Intratumoral spatial distribution of hypoxia and angiogenesis assessed by 18F-FAZA and 125I-Gluco-RGD autoradiography.
The hypoxia-inducible factor-1 alpha (HIF-1 alpha) 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-dTyr(4)-cyclo(-Arg-Gly-Asp-dTyr-Lys(SAA)-), or 125I-Gluco-RGD) (assessing angiogenesis via binding to alpha v beta 3 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 alpha 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 alpha v beta 3 integrin receptor expression. The HIF-1 alpha-positive (HIFpos) tumor cell fraction was not significantly influenced by breathing conditions and covered between 0% and 35% of the total tumor section surface. However, the HIFpos 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.


CONCLUSIONS
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.
Picchio
Maria
M
Nuclear Medicine Department, IBFM-CNR, Scientific Institute H. San Raffaele, Milan, Italy.
Beck
Roswitha
R
Haubner
Roland
R
Seidl
Stefan
S
Machulla
Hans-Jürgen
HJ
Johnson
Timothy D
TD
Wester
Hans-Jürgen
HJ
Reischl
Gerald
G
Schwaiger
Markus
M
Piert
Morand
M
D000818:Animals; D001345:Autoradiography; D015687:Cell Hypoxia; D005260:Female; D005462:Fluorine Radioisotopes; D005960:Glucosides; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000032:analysis; D007150:Immunohistochemistry; D007457:Iodine Radioisotopes; D051379:Mice; D009374:Neoplasms, Experimental / Q000000981:diagnostic imaging*; D009389:Neovascularization, Pathologic / Q000000981:diagnostic imaging*; D009593:Nitroimidazoles; D009842:Oligopeptides / Q000187:drug effects*; D011877:Radionuclide Imaging
D005462:Fluorine Radioisotopes; D005960:Glucosides; C497443:Hif1a protein, mouse; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007457:Iodine Radioisotopes; D009593:Nitroimidazoles; D009842:Oligopeptides; C498055:iodogluco-RGD; C498052:fluoroazomycin arabinoside