Three-dimensional vascular microenvironment landscape in human glioblastoma.

The cellular complexity of glioblastoma microenvironments is still poorly understood. In-depth, cell-resolution tissue analyses of human material are rare but highly necessary to understand the biology of this deadly tumor. Here we present a unique 3D visualization revealing the cellular composition of human GBM in detail and considering its critical association with the neo-vascular niche. Our images show a complex vascular map of human 3D biopsies with increased vascular heterogeneity and altered spatial relationship with astrocytes or glioma-cell counterparts. High-resolution analysis of the structural layers of the blood brain barrier showed a multilayered fenestration of endothelium and basement membrane. Careful examination of T cell position and migration relative to vascular walls revealed increased infiltration corresponding with tumor proliferation. In addition, the analysis of the myeloid landscape not only showed a volumetric increase in glioma-associated microglia and macrophages relative to GBM proliferation but also revealed distinct phenotypes in tumor nest and stroma. Images and data sets are available on demand as a resource for public access.

Phagocytic glioblastoma-associated microglia and macrophages populate invading pseudopalisades.

Hypoxic pseudopalisades are a pathological hallmark of human glioblastoma, which is linked to tumour malignancy and aggressiveness. Yet, their function and role in the tumour development have scarcely been explored. It is thought that pseudopalisades are formed by malignant cells escaping from the hypoxic environment, although evidence of the immune component of pseudopalisades has been elusive. In the present work, we analyse the immunological constituent of hypoxic pseudopalisades using high-resolution three-dimensional confocal imaging in tissue blocks from excised tumours of glioblastoma patients and mimic the hypoxic gradient in microfluidic platforms in vitro to understand the cellular motility. We visualize that glioblastoma-associated microglia and macrophages abundantly populate pseudopalisades, displaying an elongated kinetic morphology across the pseudopalisades, and are oriented towards the necrotic focus. In vitro experiments demonstrate that under hypoxic gradient, microglia show a particular motile behaviour characterized by the increase of cellular persistence in contrast with glioma cells. Importantly, we show that glioblastoma-associated microglia and macrophages utilize fibres of glioma cells as a haptotactic cue to navigate along the anisotropic structure of the pseudopalisades and display a high phagocytic activity at the necrotic border of the pseudopalisades. In this study, we demonstrate that glioblastoma-associated microglia and macrophages are the main immune cells of pseudopalisades in glioblastoma, travelling to necrotic areas to clear the resulting components of the prothrombotic milieu, suggesting that the scavenging features of glioblastoma-associated microglia and macrophages at the pseudopalisades serve as an essential counterpart for glioma cell invasion.

The MTOC/Golgi Complex at the T-Cell Immunological Synapse.

T cells effectively explore the tissue in search for antigens. When activated, they dedicate a big amount of energy and resources to arrange a complex structure called immunological synapse (IS), containing a particular distribution of molecules defined as supramolecular activation clusters (SMACs), and become polarized toward the target cell in a manner that channels the information specifically. This arrangement is symmetrical and requires the polarization of the MTOC and the Golgi to be operational, especially for the proper delivery of lytic granules and the recycling of molecules three dimensionally segregated at the clustered interface. Alternatively, after the productive encounter, T cells need to rearrange again to newly navigate through the tissue, changing back to a motile state called immunological kinapse (IK). In this IK state, the MTOC and the Golgi apparatus are repositioned and recruited at the back of the T cell to facilitate motility, while the established symmetry of the elements of the SMACs is broken and distributed in a different pattern. Both states, IS and IK, are interchangeable and are mainly orchestrated by the MTOC/Golgi complex, being critical for an effective immune response.

Imbalance of immunological synapse-kinapse states reflects tumor escape to immunity in glioblastoma.

Since the proper activation of T cells requires the physical interaction with target cells through the formation of immunological synapses (IS), an alteration at this level could be a reason why tumors escape the immune response. As part of their life cycle, it is thought that T cells alternate between a static phase, the IS, and a dynamic phase, the immunological kinapse (IK), depending on high or low antigen sensing. Our investigation performed in tissue samples of human glioma shows that T cells are able to establish synapsing interactions not only with glioma tumorigenic cells, but also with stromal myeloid cells. Particularly, the IS displaying a T cell receptor-rich (TCR-rich) central supramolecular activation cluster (cSMAC) is preferentially established with stromal cells, as opposed to malignant cells. Conversely, T cells in the malignant areas showed distinct morphometric parameters compared with nonneoplastic tissue - the former characterized by an elongated shape, well-suited to kinaptic dynamics. Importantly, high-resolution 3-dimensional analyses demonstrated the existence of bona-fide IK preferentially arranged in malignant areas of the tumor. This imbalance of IS/IK states between these 2 microenvironments reveals the low antigenic sensing of T cells when patrolling tumorigenic cells and reflects the immunoevasive environment of the tumor.

Studying the T Cell-Astrocyte Immune Synapse.

In this chapter, we describe the technical details to visualize and analyze effector immunological synapses between T cells and astrocytes in the brain with high-resolution confocal imaging. This procedure is critical for the optimal and even penetration of labeling antibodies within the nerve tissue to obtain accurate staining and allow a uniform three-dimensional analysis of the T cell-astrocyte interactions. We emphasize here the comprehensive exploration of the tissue and analysis with confocal microscope as well as the display of microanatomical details of the three-dimensional reconstruction for interface visualization (including peripheral and central supramolecular activation clusters, effector molecules, and other organelles such as microtubule organizing centers (MTOCs) and Golgi apparatus).