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. 2018 Mar 27;18(1):335.
doi: 10.1186/s12885-018-4238-4.

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

Susan Tsai  1   2 , Laura McOlash  3 , Katie Palen  3 , Bryon Johnson  3 , Christine Duris  4 , Qiuhui Yang  4 , Michael B Dwinell  5 , Bryan Hunt  4 , Douglas B Evans  1 , Jill Gershan  3 , Michael A James  6   7
Affiliations

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

Susan Tsai et al. BMC Cancer. .

Abstract

Background: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

Keywords: CAFs; Microenvironment; Organoid; Organotypic culture; PDAC; Pancreatic Cancer; TILs; Tumor immunology; Tumor stroma.

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Conflict of interest statement

Ethics approval and consent to participate

All procedures involving clinical samples are covered under IRB#PRO00012151 (MCW Surgical Biorepository) and PRO00028870 (preclinical chemosensitivity testing), reviewed by Medical College of Wisconsin/Froedtert Hospital Institutional Review Board #2. All patients provided written informed consent at the time of enrollment.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
Organoid and CAF Culture from Primary and Metastatic Human Pancreatic Tumors. a Micrographs of a human PDAC lung metastasis thro ugh 3 passages in organoid culture, monolayer outgrowth of tumor and fibroblast cultures. b Immunofluorescent staining of Vimentin in patient matched fibroblasts. c Micrographs of organoid culture of a human PDAC liver metastasis. d Micrographs of organoid culture of a primary human PDAC tumor collected from a rapid autopsy with associated fibroblasts. Bars, 100 μM
Fig. 2
Fig. 2
Pathologic and Immunohistochemical Characterization of Organoids. a H & E Staining and pathologic observations of organoid (tissue bank #2178). b Immunohistochemical staining with antibodies detecting the indicated proteins. Bars, 100 μM. c Immunohistochemical staining of tumor tissues
Fig. 3
Fig. 3
Immunofluorescent Characterization of Organoids. a IF staining of representative 1914 organoid and Panc1 spheroid as described using the indicated antibodies. b H&E on MCW670 spheroids. c IF staining of Laminin α5 in sections of 3D spheroids and organoids. Bars, 100 μM. d IF staining of CK19 in tumor tissues and αSMA in stromal fibroblasts. Arrows indicate CK19 positive tumor tissue surrounded by αSMA positive fibroblasts
Fig. 4
Fig. 4
Co-culture of PDAC Tumor Organoids, CAFs, and Lymphocytes. a RA012 primary tumor organoids and CAFs. b Micrographs and immunofluorescence for the indicated markers on co-cultured RA012 primary tumor organoids and fibroblasts. c Number of viable cells in culture at different time points. d Percentage of T cell subsets in culture with CD3/26 activation on day 6. e Micrographs of T-lymphocytes in co-culture with organoids or empty Matrigel domes at the Matrigel boundary. f Micrographs of fixed cells and immunofluorescence using anti-CD3 and DAPI staining of T-lymphocytes in co-culture with organoids or empty Matrigel domes at the Matrigel boundary. Dotted lines represent Matrigel boundaries. Bars, 100 μM
Fig. 5
Fig. 5
Gemcitabine Response in Organoid Co-Culture Versus Organoids Alone. a Relative viability in organoid cultures treated with indicated doses of gemcitabine for 96 h. b Relative viability in organoid-fibroblast co-cultures treated with indicated doses of gemcitabine for 96 h. Error bars indicate standard deviation from the mean
Fig. 6
Fig. 6
Schematic representation of organotypic organoid co-cultures
See this image and copyright information in PMC

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