Final Report Summary - TUMOR REPROGRAMMING (Targeting Glioblastoma Reprogrammed Stem Cells)
Targeting Glioblastoma Reprogrammed Stem Cells
Glioblastoma (GBM) is the most common and most malignant type of primary brain tumor. It represents one of the deadliest human cancers, with average survival at diagnosis of about one year (Furnari et al., 2007). This poor prognosis is due to therapeutic resistance and tumor recurrence after surgical removal. Malignant gliomas are among the most vascular human tumors making them especially attractive targets for anti-angiogenic therapies. The recent characterization of the genome (Parsons et al., 2008; 2008) and transcriptome (Phillips et al., 2006; Verhaak et al., 2010) of GBM provided a high-resolution picture of the glioblastoma landscape and the major alterations driving the disease pathogenesis and biology. However, despite the progress in genetic analysis and classification of gliomas, there is still an insufficient understanding of the underlying mechanism of progression and recurrence of gliomagenesis. Mouse models of human cancers have been very instructional in understanding the basic principles of cancer biology, but they have not always been able to capture the complete extent of the human disease. Cancers arise from a single cell or a small number of cells in specific cell types, and the cellu¬lar origin of cancers is one of the major determinants of the characteristics of tumor cells. We designed lentiviral vectors that allows the induction of tumors in a region and a cell type specific manner in adult mice. Using this technology we provided an original and significant contribution in understanding the mechanism of gliomagenesis. We were able to show that gliomas can originate from differentiated cells in the central nervous system (CNS), including cortical neurons. Based on these observations we propose that any cell in the brain can be the cell of origin of the tumors. In the case of mature neurons/glia, these cells acquire the capacity to dedifferentiate/reprogram to a cell that has all the attributes of a neuroprogenitor/stem cell, which can then not only maintain its pluripotency, but also give rise to the heterogeneous cell populations observed in malignant gliomas, including endothelial cells to form new blood vessels. We were among the first ones to bring a new concept in the field of cancer: Tumor reprogramming, also known as cancer cell plasticity.
In this project, we proposed to employ the lentiviral-induced GBM mouse model together with advanced nanotechnology and immunology techniques to investigate the mechanisms of tumor reprogramming and the development of a novel therapeutic approach.
Our first aim was to analyze the genetic expression profile of tumors along disease progression. For this purpose we isolated RNA (genetic material) from tumor tissue and glioma cells and performed next generation sequencing (RNAseq analysis). We found many genes differentially expressed when comparing tumor samples to healthy control brains, as well as transformed glioma reprogrammed cells and their parental normal cell controls. Among the up-regulated genes, we identified Tenascin C (TNC), an extracellular matrix component that highly correlated with the mesenchymal, more aggressive molecular GBM subtype. In order to understand the functional role of TNC in glioma development and specifically in the process of tumor reprogramming, we used a molecular biology technique (RNAi) that allows specific silencing of the expression of TNC gene. Silencing the expression of TNC in glioma stem cells impaired cell proliferation and invasion, induced apoptosis and reduced neurosphere formation (one of the outcomes of tumor reprogramming). Moreover, silencing TNC in glioma stem cells blocked their capacity to differentiate into tumor derived endothelial cells, the cells that form new tumor blood vessels. To interrogate the impact of silencing TNC on tumor growth we used intracranial syngeneic mouse models. TNC silencing in glioma stem cells significantly decreased tumor growth and extended mouse survival. Together, our results indicate that silencing TNC conferred prolong survival and further supports the possibility of targeting TNC as a therapeutic approach to glioblastoma treatment.
Our second aim was to identify novel tumor homing peptides in order to target specifically nanoparticles to kill the tumors. In collaboration with Prof. Tambet Teesalu from the University of Tartu, Estonia, we were able to identify LinTT1 as a novel tumor-penetrating homing peptide that binds to p32 (a biomarker expressed only on the surface of tumor cells). We first assessed the relevance of p32 marker for GBM treatment by studying the distribution of p32 in a panel of GBM models. Next we prepared LinTT1 iron oxide nanoparticles, termed nanoworms (NW) and showed their homing to diverse preclinical GBM models. Finally, we evaluated the effect of LinTT1 conjugation on anti-GBM activity of NWs loaded with a therapeutic payload, D(KLAKLAK)2 peptide, that induce tumor cell death. Tumor mice treated with LinTT1- D(KLAKLAK)2 -NWs showed slower progression of tumor growth than controls, and tumor size at the end of the treatment was significantly reduced compared to the control groups. These therapy studies indicate that LinTT1 targeting may be used to potentiate the activity of anticancer nanoparticles.
