THE INTEGRATIVE GENOMIC AND FUNCTIONAL IMMUNOLOGICAL ANALYSES OF COLORECTAL CANCER INITIATING CELLS TO MODULATE STEMNESS PROPERTIES AND THE SUSCEPTIBILIY TO IMMUNE RESPONSES

We conducted a genome wide DNA methylation profiling of primary CICs-CRC and differentiated tumor cell lines-CRC, including autologous pairs. The Illumina Infinium Methylation EPIC v1.0 BeadChip Array was used to obtain DNA methylation profiles.  Idat files generated by Infinium MethylationV.1 BeadChip were analyzed using the RnBeads R package. In total, 782939 probes were retained for further differential DNA methylation analysis. Differential methylation analysis between CIC and FBS cell lines was conducted using limmaR package at the CpG sites and region levels, defined as tiling (5 kb), genes, promoters, and CpG islands. For differential methylation measures, a combined rank a total of 3529 differentially methylated sites were identified. A combined rank among the 500 best ranking regions was applied to identify differentially methylated genes (n=79) and promoters (n=31) in CRC-CICs vs. FBS cell lines. N=48 and 31 genes were hypomethylated and hypermethylated, respectively while, at the promoter levels, N=9 and 22 were hypomethylated and hypermethylated, respectively in CRC-CIC as compared to FBS cell lines (p-value ≤0.11). CICs exhibited distinct methylation patterns, compared to differentiated tumor cells (p<0.05 or 0.01). Following the data quality control and normalization, DNA extraction and purification from CICs and FBS cell lines were performed using the AllPrep DNA/RNA Mini Kit. DNA methylation has been performed, starting from a minimum of 250 ng of utilizing the Infinium Methylation EPIC V.1 BeadChip, upon bisulfite conversion of DNA through the EZ kit, followed by library construction using the Infinium Epic Bead Chip kit. The prepared library has been sequenced with sequencing depths of 50M paired end reads with the HiSeq 3000 instrument. DNA methylation profiling was performed using the Illumina EPIC array V.1.