| Objective: Chemokine receptor CXCR4and integrin αVβ3play important roles intumor progression, metastasis and angiogenesis. Imaging CXCR4and αVβ3couldprovide more insight in the roles of them in tumor. Aim of this study was to investigate99mTc-AMD-RGD as a potential double-target SPECT tracer of CXCR4and αVβ3.Methods：1. The expression intensity of CXCR4and αVβ3of A549, H460,95Dand GLC-80lung cancer cells were detected by means of confocal immunofluorescence,real time-PCR and Western blot respectively in vitro.2. Binding assay of99mTc-AMD-RGD was performed in these four types lung cancer cells at different timeintervals, and correlation between binding rate and CXCR4intensity, αVβ3intensitywere analyzed respectively.3. Blocking test was performed in A549lung cancer cellswith antagonists AMD3100and RGD2together or respectively; affinity assay of99mTc-AMD-RGD binding with CXCR4was performed in Jurkat-T cells withantagonists AMD3100, and was compared with99mTc-AMD.4. Ex vivo bio-distributionof99mTc-AMD-RGD was studied in healthy NH mice at30,60,90and120min afterinjection. Tumor uptake of tracer was measured by percent of injected dose (%ID/g) innude mice xenografted with human A549, H460,95D and GLC-80lung cancerrespectively post SPECT imaging. Correlation between tumor uptake and CXCR4intensity, β3intensity derived from human, β3intensity derived from mice wasanalyzed respectively.Results：1. By three different detection methods, CXCR4and αVβ3expressionintensity were different significantly in cultured A549, H460,95D and GLC-80lungcancer cells, they ranged from high to low as follows: A549, H460,95D and GLC-80.2.99mTc-AMD-RGD binding with four types of lung cancer cells for30min,1h,2h,4h and 6h were achieved, the radioactive binding rates reached the top at2h point, and thebio-binding rates reached the top at4h point despite of cell types; the most radioactivityof each type cells ranged from high to low as follows: A549, H460,95D and GLC-80,they were significantly different with each other (all P values were smaller than0.05);bio-binding rates of different lung cancer cells were correlated with both CXCR4andαVβ3intensity (Western blot) positively （r=0.874，P<0.01and r=0.853，P<0.01）.3.99mTc-AMD-RGD(0.068nmol/ml) binding with A549cells were inhibited by AMD3100at high concentration（6.8nmmol/ml） by32.7%, however, RGD2showed no signs ofinterference at any concentration. Affinity of99mTc-AMD-RGD for CXCR4wasslightly higher than that of99mTc-AMD (IC50values110.6±2.1umol&90.8±1.6umol).4.99mTc-AMD-RGD showed a fast blood clearance in healthy NH mice, its uptake mainlyin kidney, lung and liver had a moderate degree of uptake, heart, spleen and muscleuptake were relatively low. SPECT imaging was performed at1hour after injection of99mTc-AMD-RGD in nude mice xenografted with four types human lung cancerrespectively, the results were: H460tumor regions had obvious radioactiveaccumulation, A549tumor regions’ accumulation were slightly lower, and the sites oftumor95D and GLC-80found had no radioactivity accumulation. Various types oftumor tissue uptake and their expression intensity of CXCR4showed a significantpositive linear correlation （r=0.84，P<0.01）, however, there showed no obviouscorrelation between tumors uptake and expression intensity of β3either derived fromhuman or mice （r=0.21，P=0.794and r=-0.76，P=0.12）.Conclusions：Although99mTc-AMD-RGD was designed for double-target imagingof CXCR4and αVβ3, it binds with lung cancer cells by CXCR4specifically; αVβ3playsa negligible role than no in the binding progress.99mTc-AMD-RGD affinity for CXCR4is slightly higher than that of99mTc-AMD3100. As a SPECT tracer,99mTc-AMD-RGDhas some favorable characteristics, such as fast blood clearance, renal excretion, andsignificant uptake by tumor with high CXCR4intensity. The CXCR4molecularimaging by99mTc-AMD-RGD SPECT has high specificity and high sensitivity. |
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