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Clearance of senescent cells using senolytic drugs has been shown to represent a potential therapeutic alternative to ameliorate radiotherapy-induced xerostomia. Therefore, our aim is to develop an efficient method to screen for potent senolytic drugs in a 3D salivary gland organoid model

Since senescent cells possess anti-apoptotic properties, we selected several drugs targeting diverse apoptosis related pathways. (Sham-)irradiated salivary gland organoids were treated with different doses of candidate drugs to obtain optimal responses. As a measure of senolytic activity a Caspase-3/7 fluorogenic substrate was added together with the selected drugs to detect increases in caspase activity, resulting in bight fluorescent responses in apoptotic cells. Caspase-3/7 fluorescent activity following drug treatment was visualized in real-time using an IncuCyte S3. Using this methodology, drug concentrations that efficiently induced apoptosis in irradiated cells but were not/less toxic to unirradiated cells were used to verify the elimination of senescent cells and any corresponding changes in the organoid forming efficiency (OFE) of irradiated salivary gland cells.

We selected optimal doses for 17-DMAG, ARV-825, Dasatinib + Quercetin, TH588 and Nutlin3a that induce apoptosis only in irradiated organoids. Of these, 17-DMAG and Nutlin3a treated organoids showed increased self-renewal capacity after irradiation, with Nutlin3a having a more favorable toxicity profile. Furthermore, treatment with Nutlin3a resulted in a reduction of senescent markers in irradiated organoids, and a transcriptional increase in p53 target genes.

We conclude the screening for drugs that show senolytic activity without hampering stem cell expansion (as measured by organoid forming efficiency) may be a promising methodology for the identification of compounds with potential applications in post-radiotherapy regenerative medicine.