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

Abstract

BackgroundPatient-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.MethodsWe 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.ResultsWe 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.ConclusionsThese 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.

Optical Metabolic Imaging of Heterogeneous Drug Response in Pancreatic Cancer Patient Organoids

“…Alternatively, pancreatic organoids have emerged as an appealing method to tailor treatments by performing high-throughput drug screening directly on a patient's tumor cells (7)(8)(9). In vitro organoids recapitulate the genetic and histopathological characteristics of the original pancreatic tumor, along with its complex 3-dimensional organization (10)(11)(12)(13)(14). Organoid cultures also preserve interactions between tumor cells, immune cells (15), and fibroblasts (16), which can influence tumor drug response and are potential drug targets (17,18).…”

An Organotypic Microcosm for the Pancreatic Tumor Microenvironment

“…Additionally, normal murine pancreatic organoids induced with a Kras mutation, which occurs in 94% of PDACs, displayed typical features of early PDAC, including multilayering epithelial cells, disorganized cells, and overlapping nuclei [14][15][16]. PDAC organoids also harbor pancreatic cancer markers CK7, CK19, P53, and lack of CK20, consistent with paired tumor tissues [12,13]. Furthermore, organoids are representative models of the genetic landscape of the tumor of origin and have been indicated as a beneficial drug sensitivity model for PDAC patients [17,18].…”

“…PDAC tumor organoids represent the morphologic features of the primary tumor ( Figure 1). Compared to normal pancreatic organoids, tumor organoids contain nuclear irregularity, nucleolar prominence, and cell-cell adhesions as seen by hematoxylin and eosin staining [12,13]. Additionally, normal murine pancreatic organoids induced with a Kras mutation, which occurs in 94% of PDACs, displayed typical features of early PDAC, including multilayering epithelial cells, disorganized cells, and overlapping nuclei [14][15][16].…”

CLPTM1L/CRR9 ectodomain interaction with GRP78 at the cell surface signals for survival and chemoresistance upon ER stress in pancreatic adenocarcinoma cells

“…We previously characterized these patient‐derived organoids, which express CLPTM1L (Fig. a ) . PDAC organoids treated with CLPTM1L antagonistic antibody as monotherapy resulted in decreased organoid viability over 14 days in culture in the nanomolar range, with an IC 50 of 29 nM (Fig.…”