A Method for Serial Tissue Processing and Parallel Analysis of Aberrant Crypt Morphology, Mucin Depletion, and Beta-Catenin Staining in an Experimental Model of Colon Carcinogenesis

Visualization of aberrant crypt foci (ACF) in methylene blue-stained colon whole mounts was first described by Bird [1] and gave insight into early pathologic changes in colon epithelium associated with carcinogen treatment and the subsequent development of colon cancer. Morphologic criteria used to identify ACF include: darkly stained epithelium, slight elevation of epithelial cells above the surrounding normal mucosa, increased pericryptal zone, enlarged crypt size, and changes in crypt shape [1-4]. While numerous ACF can be found within colons of carcinogen-treated animals, tumor multiplicity is quite low with one or two carcinomas observed per animal [5]. This situation has given rise to a controversy regarding the clinical relevance of ACF [3,6,7] since they have been widely used as a surrogate endpoint for colon carcinogenesis to screen compounds for cancer chemopreventive activity in preclinical models [8]. Loss of mucin occurs in a small percentage of ACF, and these mucin-depleted foci (MDF) have been proposed to identify ACF with an increased probability of progressing to cancer, but this concept also has come under scrutiny [5,9-12]. Herein, we describe methods and evaluate the usefulness of morphometric image analysis to yield insight into this problem.Current approaches used to prepare and evaluate colon whole mounts for identification and characterization of ACF [1,12-15] have a number of issues, which are not conducive to consistent acquisition of digital images and subsequent image analysis. They include: (1) tissues mounted on filter paper or cork boards to flatten during fixation must be removed from the object for transillumination and visualization of the stained mucosa [1,16]; (2) high-quality image capture of free-floating stained specimens can be difficult or unfeasible due to specimen movement in fluid media and natural topographical changes present in the colonic tissue; (3) real-time ACF counting and scoring, using a dissecting microscope, a