Practical Review on Preclinical Human 3D Glioblastoma Models: Advances and Challenges for Clinical Translation.

Fifteen years after the establishment of the Stupp protocol as the standard of care to treat glioblastomas, no major clinical advances have been achieved and increasing patient's overall survival remains a challenge. Nevertheless, crucial molecular and cellular findings revealed the intra-tumoral and inter-tumoral complexities of these incurable brain tumors, and the essential role played by cells of the microenvironment in the lack of treatment efficacy. Taking this knowledge into account, fulfilling gaps between preclinical models and clinical samples is necessary to improve the successful rate of clinical trials. Since the beginning of the characterization of brain tumors initiated by Bailey and Cushing in the 1920s, several glioblastoma models have been developed and improved. In this review, we focused on the most widely used 3D human glioblastoma models, including spheroids, tumorospheres, organotypic slices, explants, tumoroids and glioblastoma-derived from cerebral organoids. We discuss their history, development and especially their usefulness.

Effects of VEGF blockade on the dynamics of the inflammatory landscape in glioblastoma-bearing mice.

BACKGROUND: Targeting angiogenesis has been and continues to be an attractive therapeutic modality in glioblastoma (GBM) patients. However, GBM rapidly becomes refractory to anti-VEGF therapies. Myeloid cell infiltration is an important determinant of tumor progression. Given that VEGF is a modulator of the innate immune response we sought to analyze the dynamics of this response in a mouse model of GBM undergoing anti-VEGF therapy. METHODS: We grafted GL261-DsRed cells in transgenic Thy1-CFP//LysM-EGFP//CD11c-EYFP reporter mice. We combined recurrent spectral two-photon imaging with multiparametric cytometry, immunostaining, and brain clearing to characterize at two critical stages of tumor development (day 21 and day 28 after tumor grafting) the nature and spatial distribution of the innate response in control and bevacizumab-treated mice. RESULTS: We report that at an early stage (21 day), VEGF blockade has a detectable effect on the number of microglial cells but only a mild effect on the number of infiltrating myeloid cells. At a later stage (day 28), the treatment resulted in a specific adjustment of dendritic cell subsets. In treated mice, the number of monocytes and their monocyte-derived dendritic cells (moDC) progeny was increased by approximately twofold compared to untreated mice. In agreement, by in vivo quantitative imaging, we observed that treatment increased the number of LysM-EGFP cells traveling in tumor blood vessels and doubled the densities of both infiltrated LysM-EGFP monocytes and double-labeled EGFP/EYFP moDC. The treatment also led to an increased density of conventional cDCs2 subset together with a decrease of cDCs1 subset, necessary for the development of anti-tumor immunity. Finally, we describe differential spatial cell distributions and two immune cell-traveling routes into the brain. LysM-EGFP cells distributed as a gradient from the meninges towards the tumor whereas CD11c-EYFP/MHCII+ cells were located in the basal area of the tumor. Brain clearing also revealed a flow of CD11c-EYFP cells following the corpus callosum. CONCLUSION: We uncovered new features in the dynamics of innate immune cells in GBM-bearing mice and deciphered precisely the key populations, i.e., DC subsets controlling immune responses, that are affected by VEGF blockade. Since despite differences, human pathogenesis presents similarities with our mouse model, the data provide new insights into the effect of bevacizumab at the cellular level.

Glycolipids Recognized by A2B5 Antibody Promote Proliferation, Migration, and Clonogenicity in Glioblastoma Cells.

A2B5+ cells isolated from human glioblastomas exhibit cancer stem cell properties. The A2B5 epitope belongs to the sialoganglioside family and is synthetized by the ST8 alpha-N-acetyl-neuraminidase α-2,8-sialyltransferase 3 (ST8SIA3) enzyme. Glycolipids represent attractive targets for solid tumors; therefore, the aim of this study was to decipher A2B5 function in glioblastomas. To this end, we developed cell lines expressing various levels of A2B5 either by genetically manipulating ST8SIA3 or by using neuraminidase. The overexpression of ST8SIA3 in low-A2B5-expressing cells resulted in a dramatic increase of A2B5 immunoreactivity. ST8SIA3 overexpression increased cell proliferation, migration, and clonogenicity in vitro and tumor growth when cells were intracranially grafted. Conversely, lentiviral ST8SIA3 inactivation in low-A2B5-expressing cells resulted in reduced proliferation, migration, and clonogenicity in vitro and extended mouse survival. Furthermore, in the shST8SIA3 cells, we found an active apoptotic phenotype. In high-A2B5-expressing cancer stem cells, lentiviral delivery of shST8SIA3 stopped cell growth. Neuraminidase treatment, which modifies the A2B5 epitope, impaired cell survival, proliferation, self-renewal, and migration. Our findings prove the crucial role of the A2B5 epitope in the promotion of proliferation, migration, clonogenicity, and tumorigenesis, pointing at A2B5 as an attractive therapeutic target for glioblastomas.

Inhibitor of Apoptosis Proteins Determines Glioblastoma Stem-Like Cell Fate in an Oxygen-Dependent Manner.

In glioblastomas, apoptosis inhibitor proteins (IAPs) are involved in apoptotic and nonapoptotic processes. We previously showed that IAP inhibition induced a loss of stemness and glioblastoma stem cells differentiation by activating nuclear factor-κB under normoxic conditions. Hypoxia has been shown to modulate drug efficacy. Here, we investigated how IAPs participate in glioblastoma stem-like cell maintenance and fate under hypoxia. We showed that in a hypoxic environment, IAPs inhibition by GDC-0152, a small-molecule IAPs inhibitor, triggered stem-like cell apoptosis and decreased proliferation in four human glioblastoma cell lines. We set up a three-dimensional glioblastoma spheroid model in which time-of-flight secondary ion mass spectrometry analyses revealed a decrease in oxygen levels between the periphery and core. We observed low proliferative and apoptotic cells located close to the hypoxic core of the spheres and glial fibrillary acidic protein+ cells at their periphery. These oxygen-dependent GDC-0152 antitumoral effects have been confirmed on human glioblastoma explants. Notably, serine-threonine kinase activation analysis revealed that under hypoxic conditions, IAP inhibition activated ataxia telangiectasia and Rad3-related protein signaling. Our findings provide new insights into the dual mechanism of action of IAP inhibitors that depends on oxygen level and are relevant to their therapeutic application in tumors. Stem Cells 2019;37:731-742.