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Journal of Histochemistry and Cytochemistry, Vol. 46, 569-576, May 1998, Copyright © 1998, The Histochemical Society, Inc.

SYMPOSIUM PAPER

Vascular Permeability Factor/Vascular Endothelial Growth Factor and Vascular Stroma Formation in Neoplasia: Insights from In Situ Hybridization Studies

Lawrence F. Browna, Anthony J. Guidia, Kathi Tognazzia, and Harold F. Dvoraka
a Departments of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts

Correspondence to: Lawrence F. Brown, Dept. of Pathology, Beth Israel Deaconess Medical Center, East Campus, 330 Brookline Ave., Boston, MA 02215.


*   Summary
*Top
 *Summary
*Introduction
 *Materials and Methods
 *Results and Discussion
 *Literature Cited

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 46:569–575, 1998)

Key Words: VPF, VEGF, angiogenesis, KDR, flt-1, carcinoma


*   Introduction
*Top
 *Summary
 *Introduction
*Materials and Methods
 *Results and Discussion
 *Literature Cited

The formation of vascular stroma (angiogenesis) is essential for tumor growth beyond a minimal size (Folkman and Shing 1992 Down), and therapies targeted at inhibiting angiogenesis may prove to play an important role in control of tumor growth.

Vascular permeability factor (VPF), also known as vascular endothelial growth factor (VEGF), is a multifunctional angiogenic cytokine expressed by many tumors (reviewed in Dvorak et al. 1995 Down). VPF/VEGF binds to endothelial cells by at least two specific cell surface tyrosine kinase receptors, flt-1 and KDR (de Vries et al. 1992 Down; Terman et al. 1992 Down). VPF/VEGF induces microvascular hyperpermeability with a strength some 50,000 times that of histamine, leading to extravasation of plasma proteins and consequent proangiogenic stromal changes (Dvorak et al. 1979 Down; Senger et al. 1983 Down) . VPF/VEGF is also an endothelial cell mitogen (Ferrara and Henzel 1989 Down; Gospodarowicz et al. 1989 Down; Keck et al. 1989 Down; Leung et al. 1989 Down) and alters the synthetic profile of endothelial cells, inducing expression of proteins such as collagenase and plasminogen activators, which may play a role in angiogenesis (Pepper et al. 1991 Down; Unemori et al. 1992 Down). Angiogenesis and tumor growth can be suppressed by inhibition of VPF/VEGF or its receptors in certain experimental models (Kim et al. 1993 Down; Millauer et al. 1994 Down). In situ hybridization studies have been important in defining the role of VPF/VEGF in the formation of vascular stroma in malignancy.


*   Materials and Methods
*Top
 *Summary
 *Introduction
 *Materials and Methods
*Results and Discussion
 *Literature Cited

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 2–4 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 (Brown et al. 1992 Down). The anti-sense probe hybridizes specifically with a region of VPF/VEGF mRNA common to all known VPF/VEGF splicing variants. 35S-Labeled single-stranded anti-sense and sense RNA probes for the VPF/VEGF receptors flt-1 and KDR were designed by Robert W. Jackman and have been described previously (Brown et al. 1993a Down, Brown et al. 1993b Down).

Immunohistochemistry
Preparation of an affinity-purified rabbit antibody to the N-terminal 26 amino-acid peptide of human VPF/VEGF has been previously described (Dvorak et al. 1991 Down). The antibody bound VPF/VEGF in ELISA assays and on immunoblots (Sioussat et al. 1993 Down). Immunohistochemistry (IH) was performed on 5-µm frozen sections of the same blocks used for ISH, using an avidin–biotin–peroxidase conjugate protocol. Normal rabbit IgG diluted to an equivalent protein concentration was used as a control in place of the primary antibody.

Staining for factor VIII-related antigen utilized a rabbit polyclonal antibody (Dako; Santa Barbara, CA) with an avidin–biotin–peroxidase conjugate protocol.


*   Results and Discussion
*Top
 *Summary
 *Introduction
 *Materials and Methods
 *Results and Discussion
*Literature Cited

VPF/VEGF Expression in Invasive Carcinomas
We first used in situ hybridization studies to study expression of VPF/VEGF in an animal carcinoma model (Dvorak et al. 1991 Down). Whereas VPF/VEGF mRNA was detected only in tumor cells, immunoreactive VPF/VEGF protein could be detected in both tumor cells and blood vessels. These data suggested that VPF/VEGF was being synthesized by tumor cells and bound by endothelial cells. VPF/VEGF has been shown to bind to endothelial cells by at least two specific cell surface tyrosine kinase receptors, flt-1 and KDR (de Vries et al. 1992 Down; Terman et al. 1992 Down) .

