FITC-Conjugated Cyclic RGD Peptides as Fluorescent Probes for Staining Integrin α(v)β(3)/α(v)β(5) in Tumor Tissues

[Image: see text] This study sought to evaluate FITC-conjugated cyclic RGD peptides (FITC-RGD(2), FITC-3P-RGD(2), and FITC-Galacto-RGD(2)) as fluorescent probes for in vitro assays of integrin α(v)β(3)/α(v)β(5) expression in tumor tissues. FITC-RGD(2), FITC-3P-RGD(2), and FITC-Galacto-RGD(2) were prepared, and their integrin α(v)β(3)/α(v)β(5) binding affinity was determined using the displacement assay against (125)I-echistatin bound to U87MG glioma cells. IC(50) values of FITC-Galacto-RGD(2), FITC-3P-RGD(2), and FITC-RGD(2) were calculated to be 28 ± 8, 32 ± 7, and 89 ± 17 nM, respectively. The integrin α(v)β(3)/α(v)β(5) binding affinity followed a general trend: FITC-Galacto-RGD(2) ∼ FITC-3P-RGD(2) > FITC-RGD(2). The xenografted tumor-bearing models were established by subcutaneous injection of 5 × 10(6) tumor cells into shoulder flank (U87MG, A549, HT29, and PC-3) or mammary fat pad (MDA-MB-435) of each athymic nude mouse. Three to six weeks after inoculation, the tumor size was 0.1–0.3 g. Tumors were harvested for integrin α(v)β(3)/α(v)β(5) staining, as well as hematoxylin and eosin (H&E) staining. Six human carcinoma tissues (colon cancer, pancreatic cancer, lung adenocarcinoma, squamous cell lung cancer, gastric cancer, and esophageal cancer) were obtained from recently diagnosed cancer patients. Human carcinoma slides were deparaffinized in xylene, rehydrated with ethanol, and then used for integrin α(v)β(3)/α(v)β(5) staining, as well as H&E staining. It was found that the tumor staining procedures with FITC-conjugated cyclic RGD peptides were much simpler than those with the fluorescence-labeled integrin α(v)β(3) antibodies. Since FITC-RGD(2), FITC-3P-RGD(2), and FITC-Galacto-RGD(2) were able to co-localize with the fluorescence-labeled integrin β(3) antibody, their tumor localization and tumor cell binding are integrin α(v)β(3)-specific. Quantification of the fluorescent intensity in five xenografted tumors (U87MG, MDA-MB-435, A549, HT29, and PC-3) and six human carcinoma tissues revealed an excellent linear relationship between the relative integrin α(v)β(3)/α(v)β(5) expression levels determined with FITC-Galacto-RGD(2) and those obtained with the fluorescence-labeled anti-human integrin β(3) antibody. There was also an excellent linear relationship between the tumor uptake (%ID/g) of (99m)Tc-3P-RGD(2) (an integrin α(v)β(3)/α(v)β(5)-targeted radiotracer) and the relative integrin α(v)β(3)/α(v)β(5) expression levels from the quantification of fluorescent intensity in the tumor tissues stained with FITC-Galacto-RGD(2). These results suggest that FITC-conjugated cyclic RGD peptides might be useful to correlate the in vitro findings with the in vivo imaging data from an integrin α(v)β(3)/α(v)β(5)-targeted radiotracer. The results from this study clearly showed that the FITC-conjugated cyclic RGD peptides (particularly FITC-3P-RGD(2) and FITC-Galacto-RGD(2)) are useful fluorescent probes for assaying relative integrin α(v)β(3)/α(v)β(5) expression levels in tumor tissues.