Biomarker Discovery and Development

Colon Cancer (CC) Biomarkers

AlfaGene's Approach

 Intestinal epithelial cells (IECs) and the progenitor stem cells of the colon are the source of inflammation and malignant transformation in colorectal cancer (CRC).  AlphaGene's isolation and propagation of adult GI stem cells, with potential for differentiation into epithelial cells types representative of those found in vivo populating the GI tract, enables a much better characterization and identification of biomarkers for these stem cells and derived epithelial lineages than was previously possible. 

The first phase of this approach is the establishment of a resource panel of well-characterized adult human gastrointestinal stem cell (AHGI-SC) lines from normal/unaffected tissue, abnormal but precancerous tissue (polyps), and adenocarcinoma (cancer stem cells) derived from the colorectal region of the same individual suffering from CRC.  The AHGI-SC lines are subjected to directed differentiation to generate human intestinal primary epithelial cell (HIPEC) lines from unaffected tissues and human intestinal tumor epithelial cell (HITEC) lines from cancerous tissue.

The second phase entails utilizing these paired stem and derived epithelial cell lines from both unaffected and affected tissue regions from the same individual for biomarker discovery via differential mRNA and protein expression analysis.  Differences in mRNA expression patterns are determined using RNA expression microarrays, verified with RT-PCR and Northern analysis, and then confirmed with Western and immunohistochemical (IH) analysis of the gene product coded for by these identified mRNA transcripts.  Differential protein expression is identified via use of 2D-DIGE, iTRAQ, cICAT, and tandem GC-MS/MS and LC-MS/MS.  The identity of these potential biomarkers, as well as, verification of their differential expression, is accomplished via Western analysis and IH and immunocytochemical (IC) staining.

The third phase involves validation of these identified potential biomarkers, therapeutic targets, and drug efficacy markers.  Control and CRC tissue panels are analyzed using IH staining (and Westerns if antibodies do not work in IH staining) for these markers.  The adenocarcinoma cell lines HT29, DCD1, and CaCo2 are examined with respect to the identified markers.  Furthermore, serum and bodily fluids of control and CRC individuals are examined for the presence of these markers via Western analysis and flow cytometry.  We also test our paired stem, HIPEC and HITEC lines, tissue panels, serum, and bodily fluids with respect to reputed colon cancer biomarkers identified by other entities.


The establishment of paired stem cell lines and epithelial lineages derived from both affected (adenocarcinoma), pre-cancerous (polyps), and unaffected tissue of individuals suffering from CRC, facilitates both rapid and efficient identification of cellular, molecular, genetic, and functional changes in both adult intestinal stem cells and intestinal epithelial cells during the CRC developmental processes; thus, enabling identification of specific biomarkers, target molecules, and development of novel therapeutics.

More detailed description of AlfaGene's approach to biomarker discovery in CRC