Hypoxic stellate cells of pancreatic cancer stroma regulate extracellular matrix fiber organization and cancer cell motility

Highlights

• Pancreatic stellate cells create parallel ECM fiber architecture under hypoxia.

• ECM fibers under hypoxia enhance directionality of cancer cell movement.

• Hypoxia-induced PLOD2 regulates topographical organization of ECM fibers.

• ECM remodeling by PLOD2 promotes directional migration of pancreatic cancer cells.

Abstract

Desmoplasia and hypoxia in pancreatic cancer mutually affect each other and create a tumor-supportive microenvironment. Here, we show that microenvironment remodeling by hypoxic pancreatic stellate cells (PSCs) promotes cancer cell motility through alteration of extracellular matrix (ECM) fiber architecture. Three-dimensional (3-D) matrices derived from PSCs under hypoxia exhibited highly organized parallel-patterned matrix fibers compared with 3-D matrices derived from PSCs under normoxia, and promoted cancer cell motility by inducing directional migration of cancer cells due to the parallel fiber architecture. Microarray analysis revealed that procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2) in PSCs was the gene that potentially regulates ECM fiber architecture under hypoxia. Stromal PLOD2 expression in surgical specimens of pancreatic cancer was confirmed by immunohistochemistry. RNA interference-mediated knockdown of PLOD2 in PSCs blocked parallel fiber architecture of 3-D matrices, leading to decreased directional migration of cancer cells within the matrices. In conclusion, these findings indicate that hypoxia-induced PLOD2 expression in PSCs creates a permissive microenvironment for migration of cancer cells through architectural regulation of stromal ECM in pancreatic cancer.

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.