Three-dimensional atlas of vascular networks in human glioblastoma

• Autores: George Paul Cribaro
• Directores de la Tesis: Carlos Barcia González (dir. tes.)
• Lectura: En la Universitat Autònoma de Barcelona ( España ) en 2018
• Idioma: español
• Tribunal Calificador de la Tesis: Avelina Tortosa Moreno (presid.), Víctor J. Yuste Mateos (secret.), Judit Ribas i Fortuny (voc.)
• Programa de doctorado: Programa de Doctorado en Neurociencias por la Universidad Autónoma de Barcelona
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• Resumen

  • Glioblastoma multiforme (GBM) presents activation of neuroinflammatory markers, which promotes local glial cell activation and monocyte and lymphocyte infiltration. This inflammatory activation and local angiogenesis are thought together to promote tumor growth, and this angiogenesis appears critical to tumor survival and propagation. Blood vessel networks are believed to act as paths for alternatively activated M2 macrophages and tumorigenic cells to enter naïve brain areas. GBM blood vessels show important morphological alterations yet the significance of these changes is poorly understood.

    As such, revisiting the basic microarchitecture of glioma vasculature is opportune. Here, we analyze the microvasculature three-dimensionally (3D) in 60-µm thick, free-floating human biopsy tissue blocks, visualizing basement membrane and endothelial cell components and examining several tumor microenvironment (TME) populations. After immunohistochemical detection, samples were imaged in 3D with a laser scanning confocal microscope and the resulting 3D image stacks were analyzed with specialized software (Imaris and Fiji) to quantify relevant morpho- and topological parameters which were compared with normal tissue and correlated with tumor severity.

    Our analysis shows evidence of profound basement membrane disruption, i.e. altered collagen IV deposition and consequent vessel wall discontinuity and CD31/collagen IV disassociation, resulting in blood vessels apparently without intact endothelial cell lining. Vessels seem clearly aberrant with larger, more variable diameters, uneven, irregular collagen IV distribution and vessel wall fenestration. We identified primary tumor vascularization patterns (cooption, looping, intussusception and silent corollary vessels) and predominant branching strategies. Our data confirms that the human glioma environment utilizes multiple vasculogenesis strategies and many tumor vasculature alterations correlate positively with increased tumor severity. We established that T cells increase in GBM conditions and may enter more deeply into the tumor with increasing severity, increased penetration correlating positively with vessel wall discontinuity. TAMMs increase with tumorigenicity; MHCII+ and GFAP+ cells occupy distinct TME niches.

    We have developed a reliable and replicable sample immunostaining technique that preserves structural relationships, allowing us to study the glioma vasculature and MET cell populations and collect relevant data on spatial relationships. Such a deep analysis of human tissue is essential to understand brain diseases in their natural environment and to look beyond experimental models.