Exosomes derived from pancreatic cancer cells induce activation and profibrogenic activities in pancreatic stellate cells

https://doi.org/10.1016/j.bbrc.2017.10.141Get rights and content

Highlights

  • Effects of cancer cell-derived exosomes on stellate cell functions were examined.

  • Exosomes increased proliferation and migration in stellate cells.

  • Cancer cells increased miR-1246 and miR-1290 expression in stellate cells.

  • Exosomes and miR-1290 overexpression increased the expression of profibrogenic genes and ACTA2.

  • Exosome-mediated cell-to-cell communication regulates stellate cell functions.

Abstract

Pancreatic cancer cells (PCCs) interact with pancreatic stellate cells (PSCs), which play a pivotal role in pancreatic fibrogenesis, to develop the cancer-conditioned tumor microenvironment. Exosomes are membrane-enclosed nanovesicles, and have been increasingly recognized as important mediators of cell-to-cell communications. The aim of this study was to clarify the effects of PCC-derived exosomes on cell functions in PSCs. Exosomes were isolated from the conditioned medium of Panc-1 and SUIT-2 PCCs. Human primary PSCs were treated with PCC-derived exosomes. PCC-derived exosomes stimulated the proliferation, migration, activation of ERK and Akt, the mRNA expression of α-smooth muscle actin (ACTA2) and fibrosis-related genes, and procollagen type I C-peptide production in PSCs. Ingenuity pathway analysis of the microarray data identified transforming growth factor β1 and tumor necrosis factor as top upstream regulators. PCCs increased the expression of miR-1246 and miR-1290, abundantly contained in PCC-derived exosomes, in PSCs. Overexpression of miR-1290 induced the expression of ACTA2 and fibrosis-related genes in PSCs. In conclusion, PCC-derived exosomes stimulate activation and profibrogenic activities in PSCs. Exosome-mediated interactions between PSCs and PCCs might play a role in the development of the tumor microenvironment.

Introduction

Pancreatic cancer is the deadliest major cancer with a 5-year survival rate of 8% in the United States [1], characterized by difficulty in the early diagnosis, rapid progression, and limited response to conventional therapy [2]. Pancreatic cancer displays an extensive stromal reaction accounting for up to 90% of the tumor volume [2], [3]. It remains controversial whether the dense stroma drives the progression of pancreatic cancer or acts as a defense against pancreatic cancer [3], [4], [5]. The stroma might stimulate the aggressive behaviors of pancreatic cancer cells (PCCs) and help them escape from host immune-surveillance. On the other hand, the stroma might provide a barrier limiting the dissemination and metastasis of PCCs. Another aspect of the stroma is the very low blood perfusion and hypoxia. The dense stroma impairs drug delivery by several mechanisms such as providing a physical barrier, high interstitial pressure, compressed vessels, and a less leaky phenotype of pericytes [3], [6]. It has been established that α-smooth muscle actin (SMA)-positive pancreatic stellate cells (PSCs) play a pivotal role in cancer-associated pancreatic fibrogenesis [7], [8], [9], [10], [11], [12]. Previous studies have suggested that PSCs might promote the progression of pancreatic cancer through dynamic interactions with PCCs [7], [8], [9], [10], [11], [12]. Cytokines, growth factors and extracellular matrix components appear to regulate the crosstalk between these cell types [7], [8], [9], [10], [11], [12], [13], but the whole picture of the underlying mechanisms remains to be clarified.

It has been increasingly recognized that extracellular vesicles including exosomes are important mediators of cell-to-cell communications [14], [15], [16]. Exosomes are membrane-enclosed nanovesicles containing diverse host cell-derived bioactive molecules including proteins, lipids, and microRNAs (miRNAs) [14], [15], [16]. These constituents are transported in the exosomes to regulate the gene expression and cell functions in recipient cells. Recent studies have demonstrated the importance of exosomes in the regulation of physiological processes such as tissue repair and immune surveillance [17], [18]. Exosomes play a pivotal role in the pathogenesis of diseases including cancer [19], [20], [21]. By secreting exosomes, cancer cells reprogram cells in the tumor microenvironment and at distant sites to support the progression of cancer [19], [20], [21]. In addition, exosomes have been proposed as a source of biomarkers in bodily fluids [22], [23]. PCCs secrete large amount of exosomes, and previous studies demonstrated several actions of PCC-derived exosomes on other cell types including β-cells and immune cells [24], [25], [26], [27], [28]. The exosome-mediated communications contribute to optimize conditions for the growth and metastasis of PCCs. However, the effects of PCC-derived exosomes on PSCs remain unknown. This study aimed to clarify the effects of PCC-derived exosomes on the cell functions of PSCs.

Section snippets

Materials

Rabbit anti-CD63 antibody (#sc-15363) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit anti-CD81 antibody (#ab79559) was obtained from Abcam (Cambridge, MA). Mouse anti-GM130 antibody (#610822) was obtained from BD Biosciences (San Jose, CA). Other antibodies were purchased from Cell Signaling Technologies (Beverly, MA). Cell culture plates, dishes, and culture inserts were purchased from Thermo Fisher Scientific (Waltham, MA). Reagents and instruments for quantitative

Isolation of exosomes from pancreatic cancer cells

Exosomes were isolated from the CM of Panc-1 cells. Under transmission electron microscopy, the size of isolated vesicles ranged from 30 to 120 nm (Fig. 1A and B), corresponding to the reported size of exosomes [14], [15]. The isolated pellet was positive for the exosomal markers CD63 and CD81 [14], [15], but negative for a Golgi membrane protein, GM130 (Fig. 1C). Negative expression of GM130 excluded major contamination from cellular proteins. Similar results were obtained by the analysis of

Discussion

In this study, we showed that PCC-derived exosomes induced the activation and profibrogenic activities in PSCs, including proliferation, migration, collagen production, and α-SMA (ACTA2) mRNA expression. To our knowledge, this is the first study showing the actions of PCC-derived exosomes on human PSCs. Recent studies have shown a role of PCC-derived exosomes in the progression and associated disorders in pancreatic cancer [24], [25], [26], [27], [28]. For example, PCC-derived exosomes play a

Disclosure

The authors declare no conflict of interest.

Financial supports

This study was supported in part by JSPS (KAKENHI) (26293171, 26461029, 15H04804), the Mitsui Life Social Welfare Foundation (to A. Masamune), the Smoking Research Foundation (to A. Masamune), and the Pancreas Research Foundation of Japan (to N. Yoshida).

Acknowledgements

The authors are grateful to Shizuka Aoki for the excellent technical assistance. We also acknowledge the technical support of the Biomedical Research Core of Tohoku University Graduate School of Medicine.

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