CD133 expression is an independent prognostic marker for low survival in colorectal cancer

Abstract

Colon cancer cells have previously been demonstrated to contain a subpopulation of CD133+ tumour cells that have the ability to initiate tumour growth and are thus referred to as colon cancer-initiating cells or colon cancer stem cells (CSCs). As CD133 is currently one of the best markers to characterise colon CSCs, we analysed CD133+ tumour cells in colorectal cancer specimens using immunohistochemistry. We show that CD133 detection is specific and that the CD133 antigen is localised on the glandular-luminal surface of colon cancer cells, whereas undifferentiated tumour cells at the front of invasion are CD133-. In addition, CD133+ cells are characterised in situ by lack of CK20 expression, whereas they are positive for EpCAM. Moreover, we show that CD133 expression in colorectal cancer is an independent prognostic marker that correlates with low survival in a stratified patient collective. Our results indicate that in colorectal cancer, the CD133+ tumour cells can be detected by immunohistochemistry, which facilitates their further characterisation in situ.

Figure 1

CD133 expression is found on the glandular-luminal surface of epithelial tumour cells. Three different antibodies were compared on serial tumour sections: (A) monoclonal anti-CD133 (Cell Signalling), (B) monoclonal anti-CD133 (Miltenyi Biotech), and (C) polyclonal anti-CD133 (Santa Cruz Biotechnology). All three antibodies showed comparable staining patterns. Arrowheads indicate positive (black) and negative (white) staining; original magnification, × 200. (D) High magnification of positively stained tumour cells; original magnification, × 630.

Figure 2

Specificity of CD133 detection. Protein lysates of the CD133− DLD-1 (D1) and the CD133+ Caco-2 (C2) colon cancer cell lines were blotted and stained with the three anti-CD133 antibodies used for immunohistochemistry. All three antibodies strongly detected the CD133 antigen in Caco-2, displayed at 120 kDa, whereas DLD-1 showed no staining. Unspecific bands that were observed were considered to be negligibly weak. β-Actin was used as control. Upper panel images show full-length Western blots merged with their molecular weight markers (M).

Figure 3

(A) CD133 staining of shed intraglandular cellular debris mirrors CD133 expression of the surrounding tumour cells. Arrows indicate a CD133− tumour gland filled with CD133− debris. Arrowheads indicate a CD133+ gland with CD133+ cellular debris and apical staining of the tumour cells. Tumour glands were considered as CD133+ if either apical CD133 staining of the tumour cells or CD133+ cellular debris or both were present. (B) CD133 only marks tumour cells that are gland forming. Undifferentiated tumour ‘buds’ at the front of tumour invasion are CD133− (arrowheads). Original magnification, × 200 (A), × 400 (B).

Figure 4

CD133 and CK20 expressions are mutually exclusive. Serial sections stained for CD133 (A), CK20 (B), and EpCAM (C) demonstrate that CD133+ cells are negative for CK20, whereas CD133+ and CD133− cells express the EpCAM antigen. Original magnification, × 200.

Figure 5

Scoring of CD133 expression in colorectal cancer. Tumours that showed no staining (A) and tumours with less than 50% positively stained glands (B) were considered as CD133-low; tumours with more than 50% positively stained glands as CD133-high (C). Original magnification, × 200.

Figure 6

CD133 expression correlates with low survival. The Kaplan–Meier plot of colorectal cancer specimens (n=77) demonstrates significant (log-rank test) lower survival with CD133-high expression.