Salivary gland toxicity remains an important side effect during [177Lu]Lu-PSMA-617 therapy targeting disseminated prostate cancer. A 3D culture is expected to be more relevant to study functional damage due to the formation of acinar structures and the increased expression of AQP-5 and NKCC1. Radioprotectors, such as amifostine, could protect the salivary glands during treatment. Amifostine has to be converted to its active metabolite by alkaline phosphatase expressed in healthy cells and downregulated in cancer cells. A-253 3D salivary gland cell culture was optimized using different concentrations of Matrigel®. Optimization of NKCC1 and AQP-5 protein detection by western blot was performed using different blocking buffers and different concentrations of primary antibodies on A-549 and HK-2 cells. Alkaline phosphatase activity was measured in PC3-Flu, PC3-PIP, and A-253 cells with the Alkaline Phosphatase Diethanolamine Activity Kit. Cytotoxicity of amifostine was investigated with sulforhodamine B staining assay. The radioprotective effect of amifostine after exposure to 0, 2 or 5 Gray (Xrays) was investigated with an MTS assay. A successful 3D culture was formed. Nonspecific antibody binding was detected for the AQP-5 primary antibody, while a specific band for NKCC1 could be observed on A-549 cells and not in HK-2 cells. A-253 cells showed higher alkaline phosphatase activity compared to PC3-PIP and PC3-Flu cells (p-value<0.0001). The highest concentrations of amifostine indicated cytotoxic effects. The effects of amifostine during EBRT were inconclusive. Additional experiments are warranted to optimize AQP-5 expression and determine cytotoxic and protective effects of amifostine during EBRT.
|Qualification||Master of Science|
|Date of Award||9 Jun 2022|
|State||Published - 9 Jun 2022|