Hypoxia and VEGF mRNA expression in human tumors

Abstract

High expression of circulating plasma vascular endothelial growth factor (VEGF) in patients with cancer is an indicator of poor treatment response. Similarly, hypoxia in tumors, as measured by oxygen needle electrodes, has been found to predict for tumor-treatment failure. These two predictors may be related because hypoxia is a potent stimulator of VEGF expression in vitro. However, the demonstration of a relationship between hypoxia and VEGF in human tumors has, to date, been indirect or even negative. The purpose of this study was to test whether this unexpected result was caused by factors unique to human tumors, or whether the prior results could have been influenced by the known complexities of VEGF regulation. Therefore, we undertook a direct assessment of VEGF induction in human tumors using in situ hybridization and compared its distribution with that of hypoxia, as measured by the distribution of adducts of the hypoxia marker EF5. The distribution of both markers was assessed in relationship to the distribution of blood vessels, as measured by antibodies to CD31. Our hypothesis was that VEGF mRNA and hypoxia would colocalize, assuming that detectability of the former was not limiting. Four squamous cell carcinomas, three sarcomas and one glioblastoma multiforme were studied. When VEGF mRNA signal was detectable, its maxima colocalized with regional maxima of EF5 binding. The strongest levels of both signals were sometimes adjacent to regions of tissue necrosis. However, we were unable to predict absolute levels of EF5 binding based on absolute levels of VEGF mRNA. Conversely, for all tumors studied, regions with relatively low levels of EF5 binding had relatively low or undetectable VEGF mRNA. We found moderate EF5 binding in some keratinized cells but VEGF mRNA was not expressed by these differentiated cells. The paradigm that hypoxia and VEGF expression are linked in human tumors is supported by the data presented herein. A better understanding of the biology behind VEGF expression, including its modulation by hypoxia, is important for optimizing its use as a prognostic indicator and/or modulating its presence with biologic therapies.

Figure 1

EF5 IHC and VEGF mRNA antisense staining patterns in a cervical SCC tumor from patient #1. EF5 staining and ISH techniques were performed as described in the text. The two sections were significantly displaced from each other (approximately 200 µm). Thus, the patterns and location of the highly invaginated and corded structures are similar but not identical. (A) Regions of high EF5 fluorescent intensity are seen adjacent to regions of necrosis (N). (B) Strong VEGF mRNA intensity is seen in the cellular regions that correspond to the brightest areas of EF5 binding. Bar=500 µ.

Figure 2

EF5 IHC, VEGF mRNA antisense, hematoxylin and eosin, and VEGF mRNA sense staining patterns in a retromolar triangle SCC from patient #5. EF5 staining and ISH techniques were performed as described in the text. The sections for EF5, VEGF mRNA sense and antisense were nearly adjacent to each other. The section for ISH was counterstained for hematoxylin and eosin. (A) Moderately differentiated head and neck SCC stained for EF5. Regions of bright binding are seen adjacent to regions of necrosis (N). (B) Moderate signal for VEGF mRNA antisense are seen in cellular regions that correspond to the brightest areas of EF5 binding. The circle in the center of the figure is a bubble artifact. (C) Hematoxylin and eosin demonstrates that the tissues that stain for EF5 and VEGF mRNA antisense are viable tumor tissue. The regions that are more eosinophilic (pink) are stromal tumor tissue. (D) Staining for VEGF mRNA with the sense probe reveals no signal. Black region at 3 o'clock is an artifact. Bar=500 µ.

Figure 3

EF5 IHC, VEGF mRNA antisense, hematoxylin and eosin, and CD31/PECAM versus EF5 patterns in a retromolar triangle SCC from patient #26. EF5, CD31 staining, and ISH techniques were performed as described in the text. Sections probed for EF5 and antisense VEGF mRNA were adjacent to each other. Sections for CD31/EF5 were from the same tissue mass and counterstained with hematoxylin and eosin. (A) Regions of bright EF5 binding are seen in keratinized (K) as well as nonkeratinized (nk) tumor tissues. A region of necrosis (N) is seen adjacent to regions of high EF5 binding. (B) VEGF mRNA signal is not seen in this tissue. Keratin pearls (K) are easily identified. (C) Hematoxylin and eosin staining demonstrates complex pathologic patterns in these SCC tumors. Keratin pearls (K) and nonkeratinized tumor tissue and tumor stroma are all identified. (D) Staining for both EF5 (red) and CD31 (green) demonstrates the inverse anatomic relationship between keratinized tumor regions that bind EF5 and blood vessels. This relationship supports the hypothesis that these keratinized tumor tissues are hypoxic. Bar=500 µ.

Figure 4

EF5 IHC, VEGF mRNA antisense and sense staining patterns in a high-grade synovial cell sarcoma from patient #16. EF5 staining and ISH techniques were performed as described in the text. The two sections were adjacent to each other. (A) High VEGF mRNA signal is seen in an “H”-shaped region adjacent to areas of necrosis. (B) Regions of bright EF5 binding are seen, representing hypoxia and these correspond to the VEGF mRNA signal seen in Figure 3C. In the middle of both areas of EF5 binding, acellular regions are seen, representing microscopic areas of necrosis (N). (C) VEGF mRNA sense staining pattern corresponding to region of strongest VEGF antisense signal. Peroxidase staining is not seen, providing a negative control for comparison. Bar=500 µ.

Figure 5

Relationship between observed degree of VEGF mRNA signal (measured by ISH) and EF5 binding (measured by IHC staining, and assessed as a percentage of cube-reference binding).