Detection of colorectal adenomas using a bioactivatable probe specific for matrix metalloproteinase activity

Abstract

A significant proportion of colorectal adenomas, in particular those that lack an elevated growth component, continue to escape detection during endoscopic surveillance. Elevation of the activity of matrix metalloproteinases (MMPs), a large family of zinc endopeptidases, in adenomas serves as a biomarker of early tumorigenesis. The goal of this study was to assess the feasibility of using a newly developed near-infrared bioactivatable probe (MMPSense 680) that reports the activity of a broad array of MMP isoforms to detect early colorectal adenomas. Adenomatous polyposis coli (Apc)(+/Min-FCCC) mice that spontaneously develop multiple colorectal adenomas were injected with MMPSense 680, and the colons were imaged in an IVIS Spectrum system ex vivo. Image analyses were correlated with histopathologic findings for all regions of interest (ROIs). The biochemical basis of fluorescent signal was investigated by immunohistochemical staining of MMP-7 and -9. A strong correlation (Kendall = 0.80) was observed between a positive signal and the presence of pathologically confirmed colonic adenomas; 92.9% of the 350 ROIs evaluated were classified correctly. The correlation between two independent observers was 0.87. MMP-7 expression was localized to epithelial cells of adenomas and microadenomas, whereas staining of MMP-9 was found in infiltrating polymorphonuclear leukocytes within the adenomas. MMPSense 680 identifies colorectal adenomas, both polypoid and nonpolypoid, in Apc(+/Min-FCCC) mice with high specificity. Use of this fluorescent probe in combination with colonoscopy could aid in preventing colorectal neoplasias by providing new opportunities for early detection and therapeutic intervention.

Figure 1

Positioning of the excised colon on a ruler for image analysis. Each colon was divided into ROIs (boxes) and numbered sequentially to generate discrete areas for comparison of fluorescent signal and histopathology.

Figure 2

Representative photographs of colorectal lesions that develop in Apc^+/Min-FCCC mice. (A) Low-power view (x40) of a polypoid adenoma that projects above the surface of the nonneoplastic colonic mucosa compared with a nonpolypoid lesion (B). (B) Low-power view (x40) of a nonpolypoid adenoma (blue arrows) with a height less than twice that of the adjacent normal mucosa (yellow arrows). (C) Low-power view of a two-gland microadenoma (x100) with the boxed area displayed at a higher power (x400) in the insert. Scale bars, 100 µm.

Figure 3

Images of a representative colon excised from an Apc^+/Min-FCCC mouse. Pictures represent a white-light photograph (left panel) and fluorescent image (right panel) of the same colon. Rectangles represent separate ROIs that were positioned on the acquired image using functions written with the MATLAB Image Processing Toolbox. The fluorescence intensity is represented as a pseudocolor image (MATLAB “hot” color map) overlaid on a white-light photographic image of the colon. A ruler is visible, showing size increments in millimeters. Values of absolute fluorescence efficiency, as measured by the IVIS Spectrum, are shown on the color bar in units of 10^-5. Arrows denote pathologically confirmed adenomas.

Figure 4

Immunohistochemical staining of MMP-7 and -9 in colorectal lesions of Apc^+/Min-FCCC mice. High-power views (x400) of a large adenoma (A) and a small adenoma (B) displaying strong expression of MMP-7 in the epithelial cells (arrows) compared with nonneoplastic mucosa. (C) High-power view (x400) of an adenoma stained with anti-MMP-9 antibodies. MMP-9 is strongly expressed in the PMNLs in the stroma/lamina propria of the adenoma but not in the epithelial cells. (D) Expression of MMP-9 in the PMNLs of an ulcer bed that is nonneoplastic. Scale bars, 100 µm.