The first human tumor we studied for the expression of VPF/VEGF, flt-1, and KDR mRNAs was the highly vascular renal cell carcinoma (Brown et al. 1993a Down). VPF/VEGF mRNA was strongly expressed by the malignant epithelial cells of renal cell carcinoma (Figure 1A and Figure 1B). No labeling was seen with control sense probes, and background was low (Figure 1C and Figure 1D). Expression of mRNAs of the VPF/VEGF receptors flt-1 and KDR was strong in endothelial cells in the small vessels adjacent to carcinoma (Figure 1E and Figure 1F). Immunohistochemical staining with anti-VPF/VEGF antibodies revealed staining of both tumor cells and blood vessels (Figure 2A). This provided additional support for a model in which VPF/VEGF was expressed by tumor cells and bound to VPF/VEGF receptors expressed on endothelial cells. Strong expression of VPF/VEGF was also seen in the malignant epithelium of invasive high-grade transitional cell carcinomas of the bladder in this study.



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  		Figure 1. In situ hybridization studies of renal cell carcinoma. (A,B) Paired brightfield and polarized fluorescence photomicrographs showing strong expression of VPF/VEGF mRNA by tumor cells. (C,D) No specific labeling and low background are seen with control sense probe. (E,F) Strong expression of KDR mRNA is seen in endothelial cells of small vessels within the tumor. Bar = 25 µm.



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  		Figure 2. Immunohistochemical staining. (A) Renal cell carcinoma with light staining of tumor cells for VPF/VEGF protein and darker staining of adjacent small blood vessels. (B–D) Staining for factor VIII-related antigen (B) in normal cervix. (C,D) Marked increase in the number of vessels in high-grade dysplasia with prominent rimming pattern at the stromal–epithelial interface. Bars: A–C = 25 µm; D = 125 µm.

In an effort to extend these findings to other common human malignancies, we studied adenocarcinomas of the colon, stomach, small intestine, and pancreas (Brown et al. 1993b Down). The patterns of expression were the same as seen in renal cell carcinomas, i.e., tumor cells strongly expressed VPF/VEGF mRNA and endothelial cells in the closely associated vessels in the vascular stroma strongly expressed flt-1 and KDR. Anti-VPF/VEGF antibodies stained both tumor cells and vessels. In later studies, the same patterns of expression of VPF/VEGF and its receptors were noted in invasive ductal carcinoma of the breast (Brown et al. 1995 Down), endometrial adenocarcinoma (Guidi et al. 1996 Down), ovarian carcinoma (Abu-Jawdeh et al. 1996 Down), and invasive squamous cell carcinoma of the uterine cervix (Guidi et al. 1995 Down).

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 2–4 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 (Guidi et al. 1995 Down), strong expression of VPF/VEGF mRNA was seen not only in invasive squamous cell carcinoma but also in high grade squamous dysplasia (Figure 3A and Figure 3B). Therefore, expression of VPF/VEGF was upregulated before tumor invasion in the cervix. Furthermore, the strong expression of VPF/VEGF mRNA in high-grade dysplasia was associated with increased numbers of microvessels in the underlying stroma, which in some cases formed a nearly continuous rim at the junction of epithelium and stroma (Figure 2B–D), and the endothelial cells on these vessels strongly expressed the VPF/VEGF receptors flt-1 and KDR (Figure 3C and Figure 3D). Therefore, angiogenesis is initiated before tumor invasion in the cervix, and VPF/VEGF may play an important role in this process.



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  		Figure 3. In situ hybridization studies. (A,B) Paired brightfield and polarized fluorescence photomicrographs showing strong expression of VPF/VEGF mRNA by squamous epithelial cells in high-grade dysplasia of the cervix. (C,D) Strong expression of KDR mRNA is seen in endothelial cells of small vessels immediately beneath the dysplastic epithelium. Bar = 25 µm.

Similar findings were seen in ductal carcinoma in situ (DCIS) of the breast (Brown et al. 1995 Down). Strong expression of VPF/VEGF mRNA was seen in the epithelial cells of DCIS. The number of microvessels has been reported to be increased in DCIS, and rimming of microvessels around DCIS, similar to the pattern seen in cervical dysplasia, has also been described (Guidi et al. 1994 Down). Furthermore, the endothelial cells of these vessels strongly expressed mRNA of the VPF/VEGF receptors flt-1 and KDR.

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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  		Figure 4. In situ hybridization studies. (A,B) Paired brightfield and polarized fluorescence photomicrographs showing strong expression of VPF/VEGF mRNA by metastatic breast carcinoma cells in an axillary lymph node. (C,D) strong expression of KDR mRNA is seen in endothelial cells of small vessels adjacent to the metastatic tumor cells. Bar = 25 µm.

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 (Shweiki et al. 1992 Down). We have seen similar strong expression of VPF/VEGF expression adjacent to areas of necrosis in all of the carcinomas we have studied. Careful observation of the patterns of mRNA expression can also provide clues to factors that may regulate 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.


*   Literature Cited
*Top
 *Summary
 *Introduction
 *Materials and Methods
 *Results and Discussion
 *Literature Cited

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