The aim of my Master Thesis project, performed within the European Erasmus+ Program, was to develop new experimental strategies to compare 2D and 3D cell cultures of chondrosarcoma and chondrocyte cell lines, and to shed light on the corresponding response to low- and high-LET ionizing radiation. Indeed, chondrosarcoma is a particular chemo- and radio-resistant tumour, representing a good candidate for hadrontherapy. Different cell lines were used for this purpose, including chondrosarcoma cells, immortalized chondrocytes, and primary chondrocytes. Standard 2D cell cultures have been used for years as the main tool to obtain parameters of relevance for hadrontherapy treatment planning. However, extrapolating results to the in vivo response might not be straightforward, due to the very different complexities of such systems. To partially solve this gap, a completely new research strategy led to the development of different 3D models. In particular, the cellular and molecular radiobiology laboratory LARIA (Caen, France) developed protocols to manipulate 3D collagen scaffolds. Different 3D cell cultures, as well as conventional 2D monolayers, were investigated. Experiments were performed for the first time to elaborate new protocols to extract cells from the 3D models under investigation (collagen scaffolds and pellets). High-LET irradiations were performed at the GANIL facilities (Caen, France), providing different ion beams at various energies. Finally, a comparison between the response to these radiations and to X-rays (Cyceron, GANIL) was carried out. Survival curves were obtained in order to analyze the dose-effect relationship induced by low- and high-LET radiation. The radioresistance of 2D and 3D cultures was compared, and preliminary results showed a higher radio-sensitivity of cells grown in 3D compared to the ones grown in 2D conditions, independently from cell lines and LET used. This is an unexpected result that has to be further validated. Moreover, the response of chondrosarcoma cells irradiated with high-LET carbon ions (73.4keV/μm) was compared to the response of chondrocytes, which were irradiated with the same particles but with a higher energy, i.e. with an intermediary-LET of 34.2keV/μm. From the survival curves it is possible to estimate the surviving fractions at 2Gy (the conventional dose used in fractionated therapy schedules): it is equal to 2.8% and 17.7% respectively. These results give an insight into the radioresistance of healthy tissues and tumour in hadrontherapy. The Relative Biological Effectiveness (RBE) is another fundamental radiobiological parameter, which can be estimated from survival curves. The high-LET carbon ions impinging on the tumor have a RBE of approximately 4 (at 10% survival), while, at the intermediate-LET, the RBE is equal to approximately 2: the ions are less effective in inducing clonogenic death to the healthy cells rather than to the chondrosarcoma. This allows sparing the surrounding tissue not affected by the tumor. Finally, the RBE as a function of survival was calculated for the different cell lines in the two culture systems.
La mia Tesi di Laurea Magistrale nasce da un Progetto di ricerca effettuato nell’ambito del Programma Europeo Erasmus+: l’obiettivo è stato quello di sviluppare nuove strategie sperimentali per confrontare colture 2D e 3D di linee cellulari di condrociti sani e affetti da condrosarcoma, e di far luce sulle corrispondenti risposte dopo irraggiamenti con radiazioni a basso ed alto LET. ll condrosarcoma è in effetti un tumore particolarmente chemio- e radioresistente, il che lo rende un ottimo candidato per l’adroterapia. Diverse linee cellulari sono state utilizzate, ovvero cellule affette da condrosarcoma, condrociti immortalizzati e condrociti primari. Le colture 2D hanno costituito per anni il principale strumento per ottenere parametri rilevanti per definire piani di trattamento per l’adroterapia. Tuttavia, estendere i risultati ottenuti da queste alla risposta in vivo non è semplice né adeguato, a causa della differente complessità di tali sistemi. Per risolvere in parte questo problema, una nuova strategia di ricerca ha portato allo sviluppo di diversi modelli 3D. In particolare, il laboratorio di radiobiologia cellulare e molecolare LARIA (Caen, Francia) ha sviluppato protocolli per manipolare matrici di collagene in 3D. Sono state studiate diverse colture tridimensionali, così come quelle convenzionali in monolayer. Per la prima volta sono stati condotti esperimenti per elaborare nuovi protocolli di estrazione delle cellule dai modelli in esame (scaffolds di collagene e pellets). Sono stati eseguiti irraggiamenti ad alto LET presso la facility di GANIL (Caen, Francia), che fornisce fasci di ioni a diverse energie. Infine, è stato effettuato un confronto tra la risposta a queste radiazioni e ai raggi X (Cyceron, GANIL). Curve di sopravvivenza sono state ottenute al fine di analizzare la relazione dose-effetto indotta da radiazioni sia a basso che ad alto LET. E’ stata confrontata la radioresistenza delle colture 2D e 3D ed i risultati preliminari hanno mostrato una maggiore sensibilità delle cellule cresciute in 3D rispetto alle stesse coltivate in 2D, indipendentemente dalla linea e dal LET utilizzati. Questo è un risultato inaspettato su cui è necessario investigare ulteriormente. Inoltre, la risposta delle cellule affette da condrosarcoma irraggiate con ioni carbonio ad alto LET (73.4keV / μm) è stata confrontata con quella di condrociti esposti alle stesse particelle, ma con energia maggiore, il che comporta un LET intermedio di 34.2keV / μm. Dalle curve è possibile stimare frazioni di sopravvivenza a 2Gy (la dose utilizzata convenzionalmente in regimi di terapia frazionata) del 2,8% e 17,7% rispettivamente. Questi risultati danno un'idea della radioresistenza di tessuti sani e tumorali in adroterapia. Un altro parametro radiobiologico fondamentale che può essere ricavato dalle curve di sopravvivenza è l'efficacia biologica relativa (RBE). Gli ioni carbonio ad alto LET che colpiscono il tumore hanno una RBE di circa 4 (ad una sopravvivenza del 10%), mentre a LET intermedio il valore è pari a circa 2: gli ioni sono meno efficaci nell'indurre la morte clonogenica in cellule sane piuttosto che in quelle di condrosarcoma. Questo permette di risparmiare la parte di tessuto non affetta da tumore. E’ stata infine calcolata l'RBE in funzione della sopravvivenza, nelle linee cellulari utilizzate in entrambi i sistemi di coltura.
Modelli 3D in vitro di condrosarcoma: studio della radioresistenza dopo irraggiamenti con raggi X e ioni carbonio.
BARBIERI, SOFIA
2014/2015
Abstract
The aim of my Master Thesis project, performed within the European Erasmus+ Program, was to develop new experimental strategies to compare 2D and 3D cell cultures of chondrosarcoma and chondrocyte cell lines, and to shed light on the corresponding response to low- and high-LET ionizing radiation. Indeed, chondrosarcoma is a particular chemo- and radio-resistant tumour, representing a good candidate for hadrontherapy. Different cell lines were used for this purpose, including chondrosarcoma cells, immortalized chondrocytes, and primary chondrocytes. Standard 2D cell cultures have been used for years as the main tool to obtain parameters of relevance for hadrontherapy treatment planning. However, extrapolating results to the in vivo response might not be straightforward, due to the very different complexities of such systems. To partially solve this gap, a completely new research strategy led to the development of different 3D models. In particular, the cellular and molecular radiobiology laboratory LARIA (Caen, France) developed protocols to manipulate 3D collagen scaffolds. Different 3D cell cultures, as well as conventional 2D monolayers, were investigated. Experiments were performed for the first time to elaborate new protocols to extract cells from the 3D models under investigation (collagen scaffolds and pellets). High-LET irradiations were performed at the GANIL facilities (Caen, France), providing different ion beams at various energies. Finally, a comparison between the response to these radiations and to X-rays (Cyceron, GANIL) was carried out. Survival curves were obtained in order to analyze the dose-effect relationship induced by low- and high-LET radiation. The radioresistance of 2D and 3D cultures was compared, and preliminary results showed a higher radio-sensitivity of cells grown in 3D compared to the ones grown in 2D conditions, independently from cell lines and LET used. This is an unexpected result that has to be further validated. Moreover, the response of chondrosarcoma cells irradiated with high-LET carbon ions (73.4keV/μm) was compared to the response of chondrocytes, which were irradiated with the same particles but with a higher energy, i.e. with an intermediary-LET of 34.2keV/μm. From the survival curves it is possible to estimate the surviving fractions at 2Gy (the conventional dose used in fractionated therapy schedules): it is equal to 2.8% and 17.7% respectively. These results give an insight into the radioresistance of healthy tissues and tumour in hadrontherapy. The Relative Biological Effectiveness (RBE) is another fundamental radiobiological parameter, which can be estimated from survival curves. The high-LET carbon ions impinging on the tumor have a RBE of approximately 4 (at 10% survival), while, at the intermediate-LET, the RBE is equal to approximately 2: the ions are less effective in inducing clonogenic death to the healthy cells rather than to the chondrosarcoma. This allows sparing the surrounding tissue not affected by the tumor. Finally, the RBE as a function of survival was calculated for the different cell lines in the two culture systems.È consentito all'utente scaricare e condividere i documenti disponibili a testo pieno in UNITESI UNIPV nel rispetto della licenza Creative Commons del tipo CC BY NC ND.
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https://hdl.handle.net/20.500.14239/23408