RGD-labeled USPIO inhibits adhesion and endocytotic activity of alpha v beta3-integrin-expressing glioma cells and only accumulates in the vascular tumor compartment.
OBJECTIVE
To investigate the biologic effect of arginine-glycine-aspartic acid (RGD)-labeled ultrasmall superparamagnetic iron oxide (USPIO) (referred to as RGD-USPIO) on human umbilical vein endothelial cells (HUVECs), ovarian carcinoma (MLS) cells, and glioblastoma (U87MG) cells and on U87MG xenografts in vivo.


METHODS
All experiments were approved by the governmental review committee on animal care.USPIOs were coated with integrin-specific (RGD) or unspecific (arginine-alanine-aspartic acid [RAD]) peptides. USPIO uptake in HUVECs, MLS cells, and U87MG cells and in U87MG tumor xenografts was determined with T2 magnetic resonance (MR) relaxometry in 16 nude mice. Cells and tumors were characterized by using immunofluorescence microscopy. Trypan blue staining and lactate dehydrogenase assay were used to assess cytotoxicity. Statistical evaluation was performed by using a Mann-Whitney test or a linear mixed model with random intercept for the comparison of data from different experiments. Post hoc pairwise comparisons were adjusted according to a Tukey test.


RESULTS
HUVECs and MLS cells internalized RGD-USPIOs significantly more than unspecific probes. Controversially, U87MG cells accumulated RGD-USPIOs to a lesser extent than USPIO. Furthermore, only in U87MG cells, free RGD and alpha(v)beta(3) integrin-blocking antibodies strongly reduced endocytosis of nonspecific USPIOs. This was accompanied by a loss of cadherin-dependent intercellular contacts, which could not be attributed to cell damage. In U87MG tumors, RGD-USPIO accumulated exclusively at the neovasculature but not within tumor cells. The vascular accumulation of RGD-USPIO caused significantly higher changes of the R2 relaxation rate of tumors than observed for USPIO.


CONCLUSIONS
In glioma cells with unstable intercellular contacts, inhibition of alpha(v)beta(3) integrins by antibodies and RGD and RGD-USPIO disintegrated intercellular contacts and reduced endocytotic activity, illustrating the risk of inducing biologic effects by using molecular MR probes.
Department of Experimental Molecular Imaging, German Cancer Research Center, RWTH Aachen University, Pauwelsstrasse 20, 52074 Aachen, Germany. fkiessling@ukaachen.de
Fabian
F
Kiessling
Jochen
J
Huppert
Chunfu
C
Zhang
Jabadurai
J
Jayapaul
Stefan
S
Zwick
Eva C
EC
Woenne
Margareta M
MM
Mueller
Hanswalter
H
Zentgraf
Michael
M
Eisenhut
Yoseph
Y
Addadi
Michal
M
Neeman
Wolfhard
W
Semmler
D000818:Animals; D002448:Cell Adhesion / Q000187:drug effects*; D045744:Cell Line, Tumor / Q000378:metabolism* / Q000502:physiology*; D002470:Cell Survival; D002478:Cells, Cultured; D003287:Contrast Media / Q000493:pharmacokinetics / Q000494:pharmacology*; D003911:Dextrans / Q000493:pharmacokinetics / Q000494:pharmacology*; D004705:Endocytosis / Q000187:drug effects*; D004730:Endothelium, Vascular / Q000378:metabolism*; D005260:Female; D052203:Ferrosoferric Oxide / Q000493:pharmacokinetics / Q000494:pharmacology*; D005455:Fluorescent Antibody Technique; D005909:Glioblastoma; D005910:Glioma / Q000098:blood supply / Q000378:metabolism* / Q000503:physiopathology; D006801:Humans; D039302:Integrin alphaVbeta3 / Q000378:metabolism*; D008279:Magnetic Resonance Imaging; D058185:Magnetite Nanoparticles; D051379:Mice; D008819:Mice, Nude; D009368:Neoplasm Transplantation; D009842:Oligopeptides / Q000493:pharmacokinetics / Q000494:pharmacology*; D010051:Ovarian Neoplasms; D014471:Umbilical Veins
D003287:Contrast Media; D003911:Dextrans; D039302:Integrin alphaVbeta3; D058185:Magnetite Nanoparticles; D009842:Oligopeptides; C097921:ferumoxtran-10; C047981:arginyl-glycyl-aspartic acid; D052203:Ferrosoferric Oxide