Abstract
Pancreatic ductal adenocarcinoma (PDAC) is characterized by an acidic and fibrotic stroma. The extracellular matrix (ECM) causing the fibrosis is primarily formed by pancreatic stellate cells (PSCs). The effects of the altered biomechanics and pH landscape in the pathogenesis of PDAC, however, are poorly understood. Mechanotransduction in cells has been linked to the function of mechanosensitive ion channels such as Piezo1. Here, we tested whether this channel plays crucial roles in transducing mechanical signals in the acidic PDAC microenvironment. We performed immunofluorescence, Ca2+ influx and intracellular pH measurements in PSCs and complemented them by live-cell imaging migration experiments in order to assess the function of Piezo1 channels in PSCs. We evaluated whether Piezo1 responds to changes of extracellular and/or intracellular pH in the pathophysiological range (pH 6.6 and pH 6.9, respectively). We validated our results using Piezo1-transfected HEK293 cells as a model system. Indeed, acidification of the intracellular space severely inhibits Piezo1-mediated Ca2+ influx into PSCs. In addition, stimulation of Piezo1 channels with its activator Yoda1 accelerates migration of PSCs on a two-dimensional ECM as well as in a 3D setting. Furthermore, Yoda1-activated PSCs transmit more force to the surrounding ECM under physiological pH, as revealed by measuring the dislocation of microbeads embedded in the surrounding matrix. This is paralleled by an enhanced phosphorylation of myosin light chain isoform 9 after Piezo1 stimulation. Intriguingly, upon acidification, Piezo1 activation leads to the initiation of cell death and disruption of PSC spheroids. In summary, stimulating Piezo1 activates PSCs by inducing Ca2+ influx which in turn alters the cytoskeletal architecture. This results in increased cellular motility and ECM traction, which can be useful for the cells to invade the surroundings and to detach from the tissue. However, in the presence of an acidic extracellular pH, although net Ca2+ influx is reduced, Piezo1 activation leads to severe cell stress also limiting cellular viability. In conclusion, our results indicate a strong interdependence between environmental pH, the mechanical output of PSCs and stromal mechanics, which promotes early local invasion of PDAC cells.
Highlights
Pancreatic ductal adenocarcinoma (PDAC) is the most common primary malignant tumor of the exocrine pancreas, and credits for an abysmal prognosis among all forms of cancer
We performed Western blot to investigate Piezo1 expression in whole-cell lysates of cell populations investigated in our study: namely in untransfected HEK293 cells, in HEK293 cells transiently transfected with control plasmid or Piezo1 and in Pancreatic stellate cell (PSC) (Figure 2A)
We found that Piezo1 is abundantly expressed in PSCs, whereas overall Piezo1 was scarcely expressed in both untransfected and transiently transfected HEK293 cells
Summary
Pancreatic ductal adenocarcinoma (PDAC) is the most common primary malignant tumor of the exocrine pancreas, and credits for an abysmal prognosis among all forms of cancer. PDAC is characterized by a collagen-rich desmoplastic stroma that compresses the tumor, which thereby reaches hydrostatic pressure levels of up to 100 mmHg (DuFort et al, 2016). The desmoplastic PDAC microenvironment is markedly acidic reaching even below pH 6.5 (Cruz-Monserrate et al, 2014; Pedersen et al, 2017). In PDAC, the desmoplastic stroma is mainly produced by pancreatic stellate cells (PSCs) (Xue et al, 2018). Activated PSCs have a myofibroblast-like phenotype with expression of markers such as α-smooth muscle actin (αSMA). They are involved in fibrotic remodeling of the extracellular matrix (ECM). PSCs invade the surrounding tissues together with PDAC cells, actively facilitating PDAC metastasis (Liu et al, 2019)
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