U87MG Tumor-bearing Research Articles

Tumor Uptake of the RGD Dimeric Probe 99mTc-G3-2P4-RGD2 is Correlated with Integrin αvβ3 Expressed on both Tumor Cells and Neovasculature

Integrin αvβ3 has been well-documented as one of the key players in the process of tumor angiogenesis. Radiolabeled RGD (Arg-Gly-Asp) peptides that specifically target integrin αvβ3 have great potential for tumor early detection and noninvasively monitoring the status of tumor angiogenesis. We developed a cyclic RGD dimeric probe (99m)Tc-HYNIC-Gly3-E[PEG4-c(RGDfK)]2 ((99m)Tc-G3-2P4-RGD2) (using tricine and TPPTS as the coligands, TPPTS = trisodium triphenylphosphine-3,3',3''-trisulfonate), and investigated whether it could be used to noninvasively visualize and quantify integrin αvβ3 expression in vivo. HYNIC-Gly3-E[PEG4-c(RGDfK)]2 was synthesized and labeled with (99m)Tc. The biodistribution and planar γ-imaging studies of (99m)Tc-G3-2P4-RGD2 were performed in both U87MG (human integrin αvβ3 positive/murine integrin αvβ3 positive) and HT-29 (human integrin αvβ3 negligible /murine integrin αvβ3 positive) tumor-bearing nude mouse models. The correlation of (99m)Tc-G3-2P4-RGD2 tumor uptake values (measured by ex vivo biodistribution) with expression levels of human integrin αvβ3 or murine integrin αvβ3 (measured by Western blot) were determined in U87MG and HT-29 tumor models, respectively. (99m)Tc-G3-2P4-RGD2 exhibited increased receptor binding affinity and in vivo tumor uptake as compared with previously reported RGD dimeric tracer (99m)Tc-RGD2 (without Gly3 and PEG4 spacers). The tumor uptake of (99m)Tc-G3-2P4-RGD2 was related to the expression levels of both human integrin αvβ3 (expressed on tumor cells) and murine integrin αvβ3 (expressed on newborn tumor vasculature). Our results demonstrate that (99m)Tc-G3-2P4-RGD2 is a useful agent for integrin αvβ3 imaging. The relationship between (99m)Tc-G3-2P4-RGD2 uptake and integrin αvβ3 expression level as determined by this study would provide useful information for clinical translation of RGD probes.

Synthesis of Specific SPECT-Radiopharmaceutical for Tumor Imaging Based on Methionine: 99mTc-DTPA-bis(methionine)

Methionine-diethylenetriaminepentaaceticacid-methionine [DTPA-bis(Met)] was synthesized by covalently conjugating two molecules of methionine (Met) to DTPA and was labeled with (99m)Tc in high radiochemical purity and specific activity (166-296 MBq/micromol). Kinetic analysis showed K(m) of 12.95 +/- 3.8 nM and a maximal transport rate velocity (V(max)) of 80.35 +/- 0.42 pmol microg protein(-1) min(-1) of (99m)Tc-DTPA-bis(Met) in U-87MG cells. DTPA-bis(Met) had dissociation constants (K(d)) of 0.067 and 0.077 nM in U-87MG and BMG, respectively. (35)S-methionine efflux was trans-stimulated by (99m)Tc-labeled DTPA conjugate demonstrating concentrative transport. The blood kinetic studies showed fast clearance with t(1/2) (F) = 36 +/- 0.5 min and t(1/2) (S) = 5 h 55 min +/- 0.85 min. U-87MG and BMG tumors saturated at approximately 2000 +/- 280 nmol/kg of (99m)Tc-DTPA-bis(Met). Initial rate of transport of (99m)Tc-DTPA-bis(Met) in U-87MG tumor was found to be 4.68 x 10(-4) micromol/kg/min. The tumor (BMG cell line, malignant glioma) grafted in athymic mice were readily identifiable in the gamma images. Semiquantitative analysis from region of interest (ROI) placed over areas counting average counts per pixel with maximum radiotracer uptake on the tumor was found to be 11.05 +/- 3.99 and compared ROI with muscle (0.55 +/- 0.13). The tumor-to-contralateral muscle tissue ratio of (99m)Tc-DTPA-bis(Met) was found to be 23 +/- 3.3. Biodistribution revealed significant tumor uptake and good contrast in the U-87MG, BMG, and EAT tumor-bearing mice. In clinical trials, the sensitivity, specificity, and positive predictive values were found to be 87.8%, 92.8%, and 96.6%, respectively. (99m)Tc-DTPA-bis(Met) showed excellent tumor targeting and has promising utility as a SPECT-radiopharmaceutical for imaging methionine-dependent human tumors and to quantify the ratio of MET(+)/HCY(-).

