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Hypoxia-inducible Factor-1 Is an Intrinsic Marker for Hypoxia in Cervical Cancer Xenografts¹

Departments of Medical Biophysics [V. V., H. K. H., A. J. M., D. W. H.], Medical Oncology and Hematology, [T. N., D. W. H.], and Oncologic Pathology, [D. W. H.], Ontario Cancer Institute/Princess Margaret Hospital, Toronto, Ontario, Canada, M5G 2M9

	ABSTRACT

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The hypoxia-inducible factor 1 (HIF-1) is known to induce the expression of several proteins linked to the maintenance of oxygen homeostasis, cellular energy metabolism, and tumor progression. Its subunit (HIF-1) is stabilized under hypoxic conditions and, therefore, might represent an intrinsic marker for tissue hypoxia. Here we report on the spatial relationship between HIF-1 and the nitroimidazole hypoxia marker EF5 in cervical carcinoma xenografts, and on their spatial relationship to tumor blood vessels. EF5 was administered to mice bearing ME180 and SiHa cervical cancer xenografts. Frozen tumor tissue sections, triple-stained for HIF-1, the endothelial cell marker CD31, and EF5, were imaged using wide-field multiparameter immunofluorescence microscopy. Expression levels of EF5 and HIF-1 were similar in ME180 xenografts, but the percentage of tumor area stained with EF5 was significantly smaller than the percentage of HIF-1-positive area in SiHa tumors. In both tumor types the EF5-HIF-1 overlap was statistically significant, thus confirming their spatial and temporal colocalization. Spatial distribution analysis of EF5 and HIF-1 is consistent with different pO₂ value "thresholds" for EF5 binding and HIF-1 expression. Summarized, our results indicate that HIF-1 is a useful intrinsic marker for hypoxia in cervical carcinoma xenografts.

	Introduction

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In a number of human malignancies, hypoxia has been associated with overall poor patient outcome irrespective of the treatment modality used (1, 2, 3, 4) . These findings indicate that tumor hypoxia can contribute to the emergence of therapeutic resistance and biologically aggressive tumor phenotypes, although the precise mechanisms by which hypoxia exerts these effects remain to be fully elucidated.

A method for determinations of tumor oxygenation that has found wide clinical acceptance is polarographic O₂ Eppendorf histography (5) . Some limitations inherent to this method are its invasive nature that restricts measurements to easily accessible tumor sites and the resolution of measurements that may not reflect steep oxygen gradients that often exist in solid tumors (6) . Furthermore, intrinsic heterogeneity of tumor tissues (different proportions of normal and malignant cells, and tissue necrosis) can impede the accurate interpretation of tumor oxygenation profiles. An alternative approach to determination of tumor oxygenation is the measurement of tumor hypoxia. Nitroimidazole derivatives EF5 (7) and pimonidazole (8) can localize to hypoxic tissue regions and have been used extensively for labeling of hypoxia in both animal and human tumors. At low oxygen concentrations these markers bind to cellular macromolecules on bioreduction by nitroreductases. Subsequently, tissue-bound nitroimidazoles are detected using monoclonal antibodies. However, a potential problem associated with the use of chemical hypoxia markers is the issue of adequate sampling of (often heterogeneous) tumor tissues. On the other hand, high resolution and the ability of multiparameter spatial correlation analyses make this method very attractive for characterization of molecular mechanisms involved in cellular responses to conditions of reduced oxygenation.

HIF-1³ is a heterodimeric transcription factor composed of the HIF-1 and HIF-1ß subunits. The biological activity of HIF-1 is determined by the expression and activity of HIF-1. HIF-1 protein levels can increase rapidly in response to decreased cellular O₂ concentrations; the HIF-1 protein is just as rapidly degraded at aerobic oxygen levels (9 , 10) . HIF-1 binds to hypoxia response element DNA sequences found in promoter regions of genes involved in increased tissue delivery of O₂ or metabolic adaptation of cells to O₂ deprivation (11) . In a preceding study, we have found a positive correlation of HIF-1 expression with the pretreatment oxygenation status of human cervical carcinomas.⁴ Therefore, HIF-1 is a putative tumor hypoxia marker and might be used as a surrogate for chemical hypoxia markers in paraffin-embedded tissue specimens, thus allowing for retrospective clinical studies.

