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

在第一批资助期间，我们开发了pH敏感纳米颗粒，肿瘤细胞模型和基于阻抗的自动迁移分析。在下一步中，我们的目标是系统地研究改变细胞内和细胞外pH值（pHi和pHe）对肿瘤细胞增殖和迁移的影响。此外，实验将不仅涵盖2D单层模型，而且还涵盖3D模型（多细胞球体），以研究跨膜pH梯度对增殖和细胞动力学的影响。在项目结束时，我们希望对pHi和pHe在肿瘤生物学中的作用有新的见解。此外，我们还旨在研究两个分子方面的影响，pHe和pHi：（i）一个关键的质子转运蛋白（NHE 1），这是实质上参与产生的pH梯度的细胞膜上的作用，和（ii）质子敏感的GPCR在信号转导的pHe降低细胞内途径的可能作用。因此，我们将使用CRISPR/Cas产生的NHE 1敲除肿瘤细胞系或NHE 1的药理学抑制。然后，我们将研究pHe/pHi对细胞增殖和迁移的影响。质子敏感性GPCR的影响将使用几种已知的拮抗剂（例如Cu 2+）进行研究，并在pHe脉冲降低后进行阻抗分析。将这些结果与pH变化诱导的细胞-细胞以及细胞-基质相互作用的直接影响进行机械比较。这些体外研究将伴随着背侧皮褶腔小鼠模型中肿瘤pHe的高分辨率成像。我们还旨在将各自的体内和体外数据相关联。

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.