Radiobiological Characterization of Pancreatic Cancer Patient-Derived Organoids

Abstract

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most aggressive and lethal tumors with a 5-year survival rate of less than 10%. Neoadjuvant radio(chemo)therapy aiming tumor downsizing fails in about 70% due to high heterogeneity, strong desmoplastic stroma and intrinsic radioresistance. In this project, pancreatic cancer patient-derived organoids (PDOs) are characterized regarding radioresponse, DNA-damage, proliferation and hypoxia, and clinical patients' outcome is correlated with the preclinical radiobiological data. In contrast to 2D monolayer cultures, PDOs maintain similar appearance, organization and functionality as the original tissue and therefore might have the potential as advanced preclinical model in radiation oncology. The radiation response of nine different pancreatic cancer PDO lines was determined by 3D cell viability assay. PDOs were irradiated with 0, 2, 4, 6, and 8 Gy (CellRad, Precision, USA) 24h after seeding and the ATP-dependent viability assay was performed 72h and 7d after irradiation (RT). Changes in morphology, number, and size were investigated by microscopy at different time points after RT. PDOs were characterized immunohistochemically by y-H2AX (DNA damage), and Ki-67 (proliferation) staining. RNA sequencing data of treatment-naive PDOs were analyzed by gene set enrichment analyses (GSEA) regarding radioresistance. Preclinical results were correlated with corresponding clinical data of PDAC patients. After optimization of the experimental set-up, PDOs showed a dose-dependent decrease in viability 7d after RT and heterogeneity in radioresponse. PDO lines were classified into radiosensitive, -intermediate, and -resistant subclasses. Immunohisto-chemical staining showed a significant increase in DNA double-strand breaks after RT. A correlation between radiosensitivity and enhanced proliferation index Ki67 was observed. Based on RNA sequencing data, OXPHOS- and hypoxia-dependent genes, amongst others, were identified as pathways significantly differentially regulated between the subclasses by GSEA. Preclinical radioresistance was associated with worse survival and poor clinical outcome. The results of the preclinical experiments demonstrate the heterogeneity among PDOs in response to RT reflecting the clinical situation of patients with PDAC. The findings from the GSEA show promising aspects for further experiments to understand the role of hypoxia in PDAC and its effect on radioresistance. PDOs have the potential as a novel translational research platform in radiation oncology. Prospectively, we aim to implement the screening of the radiosensitivity of PDOs in clinical practice for the realization of truly personalized radiotherapy in PDAC patients.