Cathepsin D Expression in Colorectal Cancer: From Proteomic Discovery through Validation Using Western Blotting, Immunohistochemistry, and Tissue Microarrays

Abstract

Despite recent advances in surgical techniques and therapeutic treatments, survival from colorectal cancer (CRC) remains disappointing with some 40-50% of newly diagnosed patients ultimately dying of metastatic disease. Current staging by light microscopy alone is not sufficiently predictive of prognosis and would benefit from additional support from biomarkers in order to stratify patients appropriately for adjuvant therapy. We have identified that cathepsin D expression was significantly greater in cells from invasive front (IF) area and liver metastasis (LM) than those from main tumour body (MTB). Cathepsin D expression was subsequently examined by immunohistochemistry in tissue microarrays from 119 patients with CRC. Strong expression in tumour cells at the IF did not correlate significantly with any clinico-pathological parameters examined or patient survival. However, cathepsin D expression in cells from the MTB was highly elevated in late stage CRC and showed significant correlation with subsequent distant metastasis and shorter cancer-specific survival. We also found that macrophages surrounding tumour cells stained strongly for cathepsin D but there was no significant correlation found between cathepsin D in macrophages at IF and MTB of CRC patient with the clinic-pathological parameters examined.

Figure 1

(a) Spots of cathepsin D from samples of the main tumor body (MTB), IF area (IF), and liver metastasis (LM) on 2 D gels; (b) validation of cathepsin D profiled by 2DE western blotting of cy5 labeled LMD sample (red) and using Cy3 label antibody (green) (ECL-Plex).

Figure 2

Validation of cathepsin D expression at two different regions, main tumor body. (MTB) and invasive front (IF) area of primary tumor and liver metastasis (LM) from the same patient by immunohistochemistry (IHC) (DAB substrate, brown). Sections were counterstained with haematoxylin (blue) (20x objective).

Figure 3

Representative CRC tissue for intensity scoring of tissue microarray immunohistochemistry (DAB substrate, brown). None or weak expression of cathepsin D (a) was scored 1. (b) Moderate expression of cathepsin D in tumor cells was scored 2 and strong expression of cathepsin D (c) was scored 3. Tissue sections were counterstained with haematoxylin (blue) (20x objective).

Figure 4

Cancer-specific survival (in months) of CRC patients in association with cathepsin D expression in epithelial of main tumor.

Figure 5

The average score of cathepsin D expression in tumor cells at the MTB (solid black) and IF (blue) of CRC patients at different stage (TNM).

Figure 6

Adjacent sections of primary colorectal tumor stained for cathepsin D (a) (DAB substrate, brown). CD 68 staining (b) confirmed that very strong expression of cathepsin D in a population of cells within stromal tissue was associated with macrophage (DAB substrate, brown). Sections were counterstained with haematoxylin (blue) (40x objective).