A.E. Kennedy1, Y. Golivi1, R. Guenter1, J.B. Rose1 1University Of Alabama at Birmingham, Department Of Surgery, Birmingham, Alabama, USA
Introduction:
Pancreatic neuroendocrine tumors (PNETs) are rare cancers originating from neuroendocrine cells in the pancreas. Patients with advanced disease present a complex medical challenge due to their poor prognosis and limited treatment options. Notch signaling is a local cell-cell communication mechanism where transmembrane Notch receptors interact with neighboring cells through ligands binding, resulting in a cascade that regulates various developmental processes and cell fate decisions. Dysregulated Notch1 signaling is implicated in PNETs, affecting neuroendocrine tumor biology and potentially interacting with HIF pathways in disease progression. We hypothesized Notch1 in PNET cells provides a survival advantage in hypoxia.
Methods:
This study utilized BON cells, a commonly used cell line for PNET research. To examine the role of Notch1, the Notch1 gene was deleted in BON cells used CRISPR/Cas9 to generate a BON-Notch1-knockout (N1-KO) cell line. Wildtype (WT) and N1-KO cells were plated in duplicate at a density of 1.5*106 cells per 10cm² plate before being exposed to normoxic and hypoxic conditions (3% oxygen) for 48 hours. At the study endpoint, apoptosis and cell viability were analyzed by flow cytometry using 7-AAD and Annexin V staining. In parallel with this experiment, four 96 well plates with 8,000 cells per well and 8 replicates for each cell type were placed under normoxic and hypoxic conditions (3% oxygen). Two plates were incubated for 48 hours, and the other two for 72 hours, after which an MTT assay was performed to measure cell viability.
Results:
The MTT assay confirmed a reduction in viability in N1-KO cells compared to WT cells. WT cells showed greater survival in hypoxic conditions (48hrs: 37.8% viability, 72hrs: 39.44% viability) compared to N1-KO (48hrs: 30.95%, 72hrs: 21.52%) cells under the same conditions (48 Hours: p<0.001; 72 Hours: P<0.001). This was confirmed through flow cytometry, which showed a trend of reduced cell numbers following hypoxic treatment (plated at 1.5 × 106 cells, with an average loss of 1.2 × 106 cells in hypoxia). Flow cytometry results indicated a greater decrease in viability under hypoxia in N1KO cells compared to BON cells.
Conclusion:
Our data supports the hypothesis that Notch1 increases the ability of PNET cells to survive hypoxic conditions. This may lead to increased malignant potential of Notch1 positive NETs as they increase in size. Furthermore, it may lead to increased resistance to therapy. The mechanism of this hypoxia resistance is currently under investigation. Better understanding tumor cell adaptation to hypoxia and targeting processes of adaptation could lead to new therapeutic strategies.