Our third aim was to design homing peptide targeted-NWs and engineered lymphocytes as a novel combined strategy to treat GBM tumors. Tumor immunotherapy has become the center of attention in the past decade, with adoptive cell transfer and checkpoint blockade having striking success in the clinics. Chimeric antigen receptor (CAR) T cell therapy targeting the CD19 receptor in patients with leukemia (“liquid” cancer) has shown remarkable success, but initial attempts to use the same approach in treating “solid” tumors have been disappointing. In the previous objective we identified p32 to be expressed on the surface of glioma cells. Therefore, we generated a CAR to target p32 and evaluated the functionality of the engineered lymphocytes in vitro performing a cytotoxicity assay (killing assay) as well as activation assays (e.g. secretion of the cytokine IFN-γ. Indeed, the p32-CAR T cells were able to kill glioma cells compared to untransduced control T cells. The antitumor efficacy of this CAR was evaluated in a syngeneic mouse GBM model and in human xenograft model, showing in both models a significant survival extension in the treated groups. Interestingly, microscopy analysis of tumor sections showed reduced blood vessels in the treated group compared to control mice, supporting the potential antioangiogenic effect of these p32-CARs. Collectively, our studies identified a previously uncharacterized biomarker, p32, expressed both in glioma cells and TDECs that holds potential for serving as a novel CAR target with a dual function for cancer immunotherapy in gliomas.
Two independent manuscript with results from Aim 1 and 2 have been submitted for publication in peer-reviewed scientific journals and a third manuscript with results from aim 3 is under preparation. The projects running in my laboratory as well as the publications and members of the lab can be found in our website: https://dinorah2908.wixsite.com/dfm-lab(se abrirá en una nueva ventana)
Glioblastoma (GBM) is the most common and most malignant type of primary brain tumor. It represents one of the deadliest human cancers, with average survival at diagnosis of about one year (Furnari et al., 2007). This poor prognosis is due to therapeutic resistance and tumor recurrence after surgical removal. Malignant gliomas are among the most vascular human tumors making them especially attractive targets for anti-angiogenic therapies. The recent characterization of the genome (Parsons et al., 2008; 2008) and transcriptome (Phillips et al., 2006; Verhaak et al., 2010) of GBM provided a high-resolution picture of the glioblastoma landscape and the major alterations driving the disease pathogenesis and biology. However, despite the progress in genetic analysis and classification of gliomas, there is still an insufficient understanding of the underlying mechanism of progression and recurrence of gliomagenesis. Mouse models of human cancers have been very instructional in understanding the basic principles of cancer biology, but they have not always been able to capture the complete extent of the human disease. Cancers arise from a single cell or a small number of cells in specific cell types, and the cellu¬lar origin of cancers is one of the major determinants of the characteristics of tumor cells. We designed lentiviral vectors that allows the induction of tumors in a region and a cell type specific manner in adult mice. Using this technology we provided an original and significant contribution in understanding the mechanism of gliomagenesis. We were able to show that gliomas can originate from differentiated cells in the central nervous system (CNS), including cortical neurons. Based on these observations we propose that any cell in the brain can be the cell of origin of the tumors. In the case of mature neurons/glia, these cells acquire the capacity to dedifferentiate/reprogram to a cell that has all the attributes of a neuroprogenitor/stem cell, which can then not only maintain its pluripotency, but also give rise to the heterogeneous cell populations observed in malignant gliomas, including endothelial cells to form new blood vessels. We were among the first ones to bring a new concept in the field of cancer: Tumor reprogramming, also known as cancer cell plasticity.
In this project, we proposed to employ the lentiviral-induced GBM mouse model together with advanced nanotechnology and immunology techniques to investigate the mechanisms of tumor reprogramming and the development of a novel therapeutic approach.
