Acid-base homeostasis orchestrated by NHE1 defines the pancreatic stellate cell phenotype in pancreatic cancer
Zoltán Pethő, Karolina Najder, Stephanie Beel, Benedikt Fels, Ilka Neumann, Sandra Schimmelpfennig, Sarah Sargin, Maria Wolters, Klavs Grantins, Eva Wardelmann, Miso Mitkovski, Andrea Oeckinghaus, Albrecht Schwab
Zoltán Pethő, Karolina Najder, Stephanie Beel, Benedikt Fels, Ilka Neumann, Sandra Schimmelpfennig, Sarah Sargin, Maria Wolters, Klavs Grantins, Eva Wardelmann, Miso Mitkovski, Andrea Oeckinghaus, Albrecht Schwab
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Research Article Metabolism Oncology

Acid-base homeostasis orchestrated by NHE1 defines the pancreatic stellate cell phenotype in pancreatic cancer

Abstract

Pancreatic ductal adenocarcinoma (PDAC) progresses in an organ with a unique pH landscape, where the stroma acidifies after each meal. We hypothesized that disrupting this pH landscape during PDAC progression triggers pancreatic stellate cells (PSCs) and cancer-associated fibroblasts (CAFs) to induce PDAC fibrosis. We revealed that alkaline environmental pH was sufficient to induce PSC differentiation to a myofibroblastic phenotype. We then mechanistically dissected this finding, focusing on the involvement of the Na+/H+ exchanger NHE1. Perturbing cellular pH homeostasis by inhibiting NHE1 with cariporide partially altered the myofibroblastic PSC phenotype. To show the relevance of this finding in vivo, we targeted NHE1 in murine PDAC (KPfC). Indeed, tumor fibrosis decreased when mice received the NHE1-inhibitor cariporide in addition to gemcitabine treatment. Moreover, the tumor immune infiltrate shifted from granulocyte rich to more lymphocytic. Taken together, our study provides mechanistic evidence on how the pancreatic pH landscape shapes pancreatic cancer through tuning PSC differentiation.

Authors

Zoltán Pethő, Karolina Najder, Stephanie Beel, Benedikt Fels, Ilka Neumann, Sandra Schimmelpfennig, Sarah Sargin, Maria Wolters, Klavs Grantins, Eva Wardelmann, Miso Mitkovski, Andrea Oeckinghaus, Albrecht Schwab

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Figure 1

Environmental alkalization induces myofibroblastic PSC differentiation and proliferation.

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Environmental alkalization induces myofibroblastic PSC differentiation a...
(A) Concept of the working hypothesis. In the healthy pancreas, the marked HCO3– secretion upon each meal results in a distinct stromal acidification, keeping the PSCs in a quiescent nonfibrotic phenotype. Upon malignant transformation in early PDAC (PanIN), ductal secretion decreases, resulting in a relief of the intermittent acidity ( = relative stromal alkalization), leading to a myofibroblastic PSC differentiation. Acidic → alkaline pHe is depicted by yellow → purple colors. (B) Hallmark gene set enrichment analysis (GSEA) of RNA-Seq data from PSCs cultured at pHe7.4 versus pHe6.6 shows the top 5 differentially regulated pathways (n/N = 3/3). (C) A heatmap of RNA-Seq expression mean Z scores computed for published signature genes of immunomodulatory CAFs (left) and myofibroblastic CAFs (right). The gene (rows) Z scores for pHe6.6 and pHe7.4 are color coded. Dark green indicates higher expression Z scores (n/N = 3/3). (D) Immunocytochemistry images of PSCs cultured at pHe6.6 or pHe7.4. The activation marker αSMA (yellow) and the general PSC marker vimentin (magenta) are labeled. Nuclei are stained with DAPI (cyan). Scale bar: 50 μm. (E) Cell areas multiplied by αSMA intensity is taken as a readout for the myofibroblastic PSC phenotype. It is plotted as a function of pHe. Mean values are shown as n ≥ 142 from N = 3 mice. Half-maximum (EC50) pHe-dependent PSC activation occurs at pHe7.0. Note the logarithmic scale of the ordinate. (F) Representative Western blot (top) of p53 and GAPDH of PSCs cultured at pHe6.6 or pHe7.4, with subsequent quantification (bottom) (n/N = 3/3). (G) Representative cell cycle histogram of PSCs cultured at pHe6.6 (red) and pHe7.4 (blue), assessed by flow cytometry with propidium iodide (PI) staining. Cell populations at different stages of the cell cycle are indicated by arrows. (H) The bar chart depicts the percentage of PSCs at the G0/G1 phase of the cell cycle when cultured at pHe 6.6 (red) and pHe7.4 (blue). Data points are npHe6.6 = 3 and npHe7.4 = 5 measurements from n = 3 individual mice. Statistical tests in F and H were performed with 2-tailed unpaired Student’s t tests. A was created with BioRender.com.