In the present study, we have analyzed the spatial relationship between HIF-1 and EF5 expression in cervical cancer xenografts, and their spatial distribution in relation to tumor blood vessels. Using wide-field immunofluorescence microscopy and digital image analysis we have determined the degree of EF5-HIF-1 overlap and have mapped their distribution as a function of distance to the nearest blood vessel. Our results indicate that HIF-1 is a useful intrinsic marker for tumor hypoxia.⁵

	Materials and Methods

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Tumor Generation and Sample Preparation.
ME180 and SiHa squamous cervical carcinoma cell lines were originally obtained from the American Type Culture Collection (Manassas, VA). Cells were maintained in growth medium consisting of -MEM (Canadian Life Technologies, Inc., Burlington, Ontario, Canada) supplemented with 10% FCS (CanSera, Rexdale, Ontario, Canada) and 0.1 mg/ml kanamycin (Canadian Life Technologies, Inc.) at 37°C in a humidified atmosphere containing 95% air and 5% CO₂. Tumor cell suspension (50 µl) containing 5 x 10⁵ cells were injected into the gastrocnemius muscle of five female SCID mice/tumor type. After the tumors had reached an average size of 4–5 mm in diameter, the nitroimidazole hypoxic marker EF5 (Ben Venue Laboratories, Bedford, OH) was injected via a lateral tail vein (200 µl of a 10-mM stock solution) to give a total body concentration of 100 µM. Later (3 h), the tumors were excised and placed in vials containing the Tissue-Tek OCT compound (Sakura Finetek, Torrance, CA) and immediately frozen in liquid nitrogen.

Immunofluorescence Labeling.
Using a HM 500 OM Microtome Cryostat (Microm Laborgeraete, Walldorf, Germany), three pairs of frozen sections at a 100-µm distance were cut from five individual ME180 and SiHa tumors, respectively, and adhered to 3-aminopropyl triethoxysilane (Sigma Chemical Co., St. Louis, MO) treated glass microscope slides. The sections were fixed in 3.7% paraformaldehyde in PBS (pH 7.2) for 5 min. Nonspecific staining was blocked using a multispecies blocking reagent (Signet Laboratories, Dedham, MA). One section was stained with H&E, and the other was incubated overnight at 4°C with a monoclonal anti-HIF-1 antibody (Affinity Bioreagents, Inc., Golden, CO), diluted 1:600 in Antibody Diluent (Dako, Carpinteria, CA) and subsequently incubated with a donkey antimouse Cy3 conjugated secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) diluted 1:400. Blood vessels were visualized using a monoclonal rat antimouse CD31 antibody (BD PharMingen, San Diego, CA) diluted 1:500 followed by incubation with a donkey antirat Cy2 conjugated secondary antibody (Jackson) diluted 1:100. Finally, tissue-bound EF5 was labeled using a 1:30 dilution of the monoclonal antibody ELK3–51 (provided by Dr. Cameron Koch, University of Pennsylvania, Philadelphia, PA) directly labeled with Cy5. The sections were washed twice in PBS between the labeling steps. Antibody incubations were performed for 1 h at room temperature except where otherwise stated.

Fluorescence Microscopy and Image Acquisition.
Digital images of the H&E sections were acquired as described previously (12) and used as a guide for fluorescence image analysis. Triple-labeled sections were imaged using a cooled CCD camera (Quantix; Photometrics, Tucson, AZ) mounted on an epifluorescence microscope (Olympus, Melville, NY) fitted with a computer-controlled motorized stage (Ludl Electronic Products, Hawthorne, NY). The camera and stage were controlled through the M5+ imaging system (Imaging Research Inc., St. Catharines, Ontario, Canada). Using an UPlanFl 10x/0.3 N.A. objective (Olympus), individual fields of view were excited sequentially with blue (480/30 nm), green (535/50 nm), and red (620/60 nm) light using a filter wheel under computer control. Filter cubes (535/40 nm, 610/75 nm, and 700/75 nm) were used to collect the emitted fluorescence. Images of individual fields of view were tiled into two-dimensional arrays to generate wide area images of the entire tumor cut surface.

