Clusterin as a biomarker in murine and human intestinal neoplasia

Abstract

Early detection of colorectal cancer is critical for the management of this disease. Biomarkers for early detection of several cancers have been developed and applied clinically in recent years. We have sought to discover candidate biomarkers without the restricted choice of markers placed on microarrays, and without the biological complications of genetic and environmental heterogeneity. We have compared by cDNA subtraction two genetically matched sets of mice, one developing multiple intestinal neoplasia (C57BL/6J-ApcMin) and the other tumor-free (C57BL/6J). One prominent candidate biomarker, clusterin, was then subjected to a series of validation steps. In situ hybridization and immunohistochemistry were used to analyze clusterin expression at a cellular level on a series of murine intestinal and human colonic neoplasms. Elevated clusterin expression was characterized within certain regions of murine and human tumors regardless of tumor stage, location, or mode of initiation. The cells showing high clusterin levels generally lacked differentiation markers and adenomatous polyposis coli antigen. Tumor cells undergoing apoptosis expressed low levels of clusterin. Its specific expression patterns and correlation with cellular events during tumorigenesis make it a useful diagnostic tool in the mouse and a potential contributor to the set of biomarkers for early detection of human colon cancer.

Fig. 1.

Expression of clusterin in murine intestinal tumors. (A, E, G, I, K, M, and O) The staining (brown) of clusterin RNA with antisense probe by in situ hybridization. (C) The staining of clusterin protein with clusterin antibody by immunohistochemistry. (B, F, H, J, L, N, and P) Negative controls with sense probe. (D) Negative control lacking the primary antibody. Highly elevated clusterin RNA signal (A and B) and increased production of clusterin antigen (C and D) were detected in the tumors of small intestines. Elevated clusterin RNA signal was shown in colon tumors (E and F). Noticeable elevation of the clusterin signal is indicated by arrows (G) in the microadenomas from 29-day-old Min mice (G and H). Gradual elevation of clusterin RNA was observed in early adenomas from 29-day-old Min mice (I and J). Invasive adenocarcinomas in 270-day-old mouse (SWR × B6-Min)F₁ demonstrated highly elevated levels of clusterin (K and L). The arrows in K point to invasive tumor tissue. Up-regulation of clusterin RNA was also observed in N-ethyl-N-nitrosourea-induced adenomas (M and N) and in tumors arising from allelic silencing of Apc⁺ (O and P) in the small intestine. (Bars: 200 μm.)

Fig. 2.

Analysis of clusterin expression patterns. (A) Dual-staining immunofluorescence assay on a Min tumor section demonstrated that the WT Apc antigen (green) was present in the normal crypts, but not in tumor cells, where a high level of β-catenin antigen (red) was detected. (B) Staining of clusterin RNA (dark brown) with antisense probe in an adjacent section is similar to that of cytoplasmic β-catenin protein. The arrows in A and B point to a small exceptional region lacking both WT Apc antigen and clusterin antigen. (C–F) Expression of differentiation markers and clusterin RNA in intestinal tumors. (A, C, E, G, and I) RNA signals (dark brown) of two differentiation markers, intestinal alkaline phosphatase (C) and small proline-rich protein 2a (E), with their antisense probes. (B, D, F, H, and J) Sections adjacent to C and E were hybridized for clusterin RNA (dark brown) with the antisense probe for comparison (D and F). Solid arrows in C–F point to small exceptional regions expressing one of the differentiation markers and clusterin, whereas open arrows point to small regions lacking both of them. (G) Cells undergoing apoptosis within tumors were labeled red by fluorescent TUNEL assay, in which the nuclei were stained blue by 4′,6-diamidino-2-phenylindole (DAPI). (H) For comparison, a section adjacent to G was hybridized for clusterin RNA (dark brown) with the antisense probe. Circles indicate the regions showing a high apoptotic index, where clusterin RNA levels were relatively low. (I) A section of tumor stained for Ki67 antigen by immunofluorescence (green signal). (J) A section adjacent to I hybridized for clusterin RNA (dark brown) with the antisense probe. (Bars: 200 μm.)

Fig. 3.

Localization of clusterin protein in various human colorectal tissues. Dual-staining immunofluorescence assays (A, C, E, G, H, J, K, and L) stained clusterin antigen (green-yellow), β-catenin (red), and nuclei (blue) in the human colorectal tissues. Hematoxylin/eosin stains of hyperplastic polyp (B), tubular adenoma (D), villous adenoma (F), and invasive adenocarcinoma (I) have arrows to indicate the positions of corresponding immunostaining panels. Normal colonic crypts (A) have minimal clusterin antigen and no β-catenin in the epithelial cytoplasm. Hyperplastic polyps (C) have noticeable elevation of clusterin protein, but not β-catenin, in the epithelial cytoplasm. Tubular adenomas (E) and villous adenomas (G) have highly elevated clusterin antigen in the apical side of epithelial cytoplasm, along with increased cytoplasmicβ-catenin. Normal crypts adjacent to adenomas (H) show strong clusterin signals in the apical epithelial cytoplasm. Invasive adenocarcinomas (J) with highly elevated cytoplasmic β-catenin have detectable clusterin antigen in the cytoplasm and strong clusterin signals in the intercellular cavities of the tumor. Normal crypts close to the adenocarcinoma (within 0.5 cm from the edge) (K) show fairly strong clusterin signals in the apical cytoplasm. However, normal crypts >1 cm from the edge of the adenocarcinoma (L) have only minimal clusterin antigen in the cytoplasm. (Bars: 50 μmin A, C, E, G, H, and J–L;1mm in B, D, F, and I.)