Luminescence-based Imaging of intra- and extracellular tumor-pH and investigation of the impact of pH on tumor cell proliferation and migration in vitro

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During the first grant period, we developed pH-sensitve nanoparticles, tumor cell models, and automated impedance-based migration assays. In the next step, we aim to systematically study the impact of altered intra- and extracellular pH (pHi and pHe) on cell proliferation and migration of tumor cells. Additionally, the experiments will not only cover 2D monolayer models, but also 3D models (multicellular spheroids) to also study the impact of transmembraneous pH-gradients on proliferation and cell dynamics. At the end of the project, we hope to have new insights into the role of pHi and pHe in tumor biology. Furthermore, we also aim to study two molecular aspects concerning the impact of pHe and pHi: (i) the role of a pivotal proton transporter (NHE1), which is substantially involved in generating the pH-gradient over the cell membrane, and (ii) a possible role of proton-sensitive GPCRs in the signal transduction of decreased pHe to intracellular pathways. Therefore, we will use CRISPR/Cas-generated NHE1-knockout tumor cell lines or pharmacological inhibition of NHE1. We will then study the impact of pHe/pHi on cell proliferation and migration. The impact of proton-sensitive GPCRs will be studied using the few known antagonists (e.g. Cu2+) and also via impedimetric analysis after a pulsed decrease in pHe. These results will mechanistically be compared to a direct influence of cell-cell- as well as cell-matrix interactions induced by pH changes. These in vitro studies will be accompanied by high-resolution imaging of tumor pHe in mouse models with the dorsal skinfold chamber. We also aim to correlate the respective in vivo and in vitro data.