Knockdown of integrin α5 resulted in significantly increased motility, ANOVA (p = 0.007) while integrin α6 knockdown also increased motility significantly in one siRNA (p = 0.19 and p = 0.004), ANOVA (p = 0.04) (Fig 6B). Figure 6 A. Invasion through matrigel, laminin and fibronectin. B. Motility assay. C. Adhesion assay to matrigel, laminin and fibronectin. D. Anoikis assay of Clone #8 control, treated with scrambled

siRNA, two independent integrin ITGα5 siRNA targets and two integrin ITGα6 target siRNAs. Student’s t -test; p ≤ 0.05*, 0.01**, 0.005***. A slight decrease in adhesion to matrigel and laminin was buy Acalabrutinib observed although not significantly, while a significant reduction in adhesion to fibronectin was observed after integrin α5 siRNA treatment of Clone #8 cells (p = 0.02, p = 0.03), ANOVA (p = 0.02). Adhesion to matrigel and fibronectin was not altered with integrin α6 siRNA treatment; however adhesion to laminin was reduced (p = 0.08 Lazertinib and p = 0.01), ANOVA (p = 0.01) (Fig

6C). No significant change in anoikis response BIX 1294 datasheet was observed after either integrin α5 and α6 siRNA transfection, compared to cells treated with scrambled control (Fig 6D). Discussion One of the most lethal aspects of pancreatic cancer is its early systemic dissemination and tumour progression [24]. The inability to diagnose pancreatic cancer at an early stage has contributed to poor prognosis, as well as the difficulties in treating the metastatic disease. The exact mechanism of pancreatic invasion and metastasis has not been fully elucidated and a better understanding of these processes is essential in treating this disease. To study the inherent heterogeneity of differing sub-populations within a tumour, we isolated isogenic clonal populations from the human pancreatic cell line, MiaPaCa-2, by single CYTH4 cell cloning. Two sub-populations displaying differences in invasion were further analysed to characterise the in vitro invasive phenotype. Clone #3 was characterised as highly invasive and motile with decreased adhesion to ECM proteins. The less invasive Clone #8 displayed increased adhesion

to ECM proteins. Neither clone showed an affinity to collagen type I and IV. Grzesiak et al. [23] previously determined that the parental cell line MiaPaCa-2 does not express collagen-binding integrins α1 and α2, but showed that the cells are metastatic in an orthotopic mouse model and preferentially migrate on laminin-1. Although collagen type IV constitutes the major intrinsic component of the extracellular matrix [25], the ability of the clonal populations in our study to invade or/adhere to matrigel could be due to laminin, another major component of the ECM, and to a lesser extent fibronectin, which represents a significant step in metastasis [26]. Changes in adhesive characteristics, invasion and motility of cells have been suspected to play a role in mediating the spread of malignant cells.