Zheng Yumin, Ji Shundong, Czerwinski Andrzej, Valenzuela Francisco, Pennington Michael, Liu Shuang
Department of Nuclear Medicine, China-Japan Friendship Hospital , Beijing, 100029, China.
Bioconjug Chem. 2014 Nov 19;25(11):1925-41. doi: 10.1021/bc500452y. Epub 2014 Oct 22.
This study sought to evaluate FITC-conjugated cyclic RGD peptides (FITC-RGD2, FITC-3P-RGD2, and FITC-Galacto-RGD2) as fluorescent probes for in vitro assays of integrin αvβ3/αvβ5 expression in tumor tissues. FITC-RGD2, FITC-3P-RGD2, and FITC-Galacto-RGD2 were prepared, and their integrin αvβ3/αvβ5 binding affinity was determined using the displacement assay against (125)I-echistatin bound to U87MG glioma cells. IC50 values of FITC-Galacto-RGD2, FITC-3P-RGD2, and FITC-RGD2 were calculated to be 28 ± 8, 32 ± 7, and 89 ± 17 nM, respectively. The integrin αvβ3/αvβ5 binding affinity followed a general trend: FITC-Galacto-RGD2 ∼ FITC-3P-RGD2 > FITC-RGD2. 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 αvβ3/αvβ5 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 αvβ3/αvβ5 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 αvβ3 antibodies. Since FITC-RGD2, FITC-3P-RGD2, and FITC-Galacto-RGD2 were able to co-localize with the fluorescence-labeled integrin β3 antibody, their tumor localization and tumor cell binding are integrin αvβ3-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 αvβ3/αvβ5 expression levels determined with FITC-Galacto-RGD2 and those obtained with the fluorescence-labeled anti-human integrin β3 antibody. There was also an excellent linear relationship between the tumor uptake (%ID/g) of (99m)Tc-3P-RGD2 (an integrin αvβ3/αvβ5-targeted radiotracer) and the relative integrin αvβ3/αvβ5 expression levels from the quantification of fluorescent intensity in the tumor tissues stained with FITC-Galacto-RGD2. 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 αvβ3/αvβ5-targeted radiotracer. The results from this study clearly showed that the FITC-conjugated cyclic RGD peptides (particularly FITC-3P-RGD2 and FITC-Galacto-RGD2) are useful fluorescent probes for assaying relative integrin αvβ3/αvβ5 expression levels in tumor tissues.
本研究旨在评估异硫氰酸荧光素（FITC）偶联的环状RGD肽（FITC-RGD2、FITC-3P-RGD2和FITC-Galacto-RGD2）作为荧光探针，用于体外检测肿瘤组织中整合素αvβ3/αvβ5的表达。制备了FITC-RGD2、FITC-3P-RGD2和FITC-Galacto-RGD2，并使用针对与U87MG胶质瘤细胞结合的（125）I-echistatin的置换试验测定了它们与整合素αvβ3/αvβ5的结合亲和力。FITC-Galacto-RGD2、FITC-3P-RGD2和FITC-RGD2的半数抑制浓度（IC50）值分别计算为28±8、32±7和89±17 nM。整合素αvβ3/αvβ5的结合亲和力呈现一般趋势：FITC-Galacto-RGD2 ∼ FITC-3P-RGD2 > FITC-RGD2。通过将5×10（6）个肿瘤细胞皮下注射到每只无胸腺裸鼠的肩部侧翼（U87MG、A549、HT29和PC-3）或乳腺脂肪垫（MDA-MB-435）中，建立了异种移植荷瘤模型。接种后三至六周，肿瘤大小为0.1 - 0.3 g。收获肿瘤用于整合素αvβ3/αvβ5染色以及苏木精和伊红（H&E）染色。从最近诊断的癌症患者中获取了六种人类癌组织（结肠癌、胰腺癌、肺腺癌、肺鳞状细胞癌、胃癌和食管癌）。将人类癌组织切片在二甲苯中脱蜡，用乙醇复水，然后用于整合素αvβ3/αvβ5染色以及H&E染色。发现用FITC偶联的环状RGD肽进行肿瘤染色的程序比用荧光标记的整合素αvβ3抗体的程序简单得多。由于FITC-RGD2、FITC-3P-RGD2和FITC-Galacto-RGD2能够与荧光标记的整合素β3抗体共定位，它们在肿瘤中的定位和与肿瘤细胞的结合是整合素αvβ3特异性的。对五个异种移植肿瘤（U87MG、MDA-MB-435、A549、HT29和PC-3）和六种人类癌组织中的荧光强度进行定量分析，结果显示用FITC-Galacto-RGD2测定的相对整合素αvβ3/αvβ5表达水平与用荧光标记的抗人整合素β3抗体获得的水平之间存在良好的线性关系。（99m）Tc-3P-RGD2（一种整合素αvβ3/αvβ5靶向放射性示踪剂）在肿瘤中的摄取（%ID/g）与用FITC-Galacto-RGD2染色的肿瘤组织中荧光强度定量分析得到的相对整合素αvβ3/αvβ5表达水平之间也存在良好的线性关系。这些结果表明，FITC偶联的环状RGD肽可能有助于将体外研究结果与整合素αvβ3/αvβ5靶向放射性示踪剂的体内成像数据相关联。本研究结果清楚地表明，FITC偶联的环状RGD肽（特别是FITC-3P-RGD2和FITC-Galacto-RGD2）是用于检测肿瘤组织中相对整合素αvβ3/αvβ5表达水平的有用荧光探针。
