Original ArticlesBasement membrane destruction by pancreatic stellate cells leads to local invasion in pancreatic ductal adenocarcinoma
Introduction
Pancreatic ductal adenocarcinoma (PDAC) has the worst outcome among all common cancers, with a 5-year survival rate of only < 8% [1], mainly because of its rapid progression, difficulty in early detection, and inadequacy of available treatments. Better understanding of its pathophysiology and mechanisms of invasion and metastasis are necessary to finding new treatments that can improve its prognosis.
PDAC is characterized by desmoplasia, a desmoplastic stroma mainly composed of abundant extracellular matrix (ECM), reportedly related to cancer-treatment resistance [[2], [3], [4]]. Pancreatic stellate cells (PSCs) produce ECM and interact with pancreatic cancer cells. The active phenotype of PSCs express α-smooth muscle actin (αSMA), the myofibroblast protein, and secrete several factors that stimulate tumor growth, and facilitate cell survival and metastasis [[5], [6], [7], [8], [9]]. We previously reported heterogeneity in PSCs, such as CD10-positive PSCs, which enhance tumor progression [10], PSCs lead and promote cancer cell invasion via the function of Endo180 [11] and PSC-related autophagy which was associated with shorter survival times and disease recurrence in patients with pancreatic cancer [12]. These data suggest that PSCs are key players in invasion and metastasis by pancreatic cancer cells. In pancreatic cancers, tubular ductal adenocarcinoma is frequently seen in invasive areas. Tubular ductal adenocarcinoma is characterized by well-developed duct-forming neoplastic cells invading into an intensely desmoplastic stroma [[13], [14], [15]]. However, how the pancreatic cancer cells that form ductal structures invade the surrounding stroma is unclear. Ductal epithelial cells are separated from the surrounding stroma by a highly crosslinked and insoluble sheet-like structure called the basement membrane (BM). In pancreatic cancer progression, BM destruction is the important step before invasion to surrounding stroma. BM is also recognized as an important regulator of cell behavior [16]. In tumors, BM are significantly less crosslinked, and fragmented BMs have been associated with tumor progression and poor outcome [[16], [17], [18], [19], [20]]. BMs have been reported in pancreatic cancer tissue [[21], [22], [23]], and BM laminin expression can predicts outcome following curative resection of cancer in the pancreatic head [24]. The BM destruction is mainly carried by matrix metalloproteinases (MMPs) secreted from cancer cells or surrounding stromal cells [[25], [26], [27], [28]]. In pancreatic cancer, PSCs are reported to be an important source of MMP2 [26]. However, it has never been observed whether PSCs destroy the basement membrane.
Recent years, organoids, which are three-dimensional (3D) structures, have attracted attention. Tumor organoids can be clonally derived and recreate histoarchitectural heterogeneity observed in matched primary tumors [29]. Moreover, tumor organoids can retain patient-specific physiological changes, including hypoxia, oxygen consumption and epigenetic marks [30]. Therefore, experiments that use organoids in in vitro possibly reflect an environment close to the living body. Also, pancreatic ductal epithelial organoids established from human pluripotent stem cells have BM structures [30]. These data suggest that the PDAC BM structure can be reproduced in vitro, using organoids.
In this study, we established human PDAC organoids from human PDAC resected tissues. We co-cultured PSCs and PDAC organoids in a 3D collagen matrix and observed how the organoid destroyed BM and invaded the collagen matrix, using time-lapse imaging. We also investigated factors involved in BM destruction and elucidated the mechanisms of PSC-induced BM destruction.
Section snippets
Materials and methods
Detailed information is provided in the Supplementary Materials and methods.
Human PDAC organization includes basement membranes
To investigate the presence of PDAC with BM structure, we performed fluorescent immunostaining of PDAC tissue with basal markers and found that the BM stained with Laminin α5 was present when PDAC maintained a duct structure (Fig. 1A). However, the BM structure disappeared when PDAC lost the duct structure and invaded into surround stroma. (Fig. 1B).
Human PDAC organoids include basement membranes
To investigate how the BM of PDAC is destroyed, we established two PDAC organoid lines (PDAC1 and PDAC2) by 3D culture, from specimens resected in
Discussion
Invasive tubular ductal adenocarcinoma is the most frequent histopathological type of pancreatic cancer, and its histopathological features show neoplastic cells forming ductal structures and invading into abundant desmoplastic stroma [14,15,37]. However, how the invasive ductal carcinoma cells with ductal structures invade the surrounding stroma is unclear. In this study, we have shown, for first time, the invasion mechanisms of the invasive ductal adenocarcinoma in pancreatic cancer, using
Funding
This work was supported in part by Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research (B) and (C) and Scientific Research on Innovative Areas (Grant numbers 17H04284, 17K19602, 15K10185, 16K15621,16K10601, 16H05417, 15H04933, 16H05418, 17K19605).
Conflicts of interest
None.
Acknowledgements
The authors thank E. Manabe, S. Sadatomi (Department of Surgery and Oncology, Kyushu University Hospital), and members of the Research Support Center and Department of Anatomic Pathology, Graduate School of Medical Sciences, Kyushu University, for their expert technical assistance. We also thank Edanz Group (www.edanzediting.com/ac) for editing a draft of this manuscript.
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