An Engineered Knottin Peptide Labeled with 18F for PET Imaging of Integrin Expression

Knottins are small constrained polypeptides that share a common disulfide-bonded framework and a triple-stranded beta-sheet fold. Previously, directed evolution of the Ecballium elaterium trypsin inhibitor (EETI-II) knottin led to the identification of a mutant that bound to tumor-specific alpha(v)beta(3) and alpha(v)beta(5) integrin receptors with low nanomolar affinity. The objective of this study was to prepare and evaluate a radiofluorinated version of this knottin (termed 2.5D) for microPET imaging of integrin positive tumors in living subjects. Knottin peptide 2.5D was prepared by solid-phase synthesis and folded in vitro, and its free N-terminal amine was reacted with N-succinimidyl-4-18/19F-fluorobenzoate (18/19F-SFB) to produce the fluorinated peptide 18/19F-FB-2.5D. The binding affinities of unlabeled knottin peptide 2.5D and 19F-FB-2.5D to U87MG glioblastoma cells were measured by competition binding assay using 125I-labeled echistatin. It was found that unlabeled 2.5D and 19F-FB-2.5D competed with 125I-echistatin for binding to cell surface integrins with IC(50) values of 20.3 +/- 7.3 and 13.2 +/- 5.4 nM, respectively. Radiosynthesis of 18F-FB-2.5D resulted in a product with high specific activity (ca. 100 GBq/micromol). Next, biodistribution and positron emission tomography (PET) imaging studies were performed to evaluate the in vivo behavior of 18F-FB-2.5D. Approximately 3.7 MBq 18F-FB-2.5D was injected into U87MG tumor-bearing mice via the tail vein. Biodistribution studies demonstrated that 18F-FB-2.5D had moderate tumor uptake at 0.5 h post injection, and coinjection of a large excess of the unlabeled peptidomimetic c(RGDyK) as a blocking agent significantly reduced tumor uptake (1.90 +/- 1.15 vs 0.57 +/- 0.14%ID/g, 70% inhibition, P < 0.05). In vivo microPET imaging showed that 18F-FB-2.5D rapidly accumulated in the tumor and quickly cleared from the blood through the kidneys, allowing excellent tumor-to-normal tissue contrast to be obtained. Collectively, 18F-FB-2.5D allows integrin-specific PET imaging of U87MG tumors with good contrast and further demonstrates that knottins are excellent peptide scaffolds for development of PET probes with potential for clinical translation.

Evaluation of 64Cu-labeled DOTA-d-Phe1-Tyr3-octreotide (64Cu-DOTA-TOC) for imaging somatostatin receptor-expressing tumors

In-111 ((111)In)-labeled octreotide has been clinically used for imaging somatostatin receptor-positive tumors, and radiolabeled octreotide analogs for positron emission tomography (PET) have been developed. Cu-64 ((64)Cu; half-life, 12.7 h) is an attractive radionuclide for PET imaging and is produced with high specific activity using a small biomedical cyclotron. The aim of this study is to produce and fundamentally examine a (64)Cu-labeled octreotide analog, (64)Cu-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-D: -Phe(1)-Tyr(3)-octreotide ((64)Cu-DOTA-TOC). (64)Cu produced using a biomedical cyclotron was reacted with DOTA-TOC for 30 min at 45 degrees C. The stability of (64)Cu-DOTA-TOC was evaluated in vitro (incubated with serum) and in vivo (blood collected after administration) by HPLC analysis. Biodistribution studies were performed in normal mice by administration of mixed solution of (64)Cu-DOTA-TOC and (111)In-DOTA-TOC and somatostatin receptor-positive U87MG tumor-bearing mice by administration of (64)Cu-DOTA-TOC or (64)Cu-1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid-octreotide ((64)Cu-TETA-OC). The tumor was imaged using (64)Cu-DOTA-TOC, (64)Cu-TETA-OC, and FDG with an animal PET scanner. (64)Cu-DOTA-TOC can be produced in amounts sufficient for clinical study with high radiochemical yield. (64)Cu-DOTA-TOC was stable in vitro, but time-dependent transchelation to protein was observed after injection into mice. In biodistribution studies, the radioactivity of (64)Cu was higher than that of (111)In in all organs except kidney. In tumor-bearing mice, (64)Cu-DOTA-TOC showed a high accumulation in the tumor, and the tumor-to-blood ratio reached as high as 8.81 +/- 1.17 at 6 h after administration. (64)Cu-DOTA-TOC showed significantly higher accumulation in the tumor than (64)Cu-TETA-OC. (64)Cu-DOTA-TOC PET showed a very clear image of the tumor, which was comparable to that of (18)F-FDG PET and very similar to that of (64)Cu-TETA-OC. (64)Cu-DOTA-TOC clearly imaged a somatostatin receptor-positive tumor and seemed to be a potential PET tracer in the clinical phase.

