Hypoxic markers
Imaging hypoxia after oxygenation-modification: Comparing [18F]FMISO autoradiography with pimonidazole immunohistochemistry in human xenograft tumors

https://doi.org/10.1016/j.radonc.2006.07.023Get rights and content

Abstract

Purpose

Hypoxia is one of the reasons for radiation therapy resistance. Positron emission tomography using 18F-labeled misonidazole ([18F]FMISO) is a non-invasive method of imaging tumor hypoxia. Aim of this study was to validate [18F]FMISO against the clinically most widely used hypoxic cell marker pimonidazole under different oxygenation conditions.

Materials and methods

One human head and neck squamous cell carcinoma (SCCNij3) and two human glioblastoma (E102 and E106) xenograft tumor lines were studied after injection of [18F]FMISO and pimonidazole. Control mice were compared with a second group breathing carbogen to reduce tumor hypoxia and with a third group with clamped tumors to increase hypoxia. Tumor sections were analyzed on a phosphor imaging system and consecutively stained immunohistochemically (IHC) for visualization of pimonidazole. Pixel-by-pixel analysis was performed and the hypoxic fraction, obtained after segmentation of the pimonidazole signal, was related to the mean optical density of [18F]FMISO and pimonidazole.

Results

A moderate pixel-by-pixel correlation between [18F]FMISO autoradiography and pimonidazole IHC was found for the control tumors, after carbogen breathing and after clamping for SCCNij3. For E102 and E106, mean signal intensities for pimonidazole significantly decreased after carbogen breathing and increased after clamping, mean [18F]FMISO signal intensities increased significantly after clamping and a significant correlation between the hypoxic fractions and the mean [18F]FMISO signal intensities was found.

Conclusions

[18F]FMISO autoradiography and pimonidazole immunohistochemistry can both be used to visualize treatment induced changes in tumor hypoxia. However, the response to these modifications differs widely between xenograft tumor lines.

Section snippets

Animals and tumor models

The human head and neck squamous cell carcinoma xenograft tumor line SCCNij3, and the human glioblastoma tumor lines E102 and E106 were used for these experiments. Viable 1 mm3 tumor pieces were implanted subcutaneously in the abdominal flank of athymic BALB/C nu/nu mice and tumors were used for the experiments at a diameter of 8–9 mm. All mice were kept in accordance with institutional guidelines. All experiments were approved by the Animal Experiments Committee of the Radboud University

[18F]FMISO autoradiography and pimonidazole staining

Fig. 1 depicts examples of hypoxia visualized after pimonidazole immunohistochemistry, segmentation of the pimonidazole signal and [18F]FMISO autoradiography of the SCCNij3 and E106 tumors. As results after clamping for 30 or 60 min were similar, these were pooled for the analysis. In the SCCNij3 tumors, visualization of pimonidazole revealed a significant increase of the hypoxic fraction (obtained after segmentation of the gray-value image) after clamping and a non-significant decrease of the

Discussion

A well established method for quantification of hypoxia is the use of hypoxic cell markers such as pimonidazole. Immunohistochemical detection of pimonidazole in tissue sections has been shown to predict for prognosis in advanced stage head and neck cancer [16]. A possible alternative to this method is PET imaging of the hypoxic tracer [18F]FMISO. In various solid tumors hypoxia was detected using [18F]FMISO-PET [7], [17], [25], [29]. The advantage of imaging hypoxia with a [18F]FMISO-PET scan

Conclusion

Pimonidazole labeling can be used to detect viable hypoxic cells at pO2 levels below 10 mm Hg. By visualizing pimonidazole, hypoxia can be studied with a high spatial resolution providing detailed information at the microscopic level. [18F]FMISO can be used as PET-tracer to study hypoxia at the global level in tumors in situ. In the present study, pimonidazole labeling was used for validation of [18F]FMISO autoradiography. This pimonidazole signal reflects only reduced and bound marker. The

Acknowledgements

This research was supported by EC FP6 funding (Biocare – Contract no. LSHC-CT-2004-505785) and by Grant KUN 2003-2901 of the Dutch Cancer Society. We thank Dr. J.A. Raleigh for the gift of anti-pimonidazole MAb, Dr. H. Rennen for the synthesis of [18F]FMISO and the collaborators from the Central Animal Laboratory for biotechnical assistance and animal care.

References (32)

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