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By Jamshed Arslan, Pharm. D., PhD.
Gastroesophageal adenocarcinoma originates from the glandular epithelium of the esophagus, gastroesophageal junction and stomach. The incidence of gastroesophageal adenocarcinoma is declining, but it is still one of the deadliest cancers worldwide. A key reason behind high mortality is that by the time gastroesophageal adenocarcinoma is diagnosed, the cancer has often already metastasized. The search for novel biomarkers underlying gastroesophageal adenocarcinoma development brought glypicans to the attention of a team of scientists in Germany . Glypicans are members of heparan-sulfate proteoglycans and are involved in communication between tumors and their microenvironment. Glypicans are expressed on the surface of exosomes, the nanoscale extracellular vesicles of endosomal origin that can contribute to gastroesophageal adenocarcinoma pathobiology. The researchers focused on glypican 3, due to its inconclusive role in gastroesophageal adenocarcinoma.
Analysis of glypican 3 expression by immunohistochemical examination of primary gastroesophageal adenocarcinoma tissues (tissue-glypican 3) and flow cytometry analysis of exosomes extracted from gastroesophageal adenocarcinoma patients (exosome-glypican 3) showed low exosome and high tissue expression of glypican 3 in patients. This was associated with poor survival, indicating that glypican 3 expression can serve as a diagnostic and prognostic biomarker for gastroesophageal adenocarcinoma.
To determine glypican 3 expression on exosomes, the team first extracted exosomes from the serum of gastroesophageal adenocarcinoma patients and validated through immunoblotting analysis of exosome marker proteins TSG101, CD9, CD63 and CD81. Serum samples from healthy donors and patients with non-malignant disease were used as controls. Nanoparticle tracking analysis revealed a higher concentration but reduced median size of exosomes in gastroesophageal adenocarcinoma patients compared to controls. Immunoblotting and flow cytometry showed a greater exosome-glypican 3 enrichment in controls than in the patients.
The explanation of decreased exosome-glypican 3 expression in gastroesophageal adenocarcinoma came upon analyzing glypican 3 in serum where its levels were high. This indicated that glypican 3 is mainly present in a soluble form in gastroesophageal adenocarcinoma patients. To explore glypican’s tissue expression further, the researchers set out to examine glypican 3 expression in primary tumor samples.
Lysate of HepG2, human hepatocellular carcinoma cell line, analyzed by immunoblotting-Western blot. The PVDF membrane was probed with 2 µg/mL of Primary Sheep Anti-Human Glypican 3 Antigen Affinity-purified Polyclonal Antibody [AF2119] and by Secondary HRP-conjugated Anti-Sheep IgG Secondary Antibody [HAF016]. Glypican 3 is detected at approximately 75 kDa (as indicated).
Explore Exosome Marker Proteins
To determine the relationship between glypican 3 expression on exosomes and on tissues in gastroesophageal adenocarcinoma, the researchers performed immunohistochemical analysis of tumors and adjacent healthy tissues. About 80% of cases expressed tissue-glypican 3, where high expression was correlated with higher tumor grade and dismal prognosis. On the other hand, high exosome-glypican 3 expression was associated with improved overall survival, indicating an inverse correlation between tissue-glypican 3 and exosome-glypican 3.
Since glypican 3 has been proposed as a biomarker in various cancers, the team compared exosome-glypican 3 expression with that of carcinoembryonic antigen (CEA) and other gastroesophageal adenocarcinoma-specific biomarkers. Statistical analyses revealed that exosome-glypican 3 was a better biomarker in differentiating patient samples from the controls than other routine biomarkers. Likewise, expression of biomarkers other than exosome-glypican 3 had no correlation with overall survival. In summary, glypican 3 expression (tissue-glypican 3 and exosome-glypican 3) can be a diagnostic and prognostic biomarker for gastroesophageal adenocarcinoma.
This study offers evidence that glypican 3 may serve as a better biomarker for gastroesophageal adenocarcinoma than current standards (e.g., CEA, CA 72-4 and CA 19-9). The authors point out that the prior notion about glypican 3 being a tumor suppressor gene may be faulty. This is because previous reports relied on RNA expression, rather than protein, in gastric tumors. This study sheds light on the importance of functional expression of glypican 3 and implicates a role of post-translational modifications as a factor underscoring discrepancies between current and previous reports. Overall, these findings hold great promise for the pathophysiological evaluation of gastroesophageal adenocarcinoma, as it offers an improved diagnostic and prognostic biomarker for gastroesophageal adenocarcinoma and provides a more accurate insight on the nature of glypican 3.
Jamshed Arslan, Pharm D, PhD
Dr. Arslan is an Assistant Professor at Dow University of Health Sciences, Pakistan,
where he teaches Pharmacology to future pharmacists.
References
Rahbari, M., Pecqueux, M., Aust, D., Stephan, H., Tiebel, O., Chatzigeorgiou, A., … Kahlert, C. (2019). Expression of Glypican 3 Is an Independent Prognostic Biomarker in Primary Gastro-Esophageal Adenocarcinoma and Corresponding Serum Exosomes. Journal of Clinical Medicine. https://doi.org/10.3390/jcm8050696