Differential effect of sunitinib on the distribution of temozolomide in an orthotopic glioma model

Normalization of tumor vasculature by antiangiogenic agents may improve the delivery of cytotoxic drugs to the tumor, leading to more effective therapy. In this study, we used pharmacokinetic and pharmacodynamic approaches to investigate how sunitinib at different dose levels affects brain distribution of temozolomide (TMZ), and to ascertain the relationship between intratumoral TMZ concentrations and tumor vascularity in an orthotopic human glioma model. Three groups of intracerebral U87MG tumor-bearing mice were given either vehicle or sunitinib at 20 mg/kg or 60 mg/kg per day for 7 days before receiving a steady-state regimen of TMZ that consisted of an intravenous bolus and a 3-h intraarterial infusion. TMZ concentrations in plasma, normal brain, and brain tumor were determined, and several biomarkers related to the antiangiogenic activity of sunitinib were examined. TMZ distribution in the normal brain as indicated by the brain-to-plasma steady-state TMZ concentration ratios was analogous across the three treatment groups. The brain tumor-to-plasma steady-state TMZ concentration (ss C(t)/C(p)) ratio was significantly increased in the 20 mg/kg sunitinib group (0.98 +/- 0.17) compared with the control (0.76 +/- 0.17) and 60 mg/kg sunitinib (0.68 +/- 0.09) groups. The ss C(t)/C(p) ratios were significantly correlated with the vascular normalization index (VNI), derived from the expression of CD31, collagen IV, and alpha-smooth muscle actin, which represents the fraction of functioning vessels out of the total tumor vessels. In conclusion, the effect of sunitinib on the brain tumor distribution of TMZ was dose dependent and indicated that optimal tumor exposure was achieved at a lower dose and was associated with the VNI.

Dual-Function Probe for PET and Near-Infrared Fluorescence Imaging of Tumor Vasculature

To date, the in vivo imaging of quantum dots (QDs) has been mostly qualitative or semiquantitative. The development of a dual-function PET/near-infrared fluorescence (NIRF) probe can allow for accurate assessment of the pharmacokinetics and tumor-targeting efficacy of QDs. A QD with an amine-functionalized surface was modified with RGD peptides and 1,4,7,10-tetraazacyclodocecane-N,N',N'',N'''-tetraacetic acid (DOTA) chelators for integrin alpha(v)beta(3)-targeted PET/NIRF imaging. A cell-binding assay and fluorescence cell staining were performed with U87MG human glioblastoma cells (integrin alpha(v)beta(3)-positive). PET/NIRF imaging, tissue homogenate fluorescence measurement, and immunofluorescence staining were performed with U87MG tumor-bearing mice to quantify the probe uptake in the tumor and major organs. There are about 90 RGD peptides per QD particle, and DOTA-QD-RGD exhibited integrin alpha(v)beta(3)-specific binding in cell cultures. The U87MG tumor uptake of (64)Cu-labeled DOTA-QD was less than 1 percentage injected dose per gram (%ID/g), significantly lower than that of (64)Cu-labeled DOTA-QD-RGD (2.2 +/- 0.3 [mean +/- SD] and 4.0 +/- 1.0 %ID/g at 5 and 18 h after injection, respectively; n = 3). Taking into account all measurements, the liver-, spleen-, and kidney-to-muscle ratios for (64)Cu-labeled DOTA-QD-RGD were about 100:1, 40:1, and 1:1, respectively. On the basis of the PET results, the U87MG tumor-to-muscle ratios for DOTA-QD-RGD and DOTA-QD were about 4:1 and 1:1, respectively. Excellent linear correlation was obtained between the results measured by in vivo PET imaging and those measured by ex vivo NIRF imaging and tissue homogenate fluorescence (r(2) = 0.93). Histologic examination revealed that DOTA-QD-RGD targets primarily the tumor vasculature through an RGD-integrin alpha(v)beta(3) interaction, with little extravasation. We quantitatively evaluated the tumor-targeting efficacy of a dual-function QD-based probe with PET and NIRF imaging. This dual-function probe has significantly reduced potential toxicity and overcomes the tissue penetration limitation of optical imaging, allowing for quantitative targeted imaging in deep tissue.