Publicação
Interplay between molecular and cellular players on glioma pathophysiology
| Resumo: | Malignant gliomas are the most common primary brain tumors, accounting for 80% of all neoplasms of the central nervous system, of which glioblastoma (GBM) is the most aggressive and deadly subtype. Even with a multimodal therapy approach that includes surgery and chemo-radio-therapy, the prognosis of glioma patients remains very poor. Moreover, the etiology and clinically-relevant prognostic factors in glioma remain largely undetermined. In this context, the research summarized in this thesis focuses on i) evaluating the influence of TGF-β1 genetic variants in glioma susceptibility and patient prognosis; ii) identifying novel defining characteristics of glioma stem cells (GSCs), a subpopulation of cells that plays critical roles on tumor initiation, resistance and recurrence; and iii) unveiling new insights on the influence of mesenchymal stem cells (MSCs) in glioma behavior. Many genetic polymorphisms have been associated with glioma susceptibility and prognosis. Several studies demonstrated that polymorphisms in the TGF-β1 gene were associated with the susceptibility for different tumor types. Transforming growth factor beta (TGF-β) is known to play an important role in carcinogenesis, and its activity has been associated with poor prognosis in glioma patients. Taking into account that the relevance of single nucleotide polymorphisms (SNPs) in TGF-β1 in glioma is not known, we evaluated two SNPs (-509C/T and 869T/C) in this gene in glioma risk and patient prognosis. A case-control study involving 138 Caucasian cancer-free control and 118 glioma patients from Portugal was performed. We showed that TGF-β1 -509C/T and 869T/C variants were not significantly associated with glioma risk. Importantly, we demonstrated that both homozygous -509TT and 869CC genotypes were associated with longer overall survival of GBM patients. Our data suggested that TGF-β1 -509C/T and 869T/C polymorphisms may be relevant prognostic biomarkers in GBM patients. A striking characteristic of malignant gliomas, particularly GBMs, is their highly heterogeneous and therapy-resistant nature. These features are partly attributed to GSCs, a subpopulation of cancer cells with stem cell features that are involved in tumor initiation, progression, and recurrence, making them crucial therapeutic targets. Their isolation has been challenging as the markers typically used lack sufficient specificity and sensitivity. Thus, we also investigated here if intracellular autofluorescence, a biomarker of epithelial cancer stem cells, could be used as a biomarker to improve GSCs identification and isolation. We found that both established and patient-derived primary GBM cells presented a subpopulation of autofluorescent cells (Fluo+). Moreover, we showed that Fluo+ cells had typical features of GSCs, including higher expression of stem cell protein markers and pluripotency-associated genes, enriched capacity to grow as neurospheres, and long-term self-renewal ability. Additionally, treatments with temozolomide (TMZ) or radiation led to a significant increase in the percentage of Fluo+ cells. Importantly, in vivo studies showed that mice with intracranial tumors derived from Fluo+ GBM cells presented a significantly shorter overall survival than those with non-autofluorescent cells (Fluo-) GBM cells. Finally, the underlying mechanism of the autofluorescent phenotype was due to the uptake, and accumulation of riboflavin in GSCs, by the ABCG2 transporters, exclusively in cells with GSCs’ features. Together, our data revealed autofluorescence as a novel and useful biomarker for GSCs. In the light of the lack of curative therapies for malignant glioma, many exploratory therapeutic strategies have been analyzed. Among these, MSCs have been studied as a new approach for the treatment of malignant gliomas, due to their inherent capacity of homing to glioma, and their ability to be engineered to deliver anti-tumoral agents. In the third experimental work included in this thesis, we assessed the impact of the secretome of MSCs on hallmark characteristics of GBM cells, such as cell viability, migration, proliferation, tumor growth, as well as chemotherapy response. Using in vitro approaches, we observed that GBM cells exposed to conditioned media (CM) from human umbilical cord perivascular cells (HUCPVCs, a MSC population) presented an increased cellular viability, proliferation and migration, while not affecting the sensitivity of GBM cells to TMZ treatment. Additionally, in the in vivo CAM assay, we found that CM from HUCPVCs promoted GBM tumor growth. Finally, proteomic analyses to characterize the secretome of HUCPVCs identified several proteins involved in promotion of cell survival, proliferation and migration, revealing novel putative molecular mediators for the effects observed in GBM cells exposed to HUCPVCs CM. Our data highlights that caution must be taken regarding the use of MSCs as stem-cell based therapies for GBM. In summary, the thesis presented here contributes to the better understanding of several dimensions of glioma, from factors that may be causative, to those that influence their pathophysiology and progression, to therapeutics insights. Particularly, assessing TGF-β1 SNPs -509C/T and 869T/C variants may be clinically relevant for GBM patients; using autofluorescence as a biomarker for GSCs identification may be important to develop new GSCs-specific therapies; and using MSCs as stem cell-based therapies for GBM does not seem to be a safe choice. |
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| Autores principais: | Castro, Joana Isabel Martins Cosme Vieira |
| Assunto: | Ciências Médicas::Ciências da Saúde |
| Ano: | 2017 |
