Abstract 5726: Targeting canine bladder cancer with Nimbolide, a Neem ( <i>Azadirachta indica</i> ) limonoid

Abstract Background: The 5-year relative survival rate for Bladder Cancer (BlCa) patients with distant metastases is about 9%. Hence, novel and innovative therapies for metastatic BlCa are required. Dogs were found to develop muscle-invasive BlCa (MIBC) spontaneously and 10% progress to distant metastases at the time of diagnoses. Currently most dogs with MIBC are treated with non-steroidal anti-inflammatory drugs (NSAIDs); however, &amp;lt;25% respond to them. Our quest was to identify low-cost natural compounds of low toxicity that can be used in dogs. Nimbolide, a limonoid extracted from Neem leaves, was found to have anti-tumor effects in human BlCa cell lines. Hence, we tested whether this natural product will be effective in canine BlCa cell lines. Methods: We obtained two canine BlCa cell lines - K9TCC-PU-AXC (AXC), that formed tumors in nude mice and K9TCC-PU-Pu (PuPu), which did not, from Dr. Deborah Knapp, Purdue University. Cell viability was estimated by MTT assay and by live/dead cell staining on 3D-printed spheroids. Flow cytometry was used to estimate the rate of apoptosis. Autophagy was estimated by the expression of LC3B and p62 whereas EMT was estimated by the expression of vimentin, Snail-1, Snail-2, and cell migration was estimated by scratch assay using a Cytation 5 platform. Results: Nimbolide was more effective in suppressing proliferation in PuPu cells (IC50 = 0.656 µM) compared to AXC cells (IC50 = 1.2 µM). Flow cytometric analysis showed that AXC did not undergo apoptosis in response to nimbolide whereas PuPu did; however, nimbolide caused autophagy in both cells. On the other hand, nimbolide prevented epithelial mesenchymal transition (EMT) and migration in AXC, but not in PuPu cells. These effects of nimbolide were not seen in human dermal fibroblasts, underlining the specificity and selectivity of nimbolide. Conclusions: Based on these results, we intend to develop nimbolide as a therapeutic tool in the treatment of canine BlCa in future studies. Acknowledgements: The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health, the Department of Veterans Affairs or the United States Government. We are grateful for a generous award from the University of California Comprehensive Cancer Center (UCDCCC) Support Grant (P30CA093373) for this project. Citation Format: Conner Suen, Eashan Sharma, Gabriel Gilchrist, Neelu Batra, Christopher A. Lucchesi, Kenneth A. Iczkowski, Robert B. Rebhun, Paramita M. Ghosh. Targeting canine bladder cancer with Nimbolide, a Neem (Azadirachta indica) limonoid [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 5726.