S. Tabe1,2, N. Tanimizu1, K. Takeuchi1, Y. Yamamoto1,2, Y. Ueno1, M. Ohtsuka2, H. Taniguchi1 1Institute of Medical Science, University of Tokyo, Regenerative Medicine, Minato-ku, TOKYO, Japan 2Chiba University Graduate School of Medicine, Department Of General Surgery, Chiba, CHIBA, Japan
Introduction: Pancreatic ductal adenocarcinoma (PDAC) has poor prognosis. Tumor microenvironment (TME) has been implicated in aggressiveness of PDAC, such as resistance to chemotherapy, and high metastatic potential. However, precise mechanisms how TME affects PDAC characteristics remain largely unknown. One reason for limited understanding about TME is lack of good culture model recapitulating TME. We previously established an organoid culture system in which human induced pluripotent stem cells (hiPSC) derived endoderm progenitors develop epithelial tissue structures by coculturing with hiPSC mesenchymal cells (MCs) and endothelial cells (ECs) (Takebe T, Taniguchi H. et al Cell Rep 2017). We now take advantage of this technology to developed PDAC organoids that recapitulate TME. In addition, we employ macrophages in these organoids, aiming to mimic the functions of tumor-associated macrophages (TAM).
Methods: Patient derived PDAC cells were acquired from surgical specimen without chemotherapy. We co-cultured PDAC cells with hiPSC-MC and EC to generate fused PDAC organoids (FPCO). Additionally, THP-1 cells, human monocytic leukemia cell line, were induced to macrophage in the presence of phorbol-12-myristate-13-acetae (PMA) and then introduced into FPCOs. For FACS sorting, PDAC and THP-1 cells were labeled with Luciferase-GFP and Kusabira Orange (KO), respectively, by using lentivirus vector. Immunofluorescence (IF) analysis was performed for assessment of extracellular matrix (ECM) deposition, and characteristics of hiPSC-MCs and macrophages in FPCO. A luciferase assay was utilized to evaluate the response of PDAC cells to anti-cancer drugs. Following the FACS sorting, bulk RNA sequencing was performed to analyze the characteristics of PDAC cells and macrophages within the FPCO.
Results: IF revealed the Fibronectin and Tenestin C, Hyaluronic acid-binding Protein (HABP) were abundantly deposited, and hiPSC-derived MCs altered to different types of cancer associated fibroblast-like cells (CAFs): LIF+ inflammatory CAF, αSMA+ myofibroblastic CAF, and CD74+ antigen presenting CAF-like cells were identified in the interstitial region of FPCO. Macrophages in FPCOs were mostly CD11b+CD163+ cells, a typical marker of M2-like phenotype, suggesting they acquired TAM-like characteristics. Luciferase assay showed PDAC cells in FPCO became chemo-resistance more than those in conventional PDAC organoids. Bulk-RNA seq data was examined by GSEA, suggesting that ECM proteins are mainly produced by CAF-like cells, whereas proliferative capacity and EMT features of PDAC cells were augmented in the presence of macrophages.
Conclusion: Our novel PDAC organoid namely FPCO replicated the TME consisting of multiple types of CAFs. Furthermore, macrophages cocultured in FPCO acquired characteristics of TAM. Therefore, FPCO can be used for screening of anti-cancer drugs and for identifying a novel anti-PDAC treatment targeting not only PDAC cells but also CAF or TAM.