| País: | Portugal |
| Tipo de documento: | tese de doutoramento |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade do Minho |
| Idioma: | inglês |
| Origem: | RepositóriUM - Universidade do Minho |
| Resumo: | Malignant gliomas are the most common primary brain tumors, accounting for 80% of all neoplasms of the central nervous system, of which glioblastoma (GBM) is the most aggressive and deadly subtype. Even with a multimodal therapy approach that includes surgery and chemo-radio-therapy, the prognosis of glioma patients remains very poor. Moreover, the etiology and clinically-relevant prognostic factors in glioma remain largely undetermined. In this context, the research summarized in this thesis focuses on i) evaluating the influence of TGF-β1 genetic variants in glioma susceptibility and patient prognosis; ii) identifying novel defining characteristics of glioma stem cells (GSCs), a subpopulation of cells that plays critical roles on tumor initiation, resistance and recurrence; and iii) unveiling new insights on the influence of mesenchymal stem cells (MSCs) in glioma behavior. Many genetic polymorphisms have been associated with glioma susceptibility and prognosis. Several studies demonstrated that polymorphisms in the TGF-β1 gene were associated with the susceptibility for different tumor types. Transforming growth factor beta (TGF-β) is known to play an important role in carcinogenesis, and its activity has been associated with poor prognosis in glioma patients. Taking into account that the relevance of single nucleotide polymorphisms (SNPs) in TGF-β1 in glioma is not known, we evaluated two SNPs (-509C/T and 869T/C) in this gene in glioma risk and patient prognosis. A case-control study involving 138 Caucasian cancer-free control and 118 glioma patients from Portugal was performed. We showed that TGF-β1 -509C/T and 869T/C variants were not significantly associated with glioma risk. Importantly, we demonstrated that both homozygous -509TT and 869CC genotypes were associated with longer overall survival of GBM patients. Our data suggested that TGF-β1 -509C/T and 869T/C polymorphisms may be relevant prognostic biomarkers in GBM patients. A striking characteristic of malignant gliomas, particularly GBMs, is their highly heterogeneous and therapy-resistant nature. These features are partly attributed to GSCs, a subpopulation of cancer cells with stem cell features that are involved in tumor initiation, progression, and recurrence, making them crucial therapeutic targets. Their isolation has been challenging as the markers typically used lack sufficient specificity and sensitivity. Thus, we also investigated here if intracellular autofluorescence, a biomarker of epithelial cancer stem cells, could be used as a biomarker to improve GSCs identification and isolation. We found that both established and patient-derived primary GBM cells presented a subpopulation of autofluorescent cells (Fluo+). Moreover, we showed that Fluo+ cells had typical features of GSCs, including higher expression of stem cell protein markers and pluripotency-associated genes, enriched capacity to grow as neurospheres, and long-term self-renewal ability. Additionally, treatments with temozolomide (TMZ) or radiation led to a significant increase in the percentage of Fluo+ cells. Importantly, in vivo studies showed that mice with intracranial tumors derived from Fluo+ GBM cells presented a significantly shorter overall survival than those with non-autofluorescent cells (Fluo-) GBM cells. Finally, the underlying mechanism of the autofluorescent phenotype was due to the uptake, and accumulation of riboflavin in GSCs, by the ABCG2 transporters, exclusively in cells with GSCs’ features. Together, our data revealed autofluorescence as a novel and useful biomarker for GSCs. In the light of the lack of curative therapies for malignant glioma, many exploratory therapeutic strategies have been analyzed. Among these, MSCs have been studied as a new approach for the treatment of malignant gliomas, due to their inherent capacity of homing to glioma, and their ability to be engineered to deliver anti-tumoral agents. In the third experimental work included in this thesis, we assessed the impact of the secretome of MSCs on hallmark characteristics of GBM cells, such as cell viability, migration, proliferation, tumor growth, as well as chemotherapy response. Using in vitro approaches, we observed that GBM cells exposed to conditioned media (CM) from human umbilical cord perivascular cells (HUCPVCs, a MSC population) presented an increased cellular viability, proliferation and migration, while not affecting the sensitivity of GBM cells to TMZ treatment. Additionally, in the in vivo CAM assay, we found that CM from HUCPVCs promoted GBM tumor growth. Finally, proteomic analyses to characterize the secretome of HUCPVCs identified several proteins involved in promotion of cell survival, proliferation and migration, revealing novel putative molecular mediators for the effects observed in GBM cells exposed to HUCPVCs CM. Our data highlights that caution must be taken regarding the use of MSCs as stem-cell based therapies for GBM. In summary, the thesis presented here contributes to the better understanding of several dimensions of glioma, from factors that may be causative, to those that influence their pathophysiology and progression, to therapeutics insights. Particularly, assessing TGF-β1 SNPs -509C/T and 869T/C variants may be clinically relevant for GBM patients; using autofluorescence as a biomarker for GSCs identification may be important to develop new GSCs-specific therapies; and using MSCs as stem cell-based therapies for GBM does not seem to be a safe choice. |
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