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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, Ca 2+ 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 Ca 2+ 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 Ca 2+ 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 Ca 2+ 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.

Tissue acidosis plays a pivotal role in tumor progression: in particular, interstitial acidosis promotes tumor cell invasion, and is a major contributor to the dysregulation of tumor immunity and tumor stromal cells. The cell membrane and integral membrane proteins commonly act as important sensors and transducers of altered pH. Cell adhesion molecules and cation channels are prominent membrane proteins, the majority of which is regulated by protons. The pathophysiological consequences of proton-sensitive ion channel function in cancer, however, are scarcely considered in the literature. Thus, the main focus of this review is to highlight possible events in tumor progression and tumor immunity where the pH sensitivity of cation channels could be of great importance.

Neutrophil granulocytes are exposed to widely varying microenvironmental conditions when pursuing their physiological or pathophysiological functions such as fighting invading bacteria or infiltrating cancer tissue. Examples for harsh environmental challenges include among others mechanical shear stress during the recruitment from the vasculature or the hypoxic and acidotic conditions within the tumor microenvironment. Chemokine gradients, reactive oxygen species, pressure, matrix elasticity, and temperature can be added to the list of potential challenges. Transient receptor potential (TRP) channels serve as cellular sensors since they respond to many of the abovementioned environmental stimuli. The present review investigates the role of TRP channels in neutrophil granulocytes and their role in regulating and adapting neutrophil function to microenvironmental cues. Following a brief description of neutrophil functions, we provide an overview of the electrophysiological characterization of neutrophilic ion channels. We then summarize the function of individual TRP channels in neutrophil granulocytes with a focus on TRPC6 and TRPM2 channels. We close the review by discussing the impact of the tumor microenvironment of pancreatic ductal adenocarcinoma (PDAC) on neutrophil granulocytes. Since neutrophil infiltration into PDAC tissue contributes to disease progression, we propose neutrophilic TRP channel blockade as a potential therapeutic option.

Pancreatic ductal adenocarcinoma is a devastating disease with a dismal prognosis. Therapeutic interventions are largely ineffective. A better understanding of the pathophysiology is required. Ion channels contribute substantially to the “hallmarks of cancer.” Their expression is dysregulated in cancer, and they are “misused” to drive cancer progression, but the underlying mechanisms are unclear. Ion channels are located in the cell membrane at the interface between the intracellular and extracellular space. They sense and modify the tumor microenvironment which in itself is a driver of PDAC aggressiveness. Ion channels detect, for example, locally altered proton and electrolyte concentrations or mechanical stimuli and transduce signals triggered by these microenvironmental cues through association with intracellular signaling cascades. While these concepts have been firmly established for other cancers, evidence has emerged only recently that ion channels are drivers of PDAC aggressiveness. Particularly, they appear to contribute to two of the characteristic PDAC features: the massive fibrosis of the tumor stroma (desmoplasia) and the efficient immune evasion. Our critical review of the literature clearly shows that there is still a remarkable lack of knowledge with respect to the contribution of ion channels to these two typical PDAC properties. Yet, we can draw parallels from ion channel research in other fibrotic and inflammatory diseases. Evidence is accumulating that pancreatic stellate cells express the same “profibrotic” ion channels. Similarly, it is at least in part known which major ion channels are expressed in those innate and adaptive immune cells that populate the PDAC microenvironment. We explore potential therapeutic avenues derived thereof. Since drugs targeting PDAC-relevant ion channels are already in clinical use, we propose to repurpose those in PDAC. The quest for ion channel targets is both motivated and complicated by the fact that some of the relevant channels, for example, KCa3.1, are functionally expressed in the cancer, stroma, and immune cells. Only in vivo studies will reveal which arm of the balance we should put our weights on when developing channel-targeting PDAC therapies. The time is up to explore the efficacy of ion channel targeting in (transgenic) murine PDAC models before launching clinical trials with repurposed drugs.

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Neutrophil granulocytes are the first and robust responders to the chemotactic molecules released from an inflamed acidic tissue. The aim of this study was to elucidate the role of microenvironmental pH in neutrophil chemotaxis. To this end, we used neutrophils from male C57BL/6J mice and combined live cell imaging chemotaxis assays with measurements of the intracellular pH (pHi) in varied extracellular pH (pHe). Observational studies were complemented by biochemical analyses of leukotriene B4 (LTB4) production and activation of the Cdc42 Rho GTPase. Our data show that pHi of neutrophils dose-dependently adapts to a given pH of the extracellular milieu. Neutrophil chemotaxis toward C5a has an optimum at pHi ∼7.1, and its pHi dependency is almost parallel to that of LTB4 production. Consequently, a shallow pHe gradient, resembling that encountered by neutrophils during extravasation from a blood vessel (pH ∼7.4) into the interstitium (pH ∼7.2), favors chemotaxis of stimulated neutrophils. Lowering pHe below pH 6.8, predominantly affects neutrophil chemotaxis, although the velocity is largely maintained. Inhibition of the Na+/H+ exchanger 1 (NHE1) with cariporide drastically attenuates neutrophil chemotaxis at the optimal pHi irrespective of the high LTB4 production. Neutrophil migration and chemotaxis are almost completely abrogated by inhibiting LTB4 production or blocking its receptor (BLT1). The abundance of the active GTP-bound form of Cdc42 is strongly reduced by NHE1 inhibition or pHe 6.5. In conclusion, we propose that the pH dependence of neutrophil chemotaxis toward C5a is caused by a pHi-dependent production of LTB4 and activation of Cdc42. Moreover, it requires the activity of NHE1.

Here we report a novel role for TRPC6, a member of the transient receptor potential (TRPC) channel family, in the CXCL1-dependent recruitment of murine neutrophil granulocytes. Representing a central element of the innate immune system, neutrophils are recruited from the blood stream to a site of inflammation. The recruitment process follows a well-defined sequence of events including adhesion to the blood vessel walls, migration, and chemotaxis to reach the inflammatory focus. A common feature of the underlying signaling pathways is the utilization of Ca 2+ ions as intracellular second messengers. However, the required Ca 2+ influx channels are not yet fully characterized. We used WT and TRPC6 −/− neutrophils for in vitro and TRPC6 −/− chimeric mice (WT mice with WT or TRPC6 −/− bone marrow cells) for in vivo studies. After renal ischemia and reperfusion injury, TRPC6 −/− chimeric mice had an attenuated TRPC6 −/− neutrophil recruitment and a better outcome as judged from the reduced increase in the plasma creatinine concentration. In the cremaster model CXCL1-induced neutrophil adhesion, arrest and transmigration were also decreased in chimeric mice with TRPC6 −/− neutrophils. Using atomic force microscopy and microfluidics, we could attribute the recruitment defect of TRPC6 −/− neutrophils to the impact of the channel on adhesion to endothelial cells. Mechanistically, TRPC6 −/− neutrophils exhibited lower Ca 2+ transients during the initial adhesion leading to diminished Rap1 and β 2 integrin activation and thereby reduced ICAM-1 binding. In summary, our study reveals that TRPC6 channels in neutrophils are crucial signaling modules in their recruitment from the blood stream in response to CXCL1. Key point Neutrophil TRPC6 channels are crucial for CXCL1-triggered activation of integrins during the initial steps of neutrophil recruitment.