To measure gene expression levels in different CC cell populations and since CC stem cells express CD133 (AC133; O'Brien et al, 2007; Ricci-Vitiani et al, 2007), these were selected from fresh non-metastatic (TNM stages 1,2) and metastatic (TNM3,4) CCs as well as liver metastases (Fig 2A and Fig S2 of Supporting Information). All CCs contained CD133+ cells, which induced tumours in nude mice (CC14: 4/4; mCC11: 4/4; 105 cells injected/site) unlike their CD133− counterparts (CC14: 0/4; mCC11: 0/4; 105 cells injected/site). CD133+ cells were found to be mostly Cytokeratin+ (not shown). Early CCs (TNM1,2) had 7.8-fold larger pools of CD133+ cells on average than normal colon (11.8% ± 1.2 SEM vs. 1.5% ± 0.1 SEM, p < 0.001), and metastatic tumours (TNM3,4) harboured 40% more CD133+ cells than non-metastatic (TNM1,2) CCs (16.6% ± 1.6 SEM vs. 11.8% ± 1.2 SEM, p = 0.04) (Fig 2A).
Gene expression analyses in purified CD133+ and CD133− CC cells, shown as CD133+/CD133− average ratios after normalization (Fig 2A and Fig S2 of Supporting Information) or individually (Fig 2B), revealed ±5-fold enrichment of CD133 mRNA in CD133+ cells. This confirms the selection and its significance since the AC133 epitope used for sorting is displayed by only a fraction of CD133 protein isoforms. Analysis of CD133+ epithelial cells is important as the CD133− epithelial population (and the unfractionated pool) includes also many cell types that form the stroma.
An HH-GLI activity signature (GLI1, PTCH1, GLI2, SHH and HIP) was detected in all CCs (Fig 2A,B and Fig S2 of Supporting Information), consistent with increased SHH expression (Fig 2B; Douard et al, 2006; Oniscu et al, 2004). This signature was not enriched in CD133+ cells of normal colon or liver and it was only slightly enriched in non-metastatic tumours (TNM1,2) (Fig 2A,B). However, there was a large increase in HH-GLI activity signature levels, mostly in CD133+ cells, in metastatic TNM3,4 versus non-metastatic TNM1,2 CCs (CD133+/CD133− averages: GLI1: 1.5 in TNM1.2 vs. 2.8 in TNM3.4, p < 0.01; GLI2: 0.4 vs. 5.5, p < 0.01; HIP: 0.5 vs. 4.4, p < 0.001; Fig 2A,B). PTCH1 increased in both cell populations (0.4 vs. 4.5, p < 0.001 in CD133+ cells; 0.3 vs. 4, p < 0.001 in CD133− cells) (Fig 2A,B and Fig S2 of Supporting Information), suggesting differential regulation of HH-GLI targets. GLI1 itself and SNAIL1, also a GLI target (Li et al, 2006) involved in epithelial-to-mesenchymal transition (EMT; Batlle et al, 2000; Cano et al, 2000), similarly showed an increased CD133+/CD133− average in liver metastases versus TNM3,4 CCs (6 in liver metastases vs. 2.8 in TNM4, p < 0.01; 2 vs. 1, p = 0.01). As CD133+ cells consistently showed the highest increases in the levels of HH-GLI pathway components (Fig 2B), increases in GLI1 levels and targets thus appear to parallel the progression in carcinoma stem cells to metastatic states.
Comparison of advanced TNM4 CCs versus their metastases in the same patients (CC14 vs. mCC9; CC31 vs. mCC19) (Fig 2A and Fig S2 of Supporting Information) showed that GLI1 levels increased only in CD133+ cells (4.4 in TNM4 vs. 6 in liver metastases and 5.5 vs. 12.6 in CD133+ cells; 1.8 vs.1.8 and 2.3 vs.1.4 in CD133− cells, all ×10−4 and normalized).