Publication: Study of genetic and pharmacological inhibition of chaperone-mediated autophagy in pericytes as a key mechanism in the treatment of glioblastoma
Authors
Salinas Hidalgo, María Dolores
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Escuela Internacional de Doctorado
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Valdor Alonso, Rut
Publisher
Universidad de Murcia
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DOI
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info:eu-repo/semantics/doctoralThesis
Description
Abstract
El glioblastoma (GB) es el tumor primario maligno más agresivo del sistema nervioso central en adultos. Clasificado por la OMS como glioma difuso de grado IV, el GB se caracteriza por una marcada heterogeneidad, una infiltración agresiva y una inevitable recurrencia. Su mal pronóstico se debe a múltiples características biológicas, destacando los mecanismos de evasión inmune. Las células madre del glioma (GSCs), una subpoblación con capacidad de autorrenovación, impulsan la iniciación, progresión y recurrencia del tumor. Paralelamente, el microambiente tumoral (TME) desempeña un papel esencial en el mantenimiento del crecimiento tumoral y en la supresión de la inmunidad antitumoral. Dentro de este nicho, los pericitos (PCs), células murales vasculares situadas de los microvasos, emergen como componentes clave. Fisiológicamente esenciales para la homeostasis, los PCs son reprogramados por las células de GB, adquiriendo propiedades inmunosupresoras y pro-tumorales que facilitan la invasión tumoral. Un mecanismo central que sustenta estos cambios es la autofagia mediada por chaperonas (CMA), una vía selectiva de degradación lisosomal que regula el control de calidad proteico, el metabolismo energético, la reprogramación celular y la adaptación al estrés.
Esta tesis doctoral investiga el papel de la CMA en el mantenimiento de las propiedades mesenquimales de los PCs en condiciones fisiológicas y patológicas, explora el valor pronóstico de marcadores perivasculares dependientes de la CMA en GB, y evalúa nuevas estrategias terapéuticas dirigidas contra PCs condicionados por el GB (GBCPCs).
En el primer capítulo, se analiza cómo la CMA regula las funciones tipo MSC de los PCs durante la reparación tisular tras una lesión desmielinizante. Se demuestra que mediadores inflamatorios reducen la actividad de la CMA en PCs, suprimiendo su potencial regenerativo y promoviendo un secretoma proinflamatorio. La modulación de la CMA en PCs residentes reduce la inflamación y mejora la reparación tisular, proponiéndose como una estrategia terapéutica potencial extrapolable a otras patologías inflamatorias cerebrales.
En el segundo capítulo, se validan la lumican y la osteopontina (OPN) como biomarcadores pronósticos dependientes de la CMA en el nicho perivascular de pacientes con GB. En modelos murinos xenoinjertados y muestras humanas se observa que lumican se asocia a PCs con CMA deficiente y regresión tumoral, mientras que OPN correlaciona con PCs CMA-activos y mayor invasión tumoral. La actividad CMA inducida por el GB en PCs peritumorales se relaciona con la infiltración tumoral y la supervivencia del paciente, definiéndose tres grados de severidad. Se observan además diferencias de sexo, con mayor incidencia en varones de mediana edad pero peor pronóstico en mujeres.
En el tercer capítulo, se analiza la inhibición de la CMA en PCs como estrategia terapéutica utilizando el fosfopéptido P140, conocido por normalizar la CMA aberrante en células inmunes. En modelos murinos inmunocompetentes de GB derivados de pacientes, el tratamiento con P140 bloquea la sobreactivación de la CMA en PCs, interrumpe sus interacciones con las células tumorales y desencadena un secretoma tóxico para el GB. Se identifica la proteína tau, sustrato de CMA, como componente principal de este secretoma y marcador eficaz del éxito terapéutico y del pronóstico del paciente.
