In Vivo Fluorescence-Based Endoscopic Detection of Colon Dysplasia in the Mouse Using a Novel Peptide Probe

Colorectal cancer (CRC) is a major cause of cancer-related deaths in much of the world. Most CRCs arise from pre-malignant (dysplastic) lesions, such as adenomatous polyps, and current endoscopic screening approaches with white light do not detect all dysplastic lesions. Thus, new strategies to identify such lesions, including non-polypoid lesions, are needed. We aim to identify and validate novel peptides that specifically target dysplastic colonic epithelium in vivo. We used phage display to identify a novel peptide that binds to dysplastic colonic mucosa in vivo in a genetically engineered mouse model of colo-rectal tumorigenesis, based on somatic Apc (adenomatous polyposis coli) gene inactivation. Binding was confirmed using confocal microscopy on biopsied adenomas and excised adenomas incubated with peptide ex vivo. Studies of mice where a mutant Kras allele was somatically activated in the colon to generate hyperplastic epithelium were also performed for comparison. Several rounds of in vivo T7 library biopanning isolated a peptide, QPIHPNNM. The fluorescent-labeled peptide bound to dysplastic lesions on endoscopic analysis. Quantitative assessment revealed the fluorescent-labeled peptide (target/background: 2.17±0.61) binds ∼2-fold greater to the colonic adenomas when compared to the control peptide (target/background: 1.14±0.15), pKras mice. This work is first to image fluorescence-labeled peptide binding in vivo that is specific towards colonic dysplasia on wide-area surveillance. This finding highlights an innovative strategy for targeted detection to localize pre-malignant lesions that can be generalized to the epithelium of hollow organs.

结直肠癌（Colorectal Cancer, CRC）是全球多数地区癌症相关死亡的主要诱因。绝大多数结直肠癌起源于癌前（发育异常）病变，例如腺瘤性息肉；当前采用白光照明的内镜筛查手段无法检出所有发育异常病变。因此，亟需开发包括非息肉样病变在内的新型病变识别策略。本研究旨在鉴定并验证可在体内特异性靶向结肠发育异常上皮的新型肽类分子。
我们基于体细胞腺瘤性息肉病 coli（Adenomatous Polyposis Coli, APC）基因失活的结直肠肿瘤发生基因工程小鼠模型，采用噬菌体展示（phage display）技术，成功鉴定出一种可在体内结合结肠发育异常黏膜的新型肽段。通过共聚焦显微镜对体外与该肽段共孵育的活检腺瘤及切除腺瘤进行检测，验证了其结合活性。为开展对照研究，我们还针对结肠内体细胞激活突变型Kras等位基因以生成增生性上皮的小鼠进行了相关实验。
经过多轮体内T7噬菌体展示文库生物淘选（in vivo T7 library biopanning），我们分离得到肽段QPIHPNNM。内镜分析结果显示，该荧光标记肽段可特异性结合发育异常病变。定量评估表明，与对照肽段（靶标/背景比值：1.14±0.15）相比，荧光标记肽段（靶标/背景比值：2.17±0.61）与结肠腺瘤的结合水平约高出2倍，在Kras突变小鼠中亦观察到该现象。
本研究首次在广角内镜监测中实现了对特异性结合结肠发育异常病变的荧光标记肽段的体内成像。该发现为靶向定位癌前病变的检测策略提供了创新思路，该策略可推广至中空器官上皮的相关检测工作中。