Our first aim was to analyze the genetic expression profile of tumors along disease progression. For this purpose we isolated RNA (genetic material) from tumor tissue and glioma cells and performed next generation sequencing (RNAseq analysis). We found many genes differentially expressed when comparing tumor samples to healthy control brains, as well as transformed glioma reprogrammed cells and their parental normal cell controls. Among the up-regulated genes, we identified Tenascin C (TNC), an extracellular matrix component that highly correlated with the mesenchymal, more aggressive molecular GBM subtype. In order to understand the functional role of TNC in glioma development and specifically in the process of tumor reprogramming, we used a molecular biology technique (RNAi) that allows specific silencing of the expression of TNC gene. Silencing the expression of TNC in glioma stem cells impaired cell proliferation and invasion, induced apoptosis and reduced neurosphere formation (one of the outcomes of tumor reprogramming). Moreover, silencing TNC in glioma stem cells blocked their capacity to differentiate into tumor derived endothelial cells, the cells that form new tumor blood vessels. To interrogate the impact of silencing TNC on tumor growth we used intracranial syngeneic mouse models. TNC silencing in glioma stem cells significantly decreased tumor growth and extended mouse survival. Together, our results indicate that silencing TNC conferred prolong survival and further supports the possibility of targeting TNC as a therapeutic approach to glioblastoma treatment.
Our second aim was to identify novel tumor homing peptides in order to target specifically nanoparticles to kill the tumors. In collaboration with Prof. Tambet Teesalu from the University of Tartu, Estonia, we were able to identify LinTT1 as a novel tumor-penetrating homing peptide that binds to p32 (a biomarker expressed only on the surface of tumor cells). We first assessed the relevance of p32 marker for GBM treatment by studying the distribution of p32 in a panel of GBM models. Next we prepared LinTT1 iron oxide nanoparticles, termed nanoworms (NW) and showed their homing to diverse preclinical GBM models. Finally, we evaluated the effect of LinTT1 conjugation on anti-GBM activity of NWs loaded with a therapeutic payload, D(KLAKLAK)2 peptide, that induce tumor cell death. Tumor mice treated with LinTT1- D(KLAKLAK)2 -NWs showed slower progression of tumor growth than controls, and tumor size at the end of the treatment was significantly reduced compared to the control groups. These therapy studies indicate that LinTT1 targeting may be used to potentiate the activity of anticancer nanoparticles.
Our third aim was to design homing peptide targeted-NWs and engineered lymphocytes as a novel combined strategy to treat GBM tumors. Tumor immunotherapy has become the center of attention in the past decade, with adoptive cell transfer and checkpoint blockade having striking success in the clinics. Chimeric antigen receptor (CAR) T cell therapy targeting the CD19 receptor in patients with leukemia (“liquid” cancer) has shown remarkable success, but initial attempts to use the same approach in treating “solid” tumors have been disappointing. In the previous objective we identified p32 to be expressed on the surface of glioma cells. Therefore, we generated a CAR to target p32 and evaluated the functionality of the engineered lymphocytes in vitro performing a cytotoxicity assay (killing assay) as well as activation assays (e.g. secretion of the cytokine IFN-γ. Indeed, the p32-CAR T cells were able to kill glioma cells compared to untransduced control T cells. The antitumor efficacy of this CAR was evaluated in a syngeneic mouse GBM model and in human xenograft model, showing in both models a significant survival extension in the treated groups. Interestingly, microscopy analysis of tumor sections showed reduced blood vessels in the treated group compared to control mice, supporting the potential antioangiogenic effect of these p32-CARs. Collectively, our studies identified a previously uncharacterized biomarker, p32, expressed both in glioma cells and TDECs that holds potential for serving as a novel CAR target with a dual function for cancer immunotherapy in gliomas.
Two independent manuscript with results from Aim 1 and 2 have been submitted for publication in peer-reviewed scientific journals and a third manuscript with results from aim 3 is under preparation. The projects running in my laboratory as well as the publications and members of the lab can be found in our website: https://dinorah2908.wixsite.com/dfm-lab(se abrirá en una nueva ventana)