Abstract Intestinal cancer ranks as the second leading cause of cancer-related deaths in the United States, with an anticipated toll of 52,900 fatalities in 2025. Chronic inflammation is a recognized contributor to carcinogenesis. Patients with inflammatory bowel disease, face a 1.7-fold increased risk of developing cancer. Identifying compounds that either induce or mitigate inflammation is crucial for cancer prevention and treatment. Organoids, as in vitro 3D cell culture mimetics of specific organs, provide a more physiologically relevant and reliable human-based model for testing drug efficacy and toxicity compared to traditional 2D cell cultures or animal models. This study focuses on the development and application of bioluminescent intestinal inflammatory reporter organoids to screen and monitor the effects of various inflammatory and anti-inflammatory agents. Healthy intestinal organoids from induced pluripotent stem cells (iPSCs) or patient-derived tissues were transduced with the NF-kB nanoLuc® reporter plasmid, enabling luminescence-based monitoring of NF-kB activity. We employed lentiviral (LV) and adeno-associated viral (AAV) delivery systems to create stable and assay-ready NF-kB reporter organoids respectively. The successfully transduced organoids were assessed in both 3D and 2.5D formats to evaluate the impact of different inflammatory and anti-inflammatory compounds. To measure the activation of NF-kB, we utilized the Lumit® immunoassay to detect phosphorylated p65 levels. Protein and mRNA levels of various secreted and intracellular cytokines and chemokines were quantified through immunoassays and RT-qPCR. Stable NF-kB NanoLuc reporter organoids were established via LV transduction at a multiplicity of infection of 1000, followed by puromycin selection. Additionally, we generated transient, assay-ready reporter organoids using AAV serotype 3. Among the cytokines analyzed, the highest reporter activity was observed following treatment with a cocktail of TNFα, IL-1β, and flagellin at 3- and 6- hours post-treatment. The increase in intracellular phospho-p65 levels correlated with the observed reporter activity. Furthermore, NF-kB activation resulted in significant upregulation of other intracellular and secreted cytokines, including CXCL8, IL-1β, and TNFα. Importantly, treatment with established anti-inflammatory agents, such as Tofacitinib, led to a reduction in elevated reporter activity and downstream cytokine levels. In conclusion, the bioluminescent intestinal NF-kB reporter organoids developed through our optimized protocol represent a high-throughput platform for effectively monitoring the inflammatory effects of potential drug candidates. Citation Format: Subhashree Kumaravel, Carolina J. Sierra, Trish T. Hoang, Kevin Su, Vi Chu, Jeong-Hee Lee, Helena Borges, Thomas Machleidt. Bioluminescent intestinal reporter organoids as in vitro tools to monitor inflammatory effects of compounds 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 752.
Kumaravel et al. (2026) studied this question.