Influence of long-term acid adaptation and acute microenvironmental pH changes on Pancreatic Ductal Adenocarcinoma (PDAC) development, progression and invasion
by Jakub Mitrega
Date of Examination:2023-06-09
Date of issue:2023-08-16
Advisor:Prof. Dr. Frauke Alves
Referee:Prof. Dr. Frauke Alves
Referee:Dr. Ivan Prof Bogeski
Files in this item
Name:Jakub Mitrega thesis.pdf
This file will be freely accessible after 2024-06-08.
EnglishPancreatic ductal adenocarcinoma (PDAC) is one of the most malignant cancers worldwide and it is predicted to be the second leading cause of cancer-related deaths by 2030. PDAC development is strictly linked to the microenvironmental acidosis and p53 mutation. The aim of my project was to determine whether adaptation to acidity and acute extracellular pH (pHe) changes together with the p53 mutation affected PDAC related processes. Here I applied in vitro cell based assays and in vivo PDAC mouse models that were performed with Panc02 murine cells either expressing wild type p53 gene (lately named Panc02WT) or bearing p53 knockout mutation (lately named Panc02KO). These cells grew in 7.4 pHe (Control 7.4) and part of them underwent gradual adaptation to acidic pHe 6.7 (Adapted 6.7). For the in vitro part, in order to establish if particular features are affected by chronic acidosis or extracellular pH (pHe), the media for Control and Adapted Panc02 cells were acutely changed to 6.7 and 7.4 pHe, respectively. The obtained results from in vitro experiments show that acid adaptation increases adhesion and mobility of Panc02 cells, even more enhanced with p53 inactivation. Moreover, intracellular pH (pHi) of Adapted 6.7 cells increases in physiological pH together with elevated glycolysis as well as oxidative phosphorylation. In order to obtain more insight on glucose and mitochondrial metabolism, in vivo MRI measurements in combination with hyperpolarized pyruvate (Pyr) as contrast agent were performed on tumor bearing mice. Results showed that there is no difference in the production on lactate and alanine within the pancreatic tumors between the Panc02WT Adapted and Control group. To explore the effects of adaptation to acidity and p53 protein function on tumor development, in vivo studies were performed. Results revealed smaller tumor formation for Panc02WT Adapted cells when compared to Control, which was associated with extensive infiltration of CD45 positive immune cells. Implantation of p53 knockout cells manifested in enlarged tumor volumes and more aggressive tumor growth, observed for both Panc02KO Control and Adapted tumors. Additionally, reduced immune cell penetration and higher collagen content at the tumor border was observed in Panc02KO cells when compared to Panc02WT counterparts. Lastly, strong synergistic effect of acid adaptation and p53 inactivation manifested in higher metastatic score for Panc02KO cells when compared to Panc02WT counterparts in vivo. Chemotherapeutic treatment efficacy was assessed in vitro in the context of chronic and acute acidosis. The results showed that low pHe diminishes Panc02WT and Panc02KO cell response to gemcitabine (GEM) but not to paclitaxel (PTX). To expand these observations, in vivo studies revealed lower sensitivity of Panc02KO tumors to GEM, manifested in tumor size enlargement and increased aggressive behavior after GEM treatment when compared to Panc02WT. Histological analysis showed higher amount of collagen in Panc02KO Adapted tumor borders, when compared to Panc02WT Control and Adapted and Panc02KO Control tumors. In addition, the analysis of RNA sequencing data showed differences in expression patterns of various pathways between Panc02WT and Panc02KO cancer cells that were adapted to an acidic environment, as compared to Control cells. Specifically, genes enriched for signatures related to inflammatory pathways were found to be upregulated in tumor cells isolated from Panc02WT Adapted tumors as compared to tumor cells isolated from Control tumors, while pathways related to lipid metabolism were significantly upregulated in the Panc02KO Adapted tumor cells isolated from primary tumors that developed in vivo. In contrast, pathways related to epithelial to mesenchymal transition and Wnt signaling were found to be downregulated in Panc02WT Adapted cells as compared to Control cells, and in Panc02KO Adapted cells, there was a significant decrease in genes known to regulate the formation of tight junctions and extracellular matrix components. Furthermore, a common set of 9 genes was found to be upregulated in both Panc02WT and Panc02KO Adapted cancer cells, which may serve as potential biomarkers for long-term adaptation to an acidic environment. In summary, investigating the effects of long-term acid adaptation and p53 mutation on PDAC development in vitro experiments showed that acid adaptation increases adhesion and mobility of PDAC cells, while in vivo studies revealed smaller tumor formation after implantation of Adapted cells. RNA sequencing data showed differences in expression patterns of various pathways between Panc02 Adapted and Control cells within tumors. Overall, this study provides important insight into the impact of acid adaptation and p53 mutation on PDAC development that should be considered when developing new therapeutic approaches.
Keywords: PDAC; microenvironemntal pH; cancer; long-term acid adaptation; p53
Schlagwörter: PDAC; cancer; long-term acid adaptation; acute pH changes; p53