Intratumoral Spatial Distribution of Hypoxia and Angiogenesis Assessed by ¹⁸F-FAZA and ¹²⁵I-Gluco-RGD Autoradiography

¹ Nuclear Medicine Department, IBFM-CNR, Scientific Institute H. San Raffaele, Milan, Italy
² Nuklearmedizinische Klinik, Technische Universität München, Munich, Germany
³ Nuklearmedizinische Klinik, Technische Universität München, Munich, Germany; Universitätsklinik für Nuklearmedizin, Medizinische Universität Innsbruck, Innsbruck, Austria
⁴ Pathologisches Institut, Technische Universität München, Munich, Germany
⁵ Radiopharmazie, PET Zentrum, Universität Tübingen, Tübingen, Germany
⁶ Department of Biostatistics, University of Michigan, Ann Arbor, Michigan
⁷ Department of Radiology, Division of Nuclear Medicine, University of Michigan, Ann Arbor, Michigan

^* To whom correspondence should be addressed. E-mail: mpiert{at}umich.edu.

	Abstract

The hypoxia-inducible factor-1 (HIF-1) activates angiogenesis in response to cellular hypoxia, suggesting a spatial correlation between angiogenesis and tissue hypoxia. Methods: Using digital autoradiography of coinjected ¹⁸F-labeled azomycin arabinoside (⁸F-FAZA) (assessing regional hypoxia) and a glycosylated RGD-containing peptide (¹²⁵I-3-iodo-DTyr⁴-cyclo(-Arg-Gly-Asp-DTyr-Lys(SAA)-), or ¹²⁵I-Gluco-RGD) (assessing angiogenesis via binding to v3 integrin receptors on endothelial cells) performed on 22 EMT6 tumor xenografts, we investigated the intratumoral spatial distribution of these tracers. We applied a Bayesian bivariate image analysis using the mean tumor-to-muscle ratio as a discriminator, resulting in 4 groups: FAZA high/RGD high (Q1), FAZA low/RGD high (Q2), FAZA low/RGD low (Q3), and FAZA high/RGD low (Q4). In an additional 18 xenografts, the immunohistochemically derived HIF-1 protein distribution was compared with ¹⁸F-FAZA autoradiography. Animals were divided into groups breathing either room air or carbogen (95% oxygen, 5% CO₂) for 4 h until sacrifice. Results: Under room air conditions, roughly 60% of the tumor surface displayed a spatial coupling of ¹⁸F-FAZA and ¹²⁵I-Gluco-RGD uptake: either high (Q1) or low (Q3) uptake for both tracers, with Q1 indicating spatial association of hypoxia and angiogenesis and Q3 indicating adequate oxygenation without active angiogenesis. However, the remaining approximately 40% of the tumor surface showed discordant ¹⁸F-FAZA and ¹²⁵I-Gluco-RGD uptake, indicating that hypoxia and angiogenesis are not necessarily spatially linked to each other and highlighting substantial intratumoral heterogeneity of the ¹⁸F-FAZA and ¹²⁵I-Gluco-RGD uptake. Although carbogen breathing conditions significantly decreased the mean ¹⁸F-FAZA tumor-to-muscle ratio, no significant changes were observed for ¹²⁵I-Gluco-RGD, indicating that an acute increase in tumor oxygenation did not influence v3 integrin receptor expression. The HIF-1–positive (HIF_pos) tumor cell fraction was not significantly influenced by breathing conditions and covered between 0% and 35% of the total tumor section surface. However, the HIF_pos tumor section surface was much smaller than the tumor section surface of increased ¹⁸F-FAZA uptake, suggesting that both markers are identifying distinctly different biologic processes associated with hypoxia. Conclusion: The study revealed a substantial spatial discordance of the ¹⁸F-FAZA and ¹²⁵I-Gluco-RGD tumor distribution suggesting that hypoxia and angiogenesis are not necessarily spatially linked in malignancies. These results may prove essential in developing advanced targeted systemic chemotherapeutic approaches (such as combinations of hypoxia-activated cytotoxins and antiangiogenic drugs) for hypoxic tumors.

Key Words: tumor hypoxia, angiogenesis, HIF-1, ¹⁸F-FAZA, ¹²⁵I-Gluco-RGD

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