Biomarker Discovery and Development

Colon Cancer (CC) Biomarkers

More Detailed Description of AlfaGene's Approach to Biomarker Discovery in CRC

The cell line resources AlfaGene is creating are being utilized to identify molecular differences between normal adult stem and cancer stem cells, leading to the development of potential biomarkers, therapeutics, and efficacy markers for colorectal cancer.  This work employs AlfaGene’s novel and proprietary technology of AHGI-SC isolation and culturing (patent pending) to increase our understanding of GI cancer mechanisms and aid in the development of more efficient cancer therapies.  The overall strategy of this approach is summarized below:

 

Phase I. Isolation, establishment in long term culture, characterization, and production of serviceable quantities of 10 paired adult human stem cells from the normal/unaffected region, polyp (precancerous), and tumor affected region of the same individual afflicted with colorectal cancer (CRC).  Ten complete sets (10 + 10 + 10 = 30 cell lines) of  adult stem cell lines derived from an unaffected/”normal” region within 20-40 cm of tissue effected by an adenocarcinoma, from pre-cancerous polyps, and cancer stem cells from the adenocarcinoma, from ten individuals suffering from stage I CRC will be established.

A.  Establish 10 complete sets (normal, precancerous polyp derived and/or, stage I adenocarcinoma) of AHGI-SC lines from the colorectal region of 10 individuals suffering from CRC.

B.  Continually maintain and monitor the ability of the established AHGI-SC lines to retain their self-renewal and pluripotent capabilities by characterizing their ability, through 20 passages, to continue to propagate, differentiate, and generate non-transformed intestinal epithelial cell (IEC) lineages (enterocyte, Goblet, enteroendocrine, and Paneth), as well as, express specific stem cell markers.

 

Phase II. Characterization and production of serviceable quantities of RNA, DNA, and protein from the AHGI-SC, HIPEC, and HITEC lines established in this proposal. Characterization shall include both mRNA expression patterns via gene array analysis and differential protein expression patterns via 2D-DIGE.  Characterization shall also lead to the identified of potential biomarkers which shall be validated in Phase III.

A.  Produce large quantities of established cell lines, as well as, purify large quantities of RNA, DNA, and protein from these stem cell and derived HIPEC and HITEC lines for characterization, collaborations, and commercialization. 

B.  Differential mRNA expression pattern analysis will be performed by RNA microarray hybridization. Results will be compared and contrasted between the stem and derived IEC lines representing different cell types, tissue regions, disease stages, and disease.  The AHGI-SC, HIPEC, and HITEC sets will be derived from the same individual in order to decrease inherent variability and increase specificity.

C.  Differential protein expression analysis of the stem and HIPEC lines will be determined utilizing 2D-DIGE (iTRAQ and cICAT will be used as back-up methods) coupled with tandem LC-MS/MS (and GC-MS/MS).

D.  Identification of potential disease, disease stage, tissue region, and cell type specific biomarkers will be accomplished by comparing and contrasting the differential expression profiles from the normal adult stem cells, pre-cancerous stem cells, and cancer stem cells from the 10 CRC AHGI-SC sets.

E.  The identity and differential expression of potential region specific cellular biomarkers identified will be verified by RT-PCR, Northern analysis, Western analysis, immunocytochemical (IC) staining, immunohistochemical (IH) staining, and flow cytometry (FC).

 

Phase III. Validation of identified potential biomarkers.

A.  Immunohistochemical and Western analysis of identified potential markers in colon cancer and control tissue panels.

B.  Test for presence of identified potential biomarkers in the serum and bodily fluids of individuals suffering from colon cancer and negative controls.

C.  Immunohistochemical and Western analysis of identified potential markers in adenocarcinoma cell lines HT29, DLD1, and Caco2.

D.  Analyze and compare reputed colon cancer biomarkers identified by others utilizing the same paired stem, HIPEC, and HITEC lines and panels used in validating our identified biomarkers.

  

Phase IV. Develop diagnostic and/or therapeutic constructs (antibodies, peptides, small interfering RNA) against these molecules through collaborations with other bio-pharmaceutical companies and research university laboratories.

A.  Establish collaborations/partnerships with academic researchers or other industries to utilize our cell lines, identified biomarkers, and potential therapeutic targets.

B.  Establish collaborations with and sell licenses to bio-pharmaceutical companies to utilize our cell lines and biomarkers for research, biomarker discovery and development, therapeutic development, and drug testing and discovery in colon cancer.