Colorectal cancer (CRC) is the fourth most diagnosed cancer and the second leading cause of cancer death with 135,430 new cases and 50,260 deaths predicted to occur in the United States in 2017 “Cancer Statistics Center,” ACS Available: http://cancerstatisticscenter.cancer.org/. With current screening tests, the percentage of diagnosis occurring at localized, regional, distant, and unknown stages are 39, 35, 21, and 5 percent with corresponding 5-year survival rates of 90.1, 71.2, 13.5, and 35.5 percent, respectively “Cancer of the Colon and Rectum - Cancer Stat Facts,” NCI Available: https://seer.cancer.gov/statfacts/html/colorect.html. The high mortality rate can be attributed to the percentage of later stage diagnosis. As such, there is a demand for novel diagnostics that increase the percentage of diagnosis occurring at an early stage when the survival rate is most promising. It is possible that a novel diagnostic could be designed based on the increased expression of adhesion molecules on transforming tissue relative to normal tissue. Transforming tissue would be identified by detection constructs consisting of microparticles conjugated to ligands cognate to the adhesion molecules. The detection constructs would be administered to the epithelium of the colorectum via a spray catheter through the biopsy channel of an endoscope during colonoscopy.
The broad working hypothesis of this study is that differential expression of adhesion molecules on transforming and cancerous, relative to normal tissue, can be exploited to develop a ligand conjugated particle based in situ diagnostic assay. As a first step, the goal of this thesis was to characterize the expression of sialyl Lewis A (sLeA), sialyl Lewis X (sLeX), and CD44 on transforming and cancerous, relative to normal, cell lines and tissues.
Expression levels of CD44 proteins as well as sLeA and sLeX glycans on cancerous relative to normal human colorectal cell lines were quantified by flow cytometry. This analysis revealed that CD44v3 and CD44v5 proteins have increased expression on cancerous relative to normal human colorectal cell lines when identified by anti- CD44v3 mAb 3G5 and anti- CD44v5 mAb VFF-8. In addition, sLeA and sLeX glycans have increased expression on cancerous relative to normal human colorectal cell lines when identified by anti- sLeA mAb KM231, anti- sLeA mAb C241:5:1:4, anti- sLeX mAb CSLEX1, anti- sLeX mAb FH6, and anti- cutaneous lymphocyte antigen (CLA) mAb HECA-452.
Expression levels of sLeA and sLeX were characterized on human colorectal tissues by immunohistochemistry. The average percent staining of each core for sLeA is significantly lower on normal tissues and normal adjacent tissues compared to stage I adenocarcinoma tissues when identified by anti- sLeA mAb KM231 and anti- sLeA mAb C241:5:1:4. Results also show the average percent of staining of each tissue core for sLeX is significantly lower on normal tissues and normal adjacent tissues compared to stage I adenocarcinoma tissues when identified by anti- sLeX mAb CSLEX1. Anti- CLA mAb HECA-452 recognizes a carbohydrate domain shared by glycans including sLeA and sLeX. Results show the average percent of each core staining positive for glycans identified by anti- CLA mAb HECA-452 is significantly lower on normal tissues and normal adjacent tissues compared to stage I adenocarcinoma tissues. Interestingly, the results indicate that the average percent of glands staining positive for sLeA is significantly higher on normal tissues compared to neoplasia tissues when identified by anti- sLeA mAb C241:5:1:4.
In summary, this thesis revealed differences between the expression of CD44, sLeA, and sLeX on transforming and cancerous versus normal cell lines and tissues. These differences in adhesion molecule expression could be exploited for the design of a novel assay for the diagnosis of colorectal cancer.