W. Colley1, W. Chen1, Y. Golivi1, R. Guenter1, J. Rose1 1University Of Alabama at Birmingham, Department Of Surgery, Division Of Surgical Oncology, Birmingham, Alabama, USA
Introduction:
Pancreatic neuroendocrine tumors (pNETs) are malignancies arising from pancreatic islets. Surgery can be curative for localized pNETs, but patients with advanced disease have limited treatment options. Notch signaling is a biologic pathway currently investigated as a therapeutic target in cancer. Notch has 4 transmembrane receptors (Notch1-4) that participate in cell-cell communication and regulate growth. The gamma-secretase complex activates Notch via receptor cleavage to form the intracellular domain, which affects transcription of downstream targets such as Hes1. Gamma-secretase inhibitors (GSIs) (e.g. nirogacestat and crenigacestat) can block Notch signaling. To test the impact of GSIs in pNETs, we used in vitro 3D spheroid models instead of 2D models due to increased cell-cell contact. We hypothesized that pNET spheroids treated with GSIs will have reduced Notch signaling and lower proliferation rates.
Methods:
3D spheroids were cultured to study the effects of Notch on pNET cell proliferation and viability. Two pNET cell lines (BON, QGP) were seeded at 1,000 cells per well on agarose-coated plates to form spheroids. Proliferation was monitored over time using pixel quantification of spheroid size. Spheroids exposed to GSIs or control began treatment on day 6. Spheroids were treated with 2 doses (20μM or 40μM) of nirogacestat, crenigacestat, or DMSO controls. After 10 days, viability was measured by CellTiter-Glo 3D ATP-luminescence assays. Protein lysates were analyzed via western blot to validate expression of Notch and Hes1.
Results:
Pixel quantification showed BON and QGP spheroids without drug treatment increased in size over the 10-day period. BON and QGP spheroids treated with the highest dose of nirogacestat (40μM) showed the most significant decrease in viability versus DMSO and the lower dose (20μM). A decrease in spheroid size in cells treated with nirogacestat was observed in BON spheroids. However, BON spheroids treated with crenigacestat showed an increase in proliferation and viability at both doses. This was not observed in QGP spheroids. Western blot analysis did not show increased Notch1 or Notch3 expression in 3D compared to 2D. QGP 3D samples treated with nirogacestat did show a reduction in Hes1 compared to DMSO and crenigacestat samples.
Conclusions:
The reduction of pNET spheroid viability by nirogacestat was consistent in both cell lines, but reduction of size was cell line dependent. Crenigacestat, which selectively inhibits Notch1 in cholangiocarcinoma, unexpectedly increased viability and growth of BON spheroids. Overall, GSIs are influencing cell growth and viability, but the role of Notch signaling in this process will need to be elucidated through further experimentation.