En el cuarto capítulo, se evalúa la eliminación selectiva de GBCPCs mediante terapia con células CAR-T dirigidas frente a CD19. Los análisis transcriptómicos revelaron expresión de CD19 dependiente de la CMA inducida por GB. En muestras de pacientes, CD19 se expresa significativamente en PCs de regiones infiltradas, pero mínimamente en tejido sano. In vitro e in vivo, las células CAR-T anti-CD19 reducen de forma selectiva la viabilidad de GBCPCs sin afectar a PCs no condicionados, reprogramando el TME y potenciando las respuestas inmunes antitumorales. CD19 se propone así como un nuevo antígeno diana para eliminar PCs condicionados y frenar la progresión tumoral.
Glioblastoma (GB) is the most frequent and aggressive malignant primary tumor of the central nervous system in adults. Classified by the World Health Organization as grade IV, IDH-wild type diffuse glioma, GB exhibits [marked histological and molecular heterogeneity, aggressive infiltration of surrounding brain tissue, and nearly inevitable recurrence after treatment.] Its poor prognosis arises from several biological features, including tumor plasticity, therapy resistance, and immune evasion mechanisms. Glioma stem cells (GSCs), a subpopulation of tumor-initiating cells with self-renewal capacity, drive tumor initiation, progression, and recurrence. In parallel, the tumor microenvironment (TME) sustains tumor growth and suppresses anti-tumor immunity. Within this niche, pericytes (PCs)-vascular mural cells located on the abluminal side of microvessels-play a crucial role. Normally essential for vascular homeostasis and tissue repair, PCs are reprogrammed by GB cells, acquiring immunosuppressive and pro-tumorigenic properties that facilitate tumor invasion. A key mechanism underlying these changes is chaperone-mediated autophagy (CMA), a selective lysosomal degradation pathway regulating protein quality control, metabolism, and stress adaptation. This thesis investigates the role of CMA in maintaining the mesenchymal properties of PCs under physiological and pathological conditions, explores the prognostic value of CMA-dependent perivascular markers, and evaluates novel therapeutic strategies targeting GB-conditioned PCs (GBCPCs). Chapter I explores how CMA regulates the MSC-like functions of PCs during tissue repair following demyelinating injury. Inflammatory mediators such as IFNg downregulate CMA in PCs, suppressing stemness and promoting a pro-inflammatory secretome. We demonstrate that modulating CMA in host PCs reduces inflammation and enhances tissue repair, suggesting a therapeutic approach applicable beyond brain injury. Chapter II validates lumican and osteopontin (OPN) as CMA-dependent prognostic biomarkers in the perivascular niche of GB patients. Using xenografted mice and patient samples, we show that lumican expression associates with CMA-deficient PCs and tumor regression, while OPN correlates with CMA-active PCs and tumor invasion. GB-induced CMA activity in peritumoral PCs correlates with tumor infiltration and patient survival, defining three severity levels. Sex-based differences were also observed, with higher incidence in middle-aged men but worse prognosis in women. Lumican and OPN thus serve as predictive biomarkers for prognosis and for monitoring immune responses mediated by PCs in GB progression. Chapter III examines CMA inhibition in PCs as a therapeutic strategy using the phosphopeptide P140, known to normalize aberrant CMA in immune cells. In immunocompetent GB mouse models with patient-derived GSCs and GB cell lines, P140 administration blocked GB-induced CMA upregulation in PCs, disrupted PC-tumor interactions, and induced a secretome toxic to GB cells. We identified tau, a CMA substrate, as a key secreted component whose accumulation and release by PCs reactivated anti-tumor functions. Perivascular tau accumulation was proposed as a marker of treatment efficacy and patient prognosis. Overall, these findings validate P140 as a safe, specific therapy that restores PC anti-tumor activity and halts GB progression, while highlighting extracellular tau as a biomarker of therapeutic success.Chapter IV investigates the therapeutic potential of eliminating GBCPCs using CD19-targeted CAR-T cells. RNA-seq analyses identified CD19 expression in GBCPCs dependent on GB-induced CMA. In GB patients, CD19 expression was significantly higher in peritumoral PCs than in normal tissue. In vitro and in vivo, CD19 CAR-T cells selectively eliminated GBCPCs without affecting unconditioned PCs, reprogramming the TME and enhancing anti-tumor immunity. These results position CD19 as a novel target antigen to eradicate tumor-conditioned stromal cells and restrain GB progression.
