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 2

NHE1-mediated pH recovery is inhibited by cariporide in PSCs.

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NHE1-mediated pH recovery is inhibited by cariporide in PSCs. (A) Volcan...
(A) Volcano plot analysis of ion transporter genes (GO:0015075) derived from the RNA-Seq data of PSCs cultured at pHe7.4 and pHe6.6 (n/N = 3/3). Genes indicated by red (n = 43) and blue (n = 44) dots highlighted in rectangles are upregulated at pHe6.6 and pHe7.4, respectively. (B) Subsequent qPCR validation of ion transporter gene expression levels by the 2–ΔCT method compared with the housekeeping genes Ywhaz and 18s rRNA. Bar charts show mean expression of genes from freshly isolated quiescent PSCs (0 hours, white) and PSCs cultured at pHe7.4 (blue) or pHe6.6 (red) (n/N = 6/3). (C) Representative immunofluorescence images showing the cellular localization of NHE1 in freshly isolated quiescent PSCs (0 hours), PSCs cultured at pHe7.4 or pHe6.6 for 120 hours, or vehicle-treated KPfC-derived PSCs (PDAC-PSC) (NHE1: magenta; DAPI: blue). Scale bar: 10 μm. (D) NHE1 Western blots are shown, with the top bands at 100 kDa corresponding to the glycosylated NHE1, whereas bands with lower molecular weight (80 kDa) correspond to the unglycosylated NHE1. Lysates are from N = 3 mice each. (E) pHi recordings of WT PSCs cultured at pHe6.6 (left) and pHe7.4 (middle) or KPfC-derived CAFs (cultured at pHe7.4; right). pHi was acidified temporarily by applying the NH4+ prepulse (*) technique. NHE1-independent pHi recovery starts when pHi has reached its minimum in the presence of the Na+-free solution (“0 Na+”). NHE1-dependent pHi recovery can be observed in the last step (Ctrl) of the experiment when cariporide was added to the Na+-containing superfusion as indicated. Lines show mean pHi of npH6.6 = 35, npH6.6+CARI = 39, npH7.4 = 45, npH7.4+CARI = 68, nPDAC-CAF = 11, and nPDAC-CAF+CARI = 22 cells from N = 3 mice each. (F) Quantification of resting pHi of PSCs cultured at pHe6.6 (red) or pHe7.4 (blue) and CAFs (purple), respectively, derived from E (npH6.6 = 74, npH7.4 = 113, and nPDAC-CAF+CARI = 41 cells from N = 3 mice each). (G) Scatter plot depicts the rate of Na+-independent pHi recovery of PSCs cultured at pHe6.6 (red), pHe7.4 (blue), or CAFs (purple), derived from E (n/N see F). (H) Comparison of the rate of Na+-dependent pHi recovery of WT PSCs cultured at pHe6.6 (red) or pHe7.4 (blue), or CAFs (purple) as explained in E (n/N see E). Statistical tests in F–H were performed with 1-way ANOVA with Tukey’s post hoc test.