Image Processing and Analysis.
Grayscale images (8-bit) of the tumor vasculature, HIF-1, and EF5 were saved as TIFF files. Image processing was done using Adobe PhotoShop 5.0 (Adobe Systems Inc., San Jose, CA). Copies of the images were converted to binary format by brightness thresholding to extract structures from the background. The brightness threshold was set to a level corresponding to 2 SD above the mean brightness values found in four corner regions of the images (CD31 and HIF-1 images) and 2 SD above the mean brightness values found in nontumor tissue (EF5 images). This was followed by median filtering to remove nonspecific single-pixel fluorescence, and 2 x 2 pixel binning followed by another brightness thresholding to reduce image size and facilitate subsequent image analysis. Tissue masks were generated by contrast enhancement and brightness thresholding of EF5 fluorescence images to visualize the entire tissue. Using H&E images as orientation, tissue masks were manually edited to include only apparently viable tumor tissue. Subsequently, tissue masks were used to restrict the analyses to tumor tissue. Image analysis was done using software modules developed by one of the authors (V. V.) in the Interactive Data Language (IDL 5.1; Research Systems Inc., Boulder, CO). To be able to directly compare the sizes of areas stained with HIF-1 and EF5, HIF-1-positive areas (staining localized to nuclei) were increased to approximate the whole cell areas using the Interactive Data Language dilate operator set to a width of 3 pixels. This value was determined by incrementally dilating HIF-1 images and analyzing histograms of tumor regions that were highly positive for HIF-1 staining. The operator width that produced 100% positive pixels was used to dilate the entire image. The percentage of HIF-1- and EF5-positive areas was determined by calculating the relative numbers of HIF-1- and EF5-positive pixels. The EF5\HIF-1 and HIF-1\EF5 overlaps were determined by subscribing binary EF5 images with addresses of positive pixels from the HIF-1 binary images and vice versa.

Tumor blood vessels were used as anatomical landmarks to map the tumor tissue as a function of distance to the nearest blood vessel. Positive pixels in the binarized CD31 images were set as centers of square masks with five different side lengths: 133, 277, 409, 553, and 685 µm. The CD31 images were then convolved with the masks in descending order; the pixels belonging to the respective distance classes were assigned different brightness values. Finally, the distance mask image was restricted to tumor boundaries using the tumor tissue mask (see Fig. 1, G and H ). The distance maps were used to determine EF5 binding and HIF-1 expression in individual distance classes.

View larger version (64K): [in this window] [in a new window]   Fig. 1. Single- and multiparameter wide-field fluorescence images of HIF-1, EF5, and CD31 staining in a ME180 tumor. Wide-field images of HIF-1 (A), EF5 (B), and CD31 (C) of a triple-labeled section of a ME180 cervical carcinoma xenograft. Composite image of all three parameters (D). Regions of D shown at higher magnification to illustrate heterogeneity in EF5 and HIF-1 colocalization: E, region of EF5\HIF-1 overlap; F, HIF-1 expression in a EF5-negative region; and G, mapping of tumor area as a function of distance to nearest blood vessel. Increasing distance is represented by increasingly darker shades of blue. Blood vessels are shown in yellow. H, a region of G shown at higher magnification.

	Results

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Representative individual fluorescence images of triple-labeled frozen tumor sections (HIF-1, EF5, and CD31) and a composite of all three parameters are shown in Fig. 1, A–D . HIF-1 expression in ME180 and SiHa xenografts was localized to nuclei, and HIF-1-positive cells were mostly found in distinct clusters. HIF-1 was expressed both in areas stained with EF5 (Fig. 1E) and in proximity of blood vessels (Fig. 1F) .

HIF-1 and EF5 Colocalization Analysis.
Mean HIF-1- and EF5-positive tumor areas were quite similar in ME180 tumors (5.4% and 6.2%, respectively; P < 0.17; see Fig. 2A and Table 1 for values in individual tumors). Accordingly, the degrees of mean HIF-1\EF5 and EF5\HIF-1 spatial overlaps were similar (12.3% and 13.9%, respectively; P < 0.29; Fig. 2B ). Areas of SiHa tumors stained with EF5 were significantly smaller than areas stained with HIF-1 (1.4% and 3.5%, respectively; P < 0.01; Fig. 2A and Table 1 ). Consequently, the EF5\HIF-1 overlap was greater than the HIF-1\EF5 overlap (19.7% and 9.5%, respectively; P < 0.01; see Fig. 2B ). To determine whether the observed degree of EF5 and HIF-1 expression overlap was statistically significant, random overlap was calculated as the product of EF5 and HIF-1 expression levels (fraction EF5-positive area x fraction HIF-1-positive area). In both ME180 and SiHa xenografts the mean observed overlap was significantly higher than the random overlap (0.75% and 0.34%, P < 0.001 and 0.24% and 0.04%, P < 0.001 in ME180 and SiHa tumors, respectively; see Table 1 ), indicating that EF5 and HIF-1 were spatially and temporally colocalized.

View larger version (17K): [in this window] [in a new window]   Fig. 2. EF5 and HIF-1 expression and colocalization in ME180 and SiHa tumors. Expression of EF5 () and HIF-1 () as percentage of total tumor area is shown in the top panel. Spatial overlap of EF5 and HIF-1 (EF5 to HIF-1, ; HIF-1 to EF5, ) as percentage of total EF5- and HIF-1-positive pixels is shown in the bottom panel; bars, ± SD.

