AbstractIn its last cancer report, the National Institute for Health (NIH) indicated that 38.5 % of the population will develop a cancer during their lifetime. For 2017, it was estimated that about 1.7 million new cases would be diagnosed and more than 600,000 people will die in USA, corresponding to 64 % of survival. While this overall survival rate slowly increases over time, it masks significant disparities between the different cancer types. In fact, the death rates of several cancer types, including cancers from nervous system and pancreas, increase each year due to the inefficiency of treatment modalities for these cancers. Therefore, there is a real need for the discovery of new treatment modalities and/or for current treatment improvement. Amongst all the treatment modalities available, this thesis focuses on radiotherapy, which aims at delivering a lethal dose of ionizing radiation into the tumor. However, modern radiotherapy is still limited by the side effects caused to healthy tissues surrounding the tumor. One of the current challenges is to maximize the differential radiation dose deposited in the tumor and in normal healthy tissues (the so-called “therapeutic ratio”). For this purpose, the use of charged particles instead of classical X-ray photons is growing worldwide, ensuring a more effective tumor targeting. In the meantime, the development of nanomedicine offers the possibilities to take advantage of nanoscale materials in a range of diagnosis and therapeutic applications.
In the framework of this thesis, we have investigated the effects induced by a combination of proton irradiation and gold nanoparticles (GNPs) on various carcinoma cells. Our results demonstrate the ability of GNPs to enhance cell death upon irradiation. Thereby, a 25 % increase in cell death was observed when lung carcinoma A549 cells pre-incubated with GNPs were exposed to 225 kV X-rays and 25 keV/µm protons. Moreover, we evidenced that this radiosensitization effect vary with different physico-chemical parameters including GNP size or particle LET as well as according to the cell line of interest.
In order to maximize the cancer cell death, we investigated the mechanism(s) responsible for this enhancement effect. In this context, two different approaches were investigated. On one hand, a physico-chemical hypothesis was suggested: the interaction between ionizing radiations and GNPs leads to the emission of low-energy electrons from the GNP. These electrons interact with the surrounding medium, producing reactive oxygen species (ROS), which can damage critical biological targets. Our results showed a significant increase in the hydrogen peroxide and hydroxyl radical production in colloidal solutions upon irradiation compared to solutions that did not contain GNPs. Moreover, the use of a radical scavenger during the irradiation enabled to decrease the radiosensitization effect evidencing the key role played by ROS in the mechanism(s) responsible for it. However, simulation works highlighted that the encounter probability between charged particles and GNPs is too low to explain, on its own, the origin of this enhancement effect. Thereby, a second hypothesis was suggested: GNPs disrupt cell homeostasis predisposing it to death after irradiation. To validate this hypothesis, we investigated the effect of GNP incubation on different biological pathways. We reported that GNP incubation with lung carcinoma cells led to a time-dependent mitochondria membrane depolarization, to a decrease in ATP content and to oxidative stress. Moreover, a marked inhibition of thioredoxin reductase (TrxR) activity was observed in cells incubated with GNPs, suggesting that this enzyme is a potential GNP target. Furthermore, we reported that this TrxR activity reduction is cell type-dependent and leads to differences in cell response to X-ray irradiation. Correlation analyses demonstrated that GNP uptake and TrxR activity inhibition are associated to GNP radiosensitization effect. With all these results, we suggested a new mechanism explaining the radiosensitization effect of GNPs.
Although we demonstrated the potential of GNPs as in vitro radiosensitizers, their use for in vivo biomedical applications remains challenging due to biodistribution issues. Thereby, we developed targeted NPs, which can recognize the cancer cells. To achieving it, we grafted an antibody against EGFR, an overexpressed receptor in many types of cancers, at the GNP surface. Results obtained with this targeted GNP highlight a higher gold content in EGFR positive cells compared to EGFR negative ones. Consequently, we observed a significant enhanced effect of proton irradiation in EGFR positive cells but not in EGFR negative cells.
Finally, the last part of this thesis focused on the DNA damage and their repair in mice fibroblasts exposed to X-rays or to high-LET particles. We evidenced that the number of radiation-induced foci per Gy of radiation is LET- and mice strain-dependent, suggesting that this phenotype is driven by genetics. By associating phenotype and genetic data, we identified genetic loci associated to significant difference in radiosensitivity phenotype. These researches enable a better understanding of biological consequences associated to charged particle exposition and their genetic basis.
Altogether, these researches enable the improvement of our knowledge of the interaction between nanomaterials, cells and ionizing radiations. The new mechanism responsible for the enhancer effect that we proposed opens new research ways to maximize this amplification effect, thus increasing the chances of curability and the quality of life of patients. In addition, these results pave the way for the use of charged particles and radiosensitizing agents in a personalized medicine framework, which is expected to take off in the next decade.
|Date of Award||1 Mar 2019|
|Sponsors||Fonds de la Recherche Scientifique F.R.S.-FNRS|
|Supervisor||Stephane Lucas (Supervisor), Carine MICHIELS (Co-Supervisor), Thierry Arnould (Jury), Philippe MARTINIVE (Jury), Sylvain Costes (Jury) & Nadine Millot (Jury)|