Glioblastoma (GB) is the most frequent and aggressive malignant primary tumor of the central nervous system in adults. Classified by the World Health Organization as grade IV, IDH-wild type diffuse glioma, GB exhibits [marked histological and molecular heterogeneity, aggressive infiltration of surrounding brain tissue, and nearly inevitable recurrence after treatment.] Its poor prognosis arises from several biological features, including tumor plasticity, therapy resistance, and immune evasion mechanisms. Glioma stem cells (GSCs), a subpopulation of tumor-initiating cells with self-renewal capacity, drive tumor initiation, progression, and recurrence. In parallel, the tumor microenvironment (TME) sustains tumor growth and suppresses anti-tumor immunity. Within this niche, pericytes (PCs)-vascular mural cells located on the abluminal side of microvessels-play a crucial role. Normally essential for vascular homeostasis and tissue repair, PCs are reprogrammed by GB cells, acquiring immunosuppressive and pro-tumorigenic properties that facilitate tumor invasion. A key mechanism underlying these changes is chaperone-mediated autophagy (CMA), a selective lysosomal degradation pathway regulating protein quality control, metabolism, and stress adaptation. This thesis investigates the role of CMA in maintaining the mesenchymal properties of PCs under physiological and pathological conditions, explores the prognostic value of CMA-dependent perivascular markers, and evaluates novel therapeutic strategies targeting GB-conditioned PCs (GBCPCs). Chapter I explores how CMA regulates the MSC-like functions of PCs during tissue repair following demyelinating injury. Inflammatory mediators such as IFNg downregulate CMA in PCs, suppressing stemness and promoting a pro-inflammatory secretome. We demonstrate that modulating CMA in host PCs reduces inflammation and enhances tissue repair, suggesting a therapeutic approach applicable beyond brain injury. Chapter II validates lumican and osteopontin (OPN) as CMA-dependent prognostic biomarkers in the perivascular niche of GB patients. Using xenografted mice and patient samples, we show that lumican expression associates with CMA-deficient PCs and tumor regression, while OPN correlates with CMA-active PCs and tumor invasion. GB-induced CMA activity in peritumoral PCs correlates with tumor infiltration and patient survival, defining three severity levels. Sex-based differences were also observed, with higher incidence in middle-aged men but worse prognosis in women. Lumican and OPN thus serve as predictive biomarkers for prognosis and for monitoring immune responses mediated by PCs in GB progression. Chapter III examines CMA inhibition in PCs as a therapeutic strategy using the phosphopeptide P140, known to normalize aberrant CMA in immune cells. In immunocompetent GB mouse models with patient-derived GSCs and GB cell lines, P140 administration blocked GB-induced CMA upregulation in PCs, disrupted PC-tumor interactions, and induced a secretome toxic to GB cells. We identified tau, a CMA substrate, as a key secreted component whose accumulation and release by PCs reactivated anti-tumor functions. Perivascular tau accumulation was proposed as a marker of treatment efficacy and patient prognosis. Overall, these findings validate P140 as a safe, specific therapy that restores PC anti-tumor activity and halts GB progression, while highlighting extracellular tau as a biomarker of therapeutic success.Chapter IV investigates the therapeutic potential of eliminating GBCPCs using CD19-targeted CAR-T cells. RNA-seq analyses identified CD19 expression in GBCPCs dependent on GB-induced CMA. In GB patients, CD19 expression was significantly higher in peritumoral PCs than in normal tissue. In vitro and in vivo, CD19 CAR-T cells selectively eliminated GBCPCs without affecting unconditioned PCs, reprogramming the TME and enhancing anti-tumor immunity. These results position CD19 as a novel target antigen to eradicate tumor-conditioned stromal cells and restrain GB progression.
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