PhD defense of Alaa TAFECH de from TIMC BCM on December, the 13th, at 2pm:

" Characterization of the intracellular acidity regulation capacity of brain tumor cells in fluorescence imaging-Consequences for therapeutic optimization of temozolomide. "
 

• Place : Room 109, bâtiment Boucherle, Faculté de Médecine et Pharmacie de Grenoble, La Tronche.
   

Jury

• Angélique STEPHANOU,Chargée de recherche, CNRS Grenoble, Directrice de thèse
• Olivier  SEKSEK, Chargé de recherche HDR, CNRS Délégation Ile-de-France Sud, Rapporteur
• Annabelle BALLESTA, Chargée de recherche HDR, INSERM Paris le-de-France Centre Est, Rapporteure
• Walid RACHIDI, Professeur des Universités, UNIVERSITE GRENOBLE ALPES, Examinateur
• Antoine DELON, Professeur des Universités, UNIVERSITE GRENOBLE ALPES, Examinateur

Keywords

acidity, Warburg effect, confocal microscopy, intracellular pH regulation

Abstract

A well-known feature of tumor cells is high glycolytic activity leading to acidification of the tumor microenvironment through extensive lactate production. This acidosis promotes processes such as metastasis, aggressiveness and invasiveness that has been associated to a worse clinical prognosis. Moreover, the function and/or expression of transporters involved in regulation of intracelluar pH (pH𝑖) might be altered.

In this context, the aim of this thesis was first to characterize the capacity of a tumor to regulate intracellular acidity by measuring the pHi of two glioma cell lines: the F98 cell line (rat glioma) and the U87-MG cell line (human glioblastoma). Therefore, we have developed a new ratiometric method that allows the combination of pH-sensitive fluorescent probes and confocal microscopy to characterize the pHi regulatory capacity and pH resistance of F98 and U87 cell lines in 2D monolayer cultures and in 3D spheroids. This method makes it possible to best exploit the capacities of the confocal microscope. Our result show that the tumor regulation of acidity is not the same for the two cell lines and as a consequence, our results do not support the common idea that tumor cells behave in a similar way. On the other hand, pHi regulation appears highly cell-dependent. In 2D monolayer cultures, we have found that F98 rat glioma cells do not regulate intracellular acidity and may preserve protons inside the cells by activating the V-ATpase pump at acidic pH to bring H+ ions into lysosomes. However, we have found that human glioblastoma U87 cells are able to regulate intracellular acidity and may use the Na+ /H+ exchanger to export H+ ions outside the cells. Comparison of the measurements performed on 2D monolayer cultures and 3D spheroids exhibits some differences. Our results show that extracellular acidity may inhibit energy metabolism or protein synthesis and prevent the regulation of exchanges such as Na+/H+ in 3D spheroids. Another aim of the thesis was to assess the efficacy of Temozolomide (TMZ). TMZ is the cornerstone drug used against brain tumors. It has the particularity to be highly pH-dependent. Therefore, the effect of TMZ was studied on our two cell lines by manipulating the extracellular pH (pHe). The results has allowed us to show that drug efficiency depend on the celltype and on the pHe, which gives an argument for considering pH as a personalized therapeutic target for future research based on the combination of TMZ with pH-regulating agents. Based on our original results, we found that the use of our developed fluorescent method can be a valuable tool to assess metabolic status in glioma models and can therefore be used to characterize cells from patient biopsies to better adapt the therapy.