Immunohistochemistry was performed for protein markers CD133, CD44s, CD166, EpCAM, and ALDH1. Detailed procedures have been described elsewhere (Zlobec et al, 2007a). The following primary antibodies were used: anti-human CD133 (clone first C24B9; 1:100; Cell Signaling, Allschwil, Swizerland), anti-human CD166 (clone M0G/07; 1:200; Novocastra, Newcastle, UK), anti-human CD44s (clone DF1485; 1:50; Dako, Glostrup, Denmark), anti-human EpCAM (clone VU-1D9; 1:200; Novocastra), and anti-human ALDH1 isoform ��1(polyclonal; 1:500; AbCam, Cambridge, UK). Negative controls underwent the same protocol with the primary antibody omitted. Evaluation of immunohistochemistry For CD133, CD166, CD44s, and EpCAM, only membranous staining was considered, whereas for ALDH1, cytoplasmic immunoreactivity was evaluated (Figure 1).

Tissues were scored semi-quantitatively by evaluating the proportion of positive tumour cells over the total number of tumour cells (percentage of positive tumour cells per tissue microarray punch). Then, using receiver-operating characteristic (ROC) curve analysis (Zlobec et al, 2007b), appropriate cutoff scores for each marker were obtained. Positive staining in percentages of cells above or below the cutoff scores was classified as ��overexpression’ or ��loss’, respectively. The reliability of the cutoff score was obtained by 200 bootstrapped replications, a method which re-samples the data with replacement. Figure 1 Colorectal cancer samples with membranous positivity and corresponding negative staining for CD133 (A and B), CD166 (C and D), CD44s (E and F), EpCAM (G and H) and cytoplasmic positivity and negativity for ALDH1 (I and J).

Whole tissue sections A total of 101 whole tissue sections from corresponding colorectal cancer patients included on the tissue microarray were retrieved and immunohistochemistry for the markers found to have prognostic value was performed according to the protocol outlined above. These cases were part of a previous study used to investigate the expression of putative stem cell markers within the regions of most dense tumour budding at the invasive front of colorectal cancers only (Hostettler et al, 2010). All slides were scored in the adjacent normal mucosa, if available, tumour centre, and at the invasive tumour front separately. Differences in expression pattern were described, namely, whether increased or decreased expression was observed from the normal adjacent tissue to the tumour centre and finally to the invasive tumour front. Tumour invasion assay The colorectal cancer cell lines LS180, SW480, and Colo205 were cultured in RPMI 1640 medium supplemented, with GlutaMAX, MEM NEAA, 10m HEPES, 1m sodium pyruvate, kanamycin sulphate, and 10% FCS (all the AV-951 reagents were from Gibco, Paisley, UK).