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 4

The myofibroblastic phenotype of activated PSCs is partially reversed by cariporide but not by acidic pHe alone.

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The myofibroblastic phenotype of activated PSCs is partially reversed by...
(A) After culturing PSCs at pHe6.6 for 72 hours, medium was changed to pHe6.6 (Resting) or to pHe7.4 (PanIN-like) for another 72 hours. Lastly, the medium of pHe7.4-incubated cells was reacidified to pHe6.6 for another 72 hours (PDAC-like). Representative images of PSCs stained for αSMA (yellow), vimentin (magenta), and DAPI (cyan) are shown below each condition. Scale bar: 50 μm. (B) Scatter plot shows cell areas multiplied by αSMA fluorescence staining intensity (logarithmic scale) under conditions described in A. nResting = 100, nPanIN-like = 64, and nPDAC-like = 94; N = 3 mice. (C) Immunocytochemistry of PDAC-like PSCs in the absence (left) or presence (right) of cariporide (CARI). Scale bar: 50 μm. (D) Scatter plot shows cell areas multiplied by αSMA fluorescence intensity (logarithmic scale) under conditions described in C. nPDAC-like = 74, nPDAC-like+CARI = 74; N = 3 mice. (E) Intracellular pH measurements of PSCs where culture medium is reacidified after activation (pHe7.4 → pHe6.6, PDAC-like). Intracellular pH was acidified temporarily by applying the NH4+ prepulse technique, as shown in Figure 2. NHE1-dependent pH recovery can be observed when cells are superfused with Na+-containing solution (Ctrl) without (black) or with cariporide (red). Lines indicate the mean pHi of nPDAC-like = 35, nPDAC-like+CARI = 79 cells from N = 3 mice. (F) Comparison of the rate of Na+-dependent recovery of PSCs in the absence or presence of cariporide derived from E. (G) Illustration of extended working hypothesis. In manifest PDAC, acidic pHe fails to acidify pHi because of NHE1-mediated H+ extrusion (left). Therefore, PSCs and CAFs remain myofibroblastic, ultimately promoting tumor desmoplasia. However, upon NHE1 inhibition with cariporide, PSCs and CAFs fail to counterbalance the acid stress, which disrupts the myofibroblastic phenotype (right). Statistical comparison in B was performed with 1-way ANOVA with Tukey’s post hoc test, whereas in D and F, statistical comparisons were performed with 2-tailed unpaired Student’s t tests. G was created with BioRender.com.