Cancer Letters

Cancer Letters

Volume 425, 1 July 2018, Pages 65-77
Cancer Letters

Original Articles
Basement membrane destruction by pancreatic stellate cells leads to local invasion in pancreatic ductal adenocarcinoma

https://doi.org/10.1016/j.canlet.2018.03.031Get rights and content

Highlights

  • Pancreatic ductal carcinoma (PDAC) organoid reconstruct basement membrane (BM).

  • Pancreatic stellate cells (PSCs) induced BM destruction of PDAC organoid.

  • BM destruction of PDAC organoids is induced by direct contact of PSCs.

  • MMP2 and MT1MMP expression in PSCs induced BM destruction of PDAC organoid.

Abstract

Stroma invasion is an important step in pancreatic cancer progression. However, how pancreatic ductal adenocarcinoma (PDAC) with ductal structure invades the surrounding stroma has not been clear. Here, we elucidated the mechanism of stromal invasion of PDAC, using organoids. From resected PDAC specimens, we established human PDAC organoids, which developed ductal and basement membrane (BM) structures. When the organoids were co-cultured with pancreatic stellate cells (PSCs) in a collagen matrix, organoids lost their BM and ductal structures, and invaded collagen matrix more frequently than did mono-cultured organoids. Interestingly, direct contact by PSCs to PDAC organoids was observed before BM destruction. Matrix metalloproteinase (MMP) 2 or membrane type-1 MMP (MT1MMP) knockdown in PSCs significantly attenuated BM destruction by PSCs, and retained the ductal structures in organoids. Our results imply that direct contact by PSCs induces BM destruction and stromal invasion of PDAC via MMP2 which binds to MT1MMP on PSCs.

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.

References (51)

  • V. Thakur et al.

    The membrane tethered matrix metalloproteinase MT1-MMP at the forefront of melanoma cell invasion and metastasis

    Pharmacol. Res.

    (2016)
  • J. Cathcart et al.

    Targeting matrix metalloproteinases in cancer: bringing new life to old ideas

    Genes Dis

    (2015)
  • S. Ingvarsen et al.

    Targeting a single function of the multifunctional matrix metalloprotease MT1-MMP Impact on Lymphangiogenesis

    J. Biol. Chem.

    (2013)
  • M. Erkan

    The role of pancreatic stellate cells in pancreatic cancer

    Pancreatology

    (2013)
  • B.C. Özdemir et al.

    Depletion of carcinoma-associated fibroblasts and fibrosis induces immunosuppression and accelerates pancreas cancer with reduced survival

    Canc. Cell

    (2014)
  • R.L. Siegel et al.

    Cancer statistics

    CA A Cancer J. Clin.

    (2016)
  • M. Erkan et al.

    StellaTUM: current consensus and discussion on pancreatic stellate cell research

    Gut

    (2012)
  • A. Neesse et al.

    Stromal biology and therapy in pancreatic cancer: a changing paradigm

    Gut

    (2015)
  • M.G. Bachem et al.

    Pancreatic stellate cells—role in pancreas cancer

    Langenbeck's Arch. Surg.

    (2008)
  • R.F. Hwang et al.

    Cancer-associated stromal fibroblasts promote pancreatic tumor progression

    Canc. Res.

    (2008)
  • A. Vonlaufen et al.

    Pancreatic stellate cells: partners in crime with pancreatic cancer cells

    Canc. Res.

    (2008)
  • N. Ikenaga et al.

    CD10+ pancreatic stellate cells enhance the progression of pancreatic cancer

    Gastroenterology

    (2010)
  • S. Endo et al.

    Autophagy is required for activation of pancreatic stellate cells, associated with pancreatic cancer progression and promotes growth of pancreatic tumors in mice

    Gastroenterology

    (2017)
  • J. Kleeff et al.

    Pancreatic cancer

    Nat. Rev. Dis. Prim

    (2016)
  • R. Kalluri

    Basement membranes: structure, assembly and role in tumour angiogenesis

    Nat. Rev. Canc.

    (2003)
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