological processing for paraffin sections, the time before a tissue is placed into fixative, the time the tissue is in fixative, and exposure to RNA-degrading enzymes have not been controlled. In situ hybridization may therefore underestimate mRNA expression in archival paraffin-embedded tissues, but meaningful data can still be derived.
In a study of archival paraffin-embedded biopsies of the uterine cervix (
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Similar findings were seen in ductal carcinoma in situ (DCIS) of the breast (
Therefore, at least in the cervix and breast, strong expression of VPF/VEGF, flt-1, and KDR begins before tumor invasion and coincides with angiogenesis. When these tumors do invade, they may be invading a highly vascular stroma that they have induced rather than "normal" host stroma. Therefore, the formation of vascular stroma may also play a role in tumor invasion. Such findings by in situ hybridization provide insights into the process of neoplastic progression which would be difficult to obtain by other means.
VPF/VEGF Expression in Metastatic Carcinoma
In situ hybridization studies have also provided intriguing information on tumor metastases. For example, in foci of metastatic breast cancer, tumor cells strongly express VPF/VEGF (Figure 4A and Figure 4B), and there is associated vascular stroma formation with strong endothelial cell expression of VPF receptors (Figure 4C and Figure 4D). Thus, the formation of vascular stroma also occurs at sites of metastasis and VPF may play an important role in the process. Therefore, the tumor is not growing in the native tissue of the metastatic site but rather in a vascular stroma it has induced. The ability to form a vascular stroma may be important to the survival and growth of tumor cells at sites of metastases.
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Regulation of VPF/VEGF Expression
In addition to identifying the specific cell types responsible for synthesis of VPF or its receptors in tumors, in situ hybridization studies have also provided clues to factors which may regulate VPF expression. VPF/VEGF expression in glioblastoma was noted to be strongest near areas of necrosis, and in vitro experiments in the same study confirmed hypoxia as an important factor that can regulate VPF expression (
Conclusions
In situ hybridization studies have helped to establish VPF/VEGF as an important angiogenic factor in many common human carcinomas. VPF/VEGF may play an important role not only in invasive tumors but also in preinvasive lesions and in metastases.
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Literature Cited |
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