Figure 1.
CTC gene expression profiling methodology.
(A) Schematic representation of the procedure used for CTC molecular characterization. CTC were isolated from 7.5 mL of peripheral blood by immunomagnetic separation using anti-EpCAM coated magnetic beads. Isolated cells were subjected to a RNA extraction followed by a whole transcriptome amplification process (WTA). Finally amplified cDNA was hybridized onto Agilent gene expression arrays. (B) GAPDH-CD45 levels in controls and mCRC patients measured by real time PCR. Horizontal bars represent the median value of each group (*p<0.05). (C) Spearman correlation analysis between GAPDH-CD45 levels obtained both from post-array data and qPCR previous to WTA amplification. (D) SAM analysis output graph showing gene expression differences between the group of patients and controls. Dots highlighted correspond to genes with statistically significant increased levels of expression in the group of patients compared to the control background, being considered to characterize the CTC population from mCRC.
Figure 2.
Gene expression analysis and validation.
(A) Hierarchical clustering of differentially expressed genes between patients (n = 6) and controls (n = 3) (CTC specific genes). (B) RTqPCR validation of eleven genes selected from array data. CD45-normalized fold change differences between mCRC patients (n = 20) and healthy controls (n = 10) (grey bar) in the CTC enriched fraction (black bars; ***p<0.0001). Of note, no differences were observed between mCRC patients (n = 5) and controls (n = 5) in the remaining fraction after CTC immunoisolation (white bars).
Figure 3.
Gene expression of selected genes in primary tumors and metastases.
(A) Gene expression differences between lung and liver metastases from CRC patients (n = 14) and primary tumors (n = 14) for the eleven validated genes. (B) Differences in gene expression for CLU and TIMP1 between lung (n = 7) and liver (n = 7) metastases (M) compared with the primary tumor. (C) Specific up-regulation of TGFβ1, TIMP1 and CLU in the invasive front of CRC primary tumors (n = 14) compared to the non-invasive area (*p<0.05; **p<0.01; ***p<0.001).
Table 1.
Diagnostic and prognostic value of array validated genes.
Figure 4.
Schematic representation of the proposed CTC phenotype from mCRC patients.
Of the resulting microarray dataset, some of the most relevant genes are showed in relation with certain features of the process of metastasis.