Assessing Ecto-5'-Nucleotidase/CD73 Expression and Malignancy Parameters in Early- and Late- Passage C6 Glioma Cells

Glioblastoma (GB) is a highly aggressive tumor characterized by its proliferative and invasive behavior. Ecto-5'-nucleotidase (e5NT/CD73), an enzyme that hydrolyzes extracellular AMP to adenosine, plays a pivotal role in cellular processes and has been involved in tumor progression, with its upregulation observed in several cancers. C6 glioma cells, widely used in GB research, exhibit changes in morphology and biochemical properties, depending on their passage number. This study investigates malignancy-related parameters in early-passage (EPC6) and late-passage (LPC6) C6 cells, highlighting the e5NT/CD73 expression and activity. The results presented here demonstrate that the LPC6 cells showed reduced CD73 expression and lower e5NT/CD73 AMPase activity compared to the EPC6 cells. Despite a higher proliferation rate in the LPC6 cells after two days of growth, Ki67 expression analysis revealed comparable proliferation between the two cell types at 5 and 10 days. Notably, the EPC6 cells showed enhanced proliferation in response to exogenous AMP, whereas the LPC6 cells did not. Furthermore, the EPC6 cells exhibited decreased adhesion but greater colony formation than the LPC6 cells. The LPC6 cells showed a significant reduction in migration, likely due to the loss of e5NT/CD73 migratory function. In the in vivo results, all the rats injected with EPC6 cells developed tumors displaying all the histopathological features of GB, whereas the LPC6 cells formed smaller tumors. Confirming the role performed by e5NT/CD73 in glioma progression, protein silencing significantly reduced tumor growth in vivo. These findings underscore the critical role of purinergic signalling in GB progression and emphasize the need for careful monitoring of passage number and e5NT/CD73 in in vitro experiments.