Human equilibrative nucleoside transporter-1 knockdown tunes cellular mechanics through epithelial-mesenchymal transition in pancreatic cancer cells.

Yeonju Lee,Dickson K. Kirui,Haifa Shen,Eugene J. Koay,Fazle Hussain,Weijia Zhang,Mauro Ferrari,Lidong Qin,Xin-Yuan Guan

doi:10.1371/journal.pone.0107973

Abstract

We report cell mechanical changes in response to alteration of expression of the human equilibrative nucleoside transporter-1 (hENT1), a most abundant and widely distributed plasma membrane nucleoside transporter in human cells and/or tissues. Modulation of hENT1 expression level altered the stiffness of pancreatic cancer Capan-1 and Panc 03.27 cells, which was analyzed by atomic force microscopy (AFM) and correlated to microfluidic platform. The hENT1 knockdown induced reduction of cellular stiffness in both of cells up to 70%. In addition, cellular phenotypic changes such as cell morphology, migration, and expression level of epithelial-mesenchymal transition (EMT) markers were observed after hENT1 knockdown. Cells with suppressed hENT1 became elongated, migrated faster, and had reduced E-cadherin and elevated N-cadherin compared to parental cells which are consistent with epithelial-mesenchymal transition (EMT). Those cellular phenotypic changes closely correlated with changes in cellular stiffness. This study suggests that hENT1 expression level affects cellular phenotype and cell elastic behavior can be a physical biomarker for quantify hENT1 expression and detect phenotypic shift. Furthermore, cell mechanics can be a critical tool in detecting disease progression and response to therapy.

Highlights

Pancreatic adenocarcinoma (PDAC) is one of the most lethal human cancers with an extremely poor prognosis [1]
Cell stiffness affects the extent of cantilever deflection upon interaction with the surface of adherent cells [19]
A constant Young’s modulus of Panc 03.27 cells was obtained within the indentation forces up to 200 pN, indicating the measurement of cellular stiffness using atomic force microscopy (AFM) is only valid at small deformations of the living cells

Summary

Introduction

Pancreatic adenocarcinoma (PDAC) is one of the most lethal human cancers with an extremely poor prognosis [1]. Gemcitabine (29,29difluorodeoxycytidine) is one of efficient anticancer agents for pancreatic cancer [1]. It is a cytotoxic pyrimidine deoxynucleoside analogue that is transported into the cellular compartment through the primary transport protein, human equilibrative nucleoside transporter-1 (hENT1), and eventually inhibits DNA replication. The hENT1 expression level in pancreatic cancer cells has previously been correlated to therapeutic efficacy where cells with higher hENT1 expression were shown to respond better to gemcitabine. Cellular level studies have shown that pancreatic cancer cells with low hENT1 expression are highly resistant to gemcitabine [4]. Clinical studies have established that hENT1 expression affect how patients respond to treatment where patients whose tumor expressed low hENT1 biomarker responded poorly to gemcitabine therapy [3,5]

Methods

Results

Conclusion