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  • br Introduction br Pancreatic cancer is notorious for its


    1. Introduction
    Pancreatic cancer is notorious for its poor prognosis with a five-year survival rate of approximately 7.5% and is expected to be the second leading cause of cancer death in USA by the year 2030 [1–3]. Pan-creatic ductal adenocarcinoma (PDAC) accounts for 90% of the pan-creatic cancer and is a unique stroma-rich, desmoplastic type of cancer where the stroma represents as high as 90% of the tumor volume [4]. Stromal components include the cancer-associated fibroblasts (CAFs), immune cells, and extracellular matrix (ECM), as well as many soluble factors that promote the aggressiveness of PDAC. Pancreatic stellate Aztreonam (PSCs) are the major source of CAFs and ECM in PDAC [5–7]. In vivo models and in vitro co-cultures reveal that PSCs create hypoxic and fibrotic microenvironments and increase the growth and metastasis of tumor [8,9]. PSCs play direct and indirect roles in chemo-resistance
    through modulation of the metabolism, sensitivity, and delivery of drug by excessive ECM deposition [10,11].
    Most of the currently available in vitro cancer cell models [e.g., two-dimensional (2D) monolayer culture] do not truly reflect the tissue environment that have both cell-matrix and cell-cell interactions. They neither reproduce the morphological organization or pathophysiologic features of carcinomas in vivo [12,13]. In recent years, three-dimen-sional (3D) spheroid culture systems have attracted the attention in the areas of regenerative medicine, tumor biology, drug screening, tissue engineering, and toxicology [14]. 3D cell culture, compared to the conventional 2D cell culture, may provide a more physiologically re-levant condition. There are several protocols to generate 3D spheroids, such as hanging drop plates, ultra-low attachment (ULA) plates, and general culture plates coated with biomaterials or synthetic polymers [15]. Although there are a few 3D tumor spheroid models, a simple,
    ∗ Corresponding author. Institute of Polymer Science and Engineering, National Taiwan University, No. 1, Sec. 4 Roosevelt Road, Taipei, 10617, Taiwan, ROC. ∗∗ Corresponding author. Department of Surgery, National Taiwan University Hospital, Chung-Shan South Rd., Taipei, 10002, Taiwan, ROC. E-mail addresses: [email protected] (Y.-W. Tien), [email protected] (S.-h. Hsu).
    high-throughput model that mimics the cellular and ECM environments for PDAC has not been reported. Meanwhile, collective cell migration is the process by which a group of cells moves in concert with collective behavior from cell-environment interactions and cell-cell communica-tion, and is an essential process during embryonic development, mor-phogenesis, and organization of multiple organisms, as well as in wound healing and cancer cell spreading [16–18]. The collective mi-gration of co-cultured cancer cells and stromal cells on a 3D platform is still poorly understood, let alone the correlation with the invasive be-havior and metastasis of cancer. A simple, rapid, and low cost platform to generate 3D biomimetic spheroids in an ECM-rich microenvironment of PDAC is highly demanded.
    Chitosan (CS) is a cationic copolymer of glucosamine and N-acet-ylglucosamine, derived from chitin, which is a second-abundant natural polysaccharide, and mostly derived from crab and shrimp shells [19–21]. Chitosan has been applied in biomedical fields because of its antibacterial activity, biocompatibility, and biodegradability. Our pre-vious study showed that neural stem cells formed neurosphere-like adherent spheroids on CS membranes [22]. Hyaluronic acid (hyalur-onan, HA), a major component of ECM, plays a critical role in pro-moting tumor growth in mice and the progression of disease [23]. The abnormal accumulation of HA in tumors is correlated with poor prog-nosis especially in patients with PDAC [24]. The HA‐rich micro-environment may activate tumor progression by increased cell pro-liferation, migration, invasion, metastasis, angiogenesis, and less sensitivity towards chemotherapy [25,26]. Recently a number of on-going HA-targeted therapy clinical trials, such as depleting HA in tumor stroma or blocking HA signaling/synthesis could provide a promising therapeutic effect against PDAC [27,28]. To mimic tumor spheroid, the HA-rich microenvironment of tumor growth is an attractive strategy, particularly in PDAC, which is characterized by a high stromal content and associated with an extremely poor prognosis [24].
    Understanding the dynamics of cell assembly is a crucial step for studying developmental processes of cancer. Cell-cell interaction, merging, and assembly into spheroids may reflect the tumor formation in an endogenous 3D microenvironment. In the present study, we co-cultured pancreatic cancer cells and PSCs on CS-HA substrates and monitored by real-time imaging their self-assembly into PDAC-like tumor spheroids. The HA-rich CS-HA plates were employed to mimic the microenvironment of PDAC. We examined the cellular interaction, migration, and drug resistance to verify if the tumor-like spheroids could mimic the cancer microenvironment of PDAC and serve as a drug screening platform in the future.