Development of primary human pancreatic cancer organoids, matched stromal and immune cells and 3D tumor microenvironment models

TLDR

Patient‑derived tumor models are the new standard for pre‑clinical drug testing and biomarker discovery, yet primary pancreatic cancer organoids and complex organotypic co‑culture models with stromal and immune components are not yet broadly implemented. The study aims to develop and characterize pancreatic cancer organoids and multi‑cell‑type organotypic co‑culture models. The authors used organoid culture, flow cytometry, cytology, immunofluorescence, and immunohistochemistry to derive organoids from resection, ascites, and autopsy sources and to create tumor‑associated fibroblast and monoculture lines. Primary human organoids displayed tumor‑like morphology, architecture, and polarity, unlike cell‑line spheroids, and the authors constructed complex organotypic models that activated myofibroblast‑like CAFs and induced tumor‑dependent lymphocyte infiltration, providing disease‑relevant 3D in‑vitro models that may aid study of tumor‑stroma/immune interactions and assessment of immunotherapies such as checkpoint inhibitors.

Abstract

Patient-derived tumor models are the new standard for pre-clinical drug testing and biomarker discovery. However, the emerging technology of primary pancreatic cancer organoids has not yet been broadly implemented in research, and complex organotypic models using organoids in co-culture with stromal and immune cellular components of the tumor have yet to be established. In this study, our objective was to develop and characterize pancreatic cancer organoids and multi-cell type organotypic co-culture models to demonstrate their applicability to the study of pancreatic cancer.We employed organoid culture methods and flow cytometric, cytologic, immunofluorescent and immunohistochemical methods to develop and characterize patient-derived pancreatic cancer organoids and multi-cell type organotypic co-culture models of the tumor microenvironment.We describe the culture and characterization of human pancreatic cancer organoids from resection, ascites and rapid autopsy sources and the derivation of adherent tumor cell monocultures and tumor-associated fibroblasts from these sources. Primary human organoids displayed tumor-like cellular morphology, tissue architecture and polarity in contrast to cell line spheroids, which formed homogenous, non-lumen forming spheres. Importantly, we demonstrate the construction of complex organotypic models of tumor, stromal and immune components of the tumor microenvironment. Activation of myofibroblast-like cancer associated fibroblasts and tumor-dependent lymphocyte infiltration were observed in these models.These studies provide the first report of novel and disease-relevant 3D in-vitro models representing pancreatic tumor, stromal and immune components using primary organoid co-cultures representative of the tumor-microenvironment. These models promise to facilitate the study of tumor-stroma and tumor-immune interaction and may be valuable for the assessment of immunotherapeutics such as checkpoint inhibitors in the context of T-cell infiltration.