Original ArticlesHypoxic stellate cells of pancreatic cancer stroma regulate extracellular matrix fiber organization and cancer cell motility
Introduction
The prognosis of pancreatic cancer remains dismal with a 5-year survival rate of 6% despite considerable progress in understanding its underlying genetic and molecular events [1]. A characteristic feature of pancreatic cancer is a dense desmoplastic stroma, which plays a crucial role in tumor aggressiveness and therapeutic resistance [2], [3]. The desmoplastic microenvironment rich in extracellular matrix (ECM) is mainly produced by the activated phenotype of pancreatic stellate cells (PSCs). Activated PSCs produce paracrine growth factors, proteolytic enzymes, and ECM components, which promote proliferation, migration, and invasion of cancer cells. Moreover, ECM in the tumor-associated stroma creates a ‘fortress-like’ hypovascular barrier that impairs the delivery of chemotherapeutics [2], [4], [5], [6].
Preclinical models have demonstrated the benefit of stromal depletion through blockade of paracrine Hedgehog signaling to improve drug delivery [4]; nonetheless, subsequent clinical trials targeting stromal desmoplasia in pancreatic cancer have failed due to paradoxical accelerated disease progression [7]. Recent experimental evidence provides insight into the failure of antistromal therapy in clinical trials, showing that stromal depletion may increase tumor aggressiveness and spread [8], [9], implying that tumor stroma may be restrictive rather than supportive of tumor growth. Consequently, these studies strongly suggest that instead of aiming to ablate desmoplasia, a more effective approach is needed for targeting pancreatic cancer stroma.
The ECM in tumor stroma plays roles in biochemical and biomechanical interactions with cells that are required for key cellular events. Several groups have observed that tumor cells preferentially invade along aligned collagen fibers [10], [11], [12]. Recent evidence indicates that ECM architecture as well as composition is altered in cancer stroma and that these changes may accelerate tumor progression [13], [14]. Rho-mediated alignment of dense collagen fibers perpendicular to the tumor boundary promotes invasion, whereas reticular collagen surrounding mammary glands restrains it [15]. However, the contribution of these stromal modifications to tumor progression and the genetic and molecular mechanisms underlying these alterations remain elusive in pancreatic cancer.
Pancreatic cancer contains an area of hypoxia, which has also been proposed as an important microenvironmental factor for tumor progression [16], [17]. Hypoxia in pancreatic cancer is formed partly through fibrogenic effects of PSCs [18], [19] as well as a result of inefficient tumor vascular supply and a high metabolic need for oxygen. Recent studies suggest that hypoxic conditions concomitantly exist in pancreatic cancer cells and surrounding stroma [20], [21]. PSCs in pancreatic cancer stroma respond to hypoxia by increasing hypoxia-inducible factor-1α (HIF-1α) protein level and producing soluble factors and ECM components [19], [22], whereas the effect of hypoxia in PSCs within tumor desmoplasia on ECM mechanical properties such as fiber alignment has not been reported.
In this study, we explored the possibility that hypoxia may be causally involved in the biomechanical properties of PSC-derived tumor stroma. In PSC-derived cancer stroma, hypoxia regulated ECM fiber architecture. We further showed that ECM produced by PSCs under hypoxia facilitated the directional migration of pancreatic cancer cells, and we link this activity to the parallel fiber architecture.
Section snippets
Patients and pancreatic tissues
Pancreatic cancer tissues were obtained from patients who underwent pancreatic resection at our institution. The study was approved by the Ethics Committee of Kyushu University and conducted according to the Ethical Guidelines for Human Genome/Gene Research enacted by the Japanese Government and the Helsinki Declaration.
Cells and culture conditions
PSCs were established in our laboratory from fresh pancreatic cancer surgical specimens using the outgrowth method [23], [24]. The isolated cells were identified as PSCs by
Generation of 3-D matrices derived from PSCs
PSCs within tumor-associated stroma produce ECM rich in fibronectin and type I collagen during desmoplasia [24]. To model stromal ECM of pancreatic cancer in vitro, we generated cell-free 3-D matrices from PSCs. PSCs were established from human pancreatic cancer tissue, and we confirmed α-SMA and GFAP expressions, which are known markers of PSCs [23], [25] (Figs. 1A and S1A). The cells were maintained in a confluent state under culture conditions for 5–6 days to obtain in vivo-like
Discussion
In this study, we found that 3-D matrices derived from PSCs under hypoxia presented higher organization of ECM fibers when compared with those derived under normoxia, leading to enhanced directional migration of cancer cells. We also determined a potential molecular mechanism underlying the enhanced organization of parallel ECM fibers and found that collagen-modifying enzyme PLOD2 in PSCs remodels the ECM through increased fiber patterned orientation. Thus, the data suggest that hypoxia in
Funding
This work was supported in part by the Ministry of Education, Culture, Sports, Science, and Technology of Japan (MEXT; KAKEN grants 26108010, 26293305, 15H04933, and 25293285).
Conflict of interest
The authors declare no conflict of interest.
Acknowledgments
The authors thank E. Manabe, S. Sadatomi, M. Ohmori (Department of Surgery and Oncology, Kyushu University Hospital), and members of Research Support Center and Department of Anatomic Pathology, Graduate School of Medical Sciences, Kyushu University, for their expert technical assistance.
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