Abstract B086: Patient-derived pancreatic tumor organoids as a tool to evaluate cancer stem cell populations and their role in therapeutic resistance
Authors: Keepers Zachery L , Roy Sanjit , Ryan William , Bhandary Binny , Lamichhane Narottam , Carrier France , Iyer Ramaswamy K , Molitoris Jason K , Regine William F , Shukla Hem D 
Published: September 2024

Abstract
Introduction: Pancreatic ductal adenocarcinoma remains one of the most lethal forms of cancer with a five-year survival rate of less than 15%. Regardless of surgery eligibility, most patients receive systemic chemo-radiation therapy, which includes FOLFIRINOX or gemcitabine and nab-paclitaxel, often followed by radiation therapy (RT) with or without 5-FU. Unfortunately, patients’ tumors frequently develop resistance to these therapies, and it’s often unclear as to why. One potential source of resistance and recurrence is cancer stem cell (CSC) perseverance following treatment. Purpose: The purpose of this study was to characterize and treat three patient-derived pancreatic tumor organoids (IDs: 8510, 7800, and 11777) with RT and LD50 doses of relevant chemotherapy drugs to evaluate the treatment response. Furthermore, we sought to examine the impact of individual and combination treatments (i.e., chemo-RT) on the population of CSCs in these organoids. Methods: Organoids were cultured in 3D BME gel domes and grown in an NCI-recommended media formulation. Genome sequencing of 505 relevant genes was performed using a PGDx kit. The dose responses of three organoids to radiation (2-12 Gy) and several chemotherapy approaches were determined via MTT assay. CSCs were identified as those cells staining positive for markers SOX2 and OCT4. The population of CSCs before and after chemotherapy, RT, and combined chemo-RT were evaluated with immunofluorescence, western blotting, and flow cytometry. Results: The data revealed a variation in the responses of tumor organoids to treatments, as demonstrated by different LD50 doses. For instance, tumor organoid 8510 exhibited higher tolerance to 5-FU and FOLFIRINOX treatments compared to organoid 7800. Interestingly, organoid 11777 showed resistance to RT, unlike organoids 8510 and 7800. Clinical genomic sequencing revealed that organoid 11777 showed unique variants of several genes known to be involved with DNA repair, including PIM1, RAD54L, and SLX4, as well as mutations in the ARID1b and TGFBR2 genes. The latter two genes may have implications for radiation resistance and could explain the unique resistance of organoid 11777 to RT. Further investigation is warranted. Regarding the population of CSCs, the immunofluorescence data has confirmed the presence of SOX2 and OCT4 in these organoids, and the immunoblot data showed an upregulation of SOX2 and OCT4 in tumor organoids treated with radiation alone. Flow cytometry analysis confirmed that the percentage of CSCs increased following radiation treatment; however, the CSC population was slightly decreased following treatment with FOLFIRINOX and significantly decreased after combination treatment with FOLFIRINOX and RT. Conclusions: Patient-derived pancreatic tumor organoids can be used as a surrogate to provide molecular insights and therapeutic sensitivity profiles in individual patients. Additionally, while the data suggests a link between CSCs and RT resistance, the combination approach results in a more pronounced inhibition of the CSC population.
Citation Format: Zachery L Keepers, Sanjit Roy, William Ryan, Binny Bhandary, Narottam Lamichhane, France Carrier, Ramaswamy K Iyer, Jason K Molitoris, William F Regine, Hem D Shukla. Patient-derived pancreatic tumor organoids as a tool to evaluate cancer stem cell populations and their role in therapeutic resistance [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B086.