Publicação
Desenvolvimento e validação de um sistema high-throughput de irradiação EBRT para estudos de radiobiologia in vitro.
| Resumo: | Prostate cancer is one of the most common types of cancer and has the sixth highest mortality rate worldwide. Current therapeutic options for this disease include surgery, chemotherapy, hormonal therapy, and external beam radiation therapy (EBRT). Radiation therapy is one of the most widely used cancer treatments and involves irradiating the tumor site with ionizing radiation to induce an anti-tumor effect through the formation of single and/or double-strand DNA breaks. Despite advancements in the modalities and equipment used in external beam X-ray radiation therapy, limitations still exist in the understanding of the biological effects associated with this type of treatment, which could be utilized for more personalized planning and treatment. In radiobiology, survival curves obtained through clonogenic assays in two-dimensional (2D) cultures are the standard method for studying the cellular response to radiation. However, these 2D models do not replicate the typical tumor architecture. To address this limitation, spheroid (3D) cellular models have gained popularity because they more accurately represent dynamic tumor architecture. Additionally, there is a need for studies using irradiation systems that closely mimic clinical conditions, to enhance the clinical translation of the results obtained.Thus, the main objective of this dissertation was to develop an efficient irradiation system for preclinical cellular models, with high clinical translatability, enabling the irradiation of prostate cancer cells in both 2D and 3D cultures. The secondary objective was to optimize the irradiation protocol to ensure reproducible positioning and conditioning, in order to study the underlying mechanisms of cellular response to external beam radiation therapy.Initially, a phantom was developed from a material with a density similar to water (epoxy resin), with dimensions of 30×30 cm and a thickness of 5 cm. This phantom was composed of two parts: an external part, which filled the space surrounding the culture plates, and an internal part, which filled the air gaps found in the lower part of the culture plates. Subsequently, a radiation therapy plan was developed, starting with the acquisition of computed tomography (CT) images of the phantom, followed by the optimization of intensity-modulated radiation therapy (IMRT) plans to cover doses of 0.25 Gy, 0.50 Gy, 0.75 Gy, 1 Gy, 2 Gy, 4 Gy, 5 Gy, 6 Gy, 8 Gy, and 10 Gy for each 6-well and 96-well culture plate. Finally, PC3 cells were irradiated in both 2D and 3D cultures. To validate the developed irradiation method, the biological effects induced by X-ray exposure in these cell cultures were assessed. For this purpose, cell survival was evaluated 8 days after irradiation in both 2D cultures and 3D (spheroid) cultures using the clonogenic assay, which allows for the assessment of the cells' ability to form colonies and survive in the long term. In addition, the viability of the spheroids (3D) was assessed 7 days after irradiation using the CellTiter-Glo® assay, which quantifies intracellular ATP levels.The results demonstrated a reduction in cell survival as a function of increasing dose, with a median lethal dose (LD50) of 3.171 Gy (95% CI: 2.837-3.525 Gy) for irradiated PC3 cells and an LD50 of 4.947 Gy (95% CI: 3.824-6.071 Gy) for irradiated PC3 spheroids. The results also demonstrated a decrease in spheroid viability with increasing dose, with a significant reduction in viability observed only for doses above 8 Gy. It is noteworthy that even at the highest dose (10 Gy), the reduction in spheroid viability was 30%. Thus, considering the results obtained, PC3 spheroids (3D) demonstrated greater radioresistance than irradiated PC3 monolayer cells (2D).In conclusion, the irradiation system developed for in vitro cell irradiation proved to be an important tool with high translational value for conducting radiobiological studies, facilitating studies of X-ray radiation response in both traditional (2D) culture models and 3D cell cultures that better reproduce the characteristics of in vivo tumors. |
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| Autores principais: | Franco, Pedro Daniel Ferreira |
| Assunto: | Metastatic prostate cancer External beam radiation therapy (EBRT) Intensity modulated radiation therapy (IMRT) Phantom Cancro da próstata metastático Radioterapia de fonte externa Radioterapia de intensidade modulada Fantoma |
