Figure 3

Chemotherapy induces mesenchymal gene expression in patient-derived colonospheres. (a) Liver metastasis-derived colonospheres were treated with 5-FU for six cycles. RNA was isolated from control (n=5) and 5-FU-treated cells (n=5), and were analyzed by gene expression profiling. The heat map shows all genes that were significantly (P<e⁻⁶) upregulated (68) or downregulated (36) in post-treatment tumor cells. See Supplementary Table 2 for a full list of genes. (b) Expression of the 5-FU-induced gene set (68 genes; Supplementary Table 2) was correlated with gene sets reflecting either an epithelial or a mesenchymal tumor phenotype in the data set of the same experiment. Correlations were assessed by using the ‘gene set versus gene sets’ option in the R2 genomics analysis and visualization platform. Gene sets reflecting a mesenchymal tumor cell phenotype positively correlate with the 5-FU-induced gene set (indicated in red), while gene sets reflecting an epithelial phenotype show a negative correlation (in blue). The P-values of the correlations are indicated as minus log10 P-values in green bars. (c) The 68 5-FU-induced genes were used to cluster the tumors of the CMS cohorts into 5-FU-induced high and low subgroups (K-means option in R2, using a two group separation) based on single gene P-values. All tumors had previously been classified into molecular subtypes. The graphs show the distribution of the CMS subtypes within the 5-FU-induced high and low subgroups. The 5-FU-induced high subgroup is enriched for mesenchymal subtypes (CMS4). (d) Kaplan–Meier curves showing the differences in relapse-free survival between the 5-FU-high and 5-FU-low subgroups in both cohorts. (e) Expression of the experimental-derived gene set of 5-FU-induced genes was compared to expression of genes upregulated in chemotherapy-exposed liver metastases by using the ‘relate 2 tracks’ option in the R2 genomics analysis and visualization platform. The XY-plot shows the correlation of the expression of both gene sets (R=0.80, P=2.0e⁻¹¹⁰) in the CIT subset of the CMS cohort. The CMS subgroups are CMS1: orange, CMS2: blue, CMS3 pink, CMS4: green.