The Cellular Behavior of Glioblastoma in a Human-specific Microenvironment Restricted; Files Only
Ganta, Nidhi (Spring 2025)
Abstract
Glioblastoma (GBM) is the most common malignant brain tumor in adults. GBMs are known for their highly invasive and proliferative properties and are associated with a dismal prognosis (~12-18 months post-diagnosis). Neuroanatomical disparities in the occurrence of GBMs suggest that the cellular and molecular profiles of the brain microenvironment impact these tumor cells. This idea has been challenging to study due to the lack of human-specific models that effectively recapitulate the biological behaviors of GBMs in region-specific microenvironments. To address these challenges, we engrafted GFP-tagged patient-derived glioma cell lines into a human induced pluripotent stem cell (hiPSC)-derived brain organoids patterned to mimic the forebrain (dorsal and ventral forebrain), the midbrain, and the hindbrain (spinal cord). Immunostaining assays were performed at 2, 7, 14, and 21 days post-engraftment to compare the extent of infiltration, as measured by the distance traveled by glioma cells in all four regions. We found that glioma cells traveled significantly more distance in forebrain organoids than the midbrain and spinal cord organoids. This suggests that glioma cells are more infiltrative in the forebrain vs. midbrain and hindbrain at baseline. Further, we aimed to determine if these regional biases in infiltration are modified with either optogenetic stimulation of the host brain organoids or in the presence of interregional interactions in a cortico-spinal assembloid system. Cortical neuron projection from the dorsal forebrain to the spinal cord in cortico-spinal assembloids supported glioma invasion, supporting the influence of neuronal activity on GBM behavior. Preliminary single-cell RNA sequencing of glioma cells revealed the enrichment of genes related to cell cycle and synaptic plasticity in an optogenetically active microenvironment. Thus, using this fully human-specific platform, we aim to gain insights into the infiltrative behaviors of GBM cells, which will advance our understanding of this devastating disease and potentially provide new therapeutic targets.
Table of Contents
Introduction ................................................................................................................ 1
Background on glioblastoma ...................................................................................................... 1
The role of the tumor microenvironment in GBM progression .................................................. 2
Different brain regions have distinct cellular and molecular properties ................................... 3
Neuronal activity promotes glioma progression......................................................................... 4
Lack of human-specific brain models to study GBM .................................................................. 5
Overarching research objective .................................................................................................. 6
Methods .......................................................................................................................................... 7
Generation of brain region-specific organoids .......................................................................... 7
Dorsal forebrain organoids ..................................................................................................... 7
Ventral forebrain organoids .................................................................................................... 7
Midbrain organoids ................................................................................................................. 8
Spinal cord organoids ............................................................................................................. 9
Glioma stem cell culture ........................................................................................................... 10
Glioma stem cell engraftments in organoids ............................................................................ 11
Fixing, cryo-embedding, and cryo-sectioning organoids ......................................................... 12
Fixing: ................................................................................................................................... 12
Cryo-embedding: .................................................................................................................. 13
Cryo-sectioning: .................................................................................................................... 13
Immunostaining......................................................................................................................... 13
Confocal imaging ...................................................................................................................... 15
Engrafted brain-region specific organoids ............................................................................ 15
Live imaging of dorsal forebrain-spinal cord assembloids ................................................... 15
Image J quantifications ............................................................................................................. 15
Distance traveled ................................................................................................................... 15
Area fraction ......................................................................................................................... 16
Cortico-spinal assembloid formation ....................................................................................... 17
Optogenetic stimulation of Dorsal Forebrain Organoids ........................................................ 18
RNA extractions and bulk RNA sequencing .............................................................................. 18
Single-cell RNA sequencing ...................................................................................................... 18
Results ...................................................................................................................... 20
Region-specific brain organoids express cellular profiles that mimic respective human brain
regions....................................................................................................................................... 20
Patient-derived glioma cells engraft hiPSC-derived dorsal forebrain organoids.................... 23
Patient-derived glioma cells are more infiltrative in the forebrain than in the midbrain and
hindbrain. .................................................................................................................................. 25
Patient-derived glioma cells invade neuron interaction in cortico-spinal assembloids. .......... 28
Patient-derived glioma cells infiltrate optogenetically activated dorsal forebrain organoids. 31
Optogenetic stimulation of dorsal forebrain organoids may not elevate neuronal activity. .... 35
Discussion..................................................................................................................................... 38
Future Directions ...................................................................................................................... 40
References .................................................................................................................................... 42
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File download under embargo until 22 May 2026 | 2025-04-09 09:52:58 -0400 | File download under embargo until 22 May 2026 |
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