| Ano: | 2024 |
| País: | Portugal |
| Tipo de documento: | dissertação de mestrado |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade de Coimbra |
| Idioma: | português |
| Origem: | Estudo Geral - Universidade de Coimbra |
| Resumo: | Prostate cancer is one of the most common types of cancer and has the sixth highest mortality rate worldwide. Current therapeutic options for this disease include surgery, chemotherapy, hormonal therapy, and external beam radiation therapy (EBRT). Radiation therapy is one of the most widely used cancer treatments and involves irradiating the tumor site with ionizing radiation to induce an anti-tumor effect through the formation of single and/or double-strand DNA breaks. Despite advancements in the modalities and equipment used in external beam X-ray radiation therapy, limitations still exist in the understanding of the biological effects associated with this type of treatment, which could be utilized for more personalized planning and treatment. In radiobiology, survival curves obtained through clonogenic assays in two-dimensional (2D) cultures are the standard method for studying the cellular response to radiation. However, these 2D models do not replicate the typical tumor architecture. To address this limitation, spheroid (3D) cellular models have gained popularity because they more accurately represent dynamic tumor architecture. Additionally, there is a need for studies using irradiation systems that closely mimic clinical conditions, to enhance the clinical translation of the results obtained.Thus, the main objective of this dissertation was to develop an efficient irradiation system for preclinical cellular models, with high clinical translatability, enabling the irradiation of prostate cancer cells in both 2D and 3D cultures. The secondary objective was to optimize the irradiation protocol to ensure reproducible positioning and conditioning, in order to study the underlying mechanisms of cellular response to external beam radiation therapy.Initially, a phantom was developed from a material with a density similar to water (epoxy resin), with dimensions of 30×30 cm and a thickness of 5 cm. This phantom was composed of two parts: an external part, which filled the space surrounding the culture plates, and an internal part, which filled the air gaps found in the lower part of the culture plates. Subsequently, a radiation therapy plan was developed, starting with the acquisition of computed tomography (CT) images of the phantom, followed by the optimization of intensity-modulated radiation therapy (IMRT) plans to cover doses of 0.25 Gy, 0.50 Gy, 0.75 Gy, 1 Gy, 2 Gy, 4 Gy, 5 Gy, 6 Gy, 8 Gy, and 10 Gy for each 6-well and 96-well culture plate. Finally, PC3 cells were irradiated in both 2D and 3D cultures. To validate the developed irradiation method, the biological effects induced by X-ray exposure in these cell cultures were assessed. For this purpose, cell survival was evaluated 8 days after irradiation in both 2D cultures and 3D (spheroid) cultures using the clonogenic assay, which allows for the assessment of the cells' ability to form colonies and survive in the long term. In addition, the viability of the spheroids (3D) was assessed 7 days after irradiation using the CellTiter-Glo® assay, which quantifies intracellular ATP levels.The results demonstrated a reduction in cell survival as a function of increasing dose, with a median lethal dose (LD50) of 3.171 Gy (95% CI: 2.837-3.525 Gy) for irradiated PC3 cells and an LD50 of 4.947 Gy (95% CI: 3.824-6.071 Gy) for irradiated PC3 spheroids. The results also demonstrated a decrease in spheroid viability with increasing dose, with a significant reduction in viability observed only for doses above 8 Gy. It is noteworthy that even at the highest dose (10 Gy), the reduction in spheroid viability was 30%. Thus, considering the results obtained, PC3 spheroids (3D) demonstrated greater radioresistance than irradiated PC3 monolayer cells (2D).In conclusion, the irradiation system developed for in vitro cell irradiation proved to be an important tool with high translational value for conducting radiobiological studies, facilitating studies of X-ray radiation response in both traditional (2D) culture models and 3D cell cultures that better reproduce the characteristics of in vivo tumors. |
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