View this table: [in this window] [in a new window]   Table 1 Spatial overlap of EF5 and HIF-1 in ME180 and SiHa cervical carcinoma xenografts

HIF-1 and EF5 Expression As a Function of Distance to the Nearest Blood Vessel.

View larger version (21K): [in this window] [in a new window]   Fig. 3. Spatial distribution of EF5 and HIF-1 relative to blood vessels. The percentage of EF5 only, HIF-1 only, and EF5\HIF-1 overlapping positive pixels is shown as a function of distance to the nearest blood vessel in ME180 (top panel) and SiHa (bottom panel) tumors; bars, ± SD.

	Discussion

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Hypoxia has been found to induce the expression of the transcription factor HIF-1 (13) . HIF-1 has been found to stimulate the expression of a variety of genes involved in oxygen transport and maintenance of cellular energy equilibrium (14) . Recent work by Jewell et al. (10) has shown that cellular HIF-1 levels rise almost instantaneously in response to conditions of reduced oxygenation. Subsequently, degradation of HIF-1 occurs within a similar time frame (min). Therefore, HIF-1 expression is potentially a marker for "real-time" determinations of hypoxia distribution, because it reflects the tumor oxygenation status at the time of biopsy or tumor removal. Chemical markers for hypoxia, e.g., EF5, continuously bind to hypoxic tissues as a function of drug availability and tissue oxygenation, and, therefore, represent cumulative or "historic" hypoxia markers.

The differences in EF5 and HIF-1 kinetics may have contributed to the relatively modest degree of spatial overlap (approximately 10–20% of total EF5- and HIF-1-positive pixels). Furthermore, dilation of HIF-1 images (to model whole cell staining) might have introduced some spatial artifacts because of the irregularity of cell shapes in tissue. We have determined that the majority of HIF-1-expressing cells are localized closer to blood vessels than the majority of EF5-positive cells. One possible explanation is that HIF-1 expression occurs at pO₂ values that are higher than those required for EF5 binding. Consequently, HIF-1 expression and EF5 binding may follow gradients in tumor oxygenation. Another possibility is that HIF-1 expression is higher in metabolically active cells more likely to be found at shorter distances to tumor blood vessels.

A number of in vitro studies indicate that not only hypoxia but also genetic alterations that occur during tumor progression (e.g., up-regulation of oncogenes, e.g., V-SRC, von Hippel-Lindau, and PI3k) may induce overexpression of HIF-1 (15, 16, 17) . Recent work by Chen et al. (18) has found that hypoxia can also induce overexpression of PI3k, thus providing an explanation for the association of increased PI3k activity and HIF-1 up-regulation. Recent clinical studies illustrate the role of von Hippel-Lindau mutation on HIF-1 expression levels in renal clear cell carcinomas (19) . Conversely, Airley et al. (20) have shown a strong correlation of tumor hypoxia and the expression of HIF-1-regulated glucose transporter GLUT-1 in cervical squamous cell carcinomas. Our results from both direct EF5\HIF-1 colocalization and HIF-1 spatial distribution analyses are consistent with a dominant role of hypoxia in determining cellular HIF-1 levels in human cervical carcinoma xenografts.

In future studies we plan to characterize HIF-1 spatial distribution patterns in relation to blood vessels in biopsies from human cervical carcinoma patients and relate these patterns to clinical parameters (e.g., tumor oxygenation as determined by Eppendorf pO₂ histography, local tumor control, and distant metastasis) to investigate the potential usefulness of HIF-1 expression as a prognostic marker in cervical carcinomas.

	FOOTNOTES

 
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.

¹ Supported by the National Cancer Institute of Canada, using funds raised by the Terry Fox Run.

² To whom requests for reprints should be addressed, at Medical Oncology and Hematology, Princess Margaret Hospital, 610 University Avenue, Toronto, Ontario, Canada, M5G 2M9. Phone: (416) 946-2262; Fax: (416) 946-2984; E-mail: david_hedley{at}pmh.toronto.on.ca

³ The abbreviations used are: HIF-1, hypoxia-inducible factor 1; EF5, [2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetamide]; PI3k, phosphatidylinositol 3R'-kinase.

⁴ H. K. Haugland, V. Vukovic, M. Pintilie, A. W. Fyles, M. Milosevic, R. P. Hill, and D. W. Hedley. Expression of hypoxia-inducible factor 1 in cervical carcinomas: correlation with tumor oxygenation and clinical outcome, submitted for publication.

⁵ Internet address: www.gnf.org/cancer/epican.

Received 5/ 7/01. Accepted 8/31/01.

	REFERENCES

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