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Glioblastoma-cortical organoids recapitulate cell state heterogeneity and intercellular transfer

ABSTRACT: Glioblastoma is characterized by heterogeneous malignant cells that are functionally integrated within the neuroglial microenvironment. Here, we model this ecosystem by growing glioblastoma into long-term cultured human cortical organoids that contain the major neuroglial cell types found in the cerebral cortex. Single-cell RNA-seq analysis suggests that, compared to matched gliomasphere models, glioblastoma cortical organoids (GCO) more faithfully recapitulate the diversity and expression programs of malignant cell states found in patient tumors. Additionally, we observe widespread transfer of glioblastoma transcripts and GFP proteins to non-malignant cells in the organoids. Mechanistically, this transfer involves extracellular vesicles and is biased towards defined glioblastoma cell states and astroglia cell types. These results extend previous glioblastoma-organoid modeling efforts and suggest widespread intercellular transfer in the glioblastoma neuroglial microenvironment.

ORGANISM(S): Mus musculus Homo sapiens

PROVIDER: GSE276838 | GEO | 2024/10/01

REPOSITORIES: GEO

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Project description:We report transcriptomes from 430 single glioblastoma cells isolated from 5 individual tumors and 102 single cells from gliomasphere cells lines generated using SMART-seq. In addition, we report population RNA-seq from the five tumors as well as RNA-seq from cell lines derived from 3 tumors (MGH26, MGH28, MGH31) cultured under serum free (CSC) and differentiated (FCS) conditions. This dataset highlights intratumoral heterogeneity with regards to the expression of de novo derived transcriptional modules and established subtype classifiers. Operative specimens from five glioblastoma patients (MGH26, MGH28, MGH29, MGH30, MGH31) were acutely dissociated, depleted for CD45+ inflammatory cells and then sorted as single cells (576 samples). Population controls for each tumor were isolated by sorting 2000-10000 cells and processed in parallel (5 population control samples). Single cells from two established cell lines, GBM6 and GBM8, were also sorted as single cells (192 samples). SMART-seq protocol was implemented to generate single cell full length transcriptomes (modified from Shalek, et al Nature 2013) and sequenced using 25 bp paired end reads. Single cell cDNA libraries for MGH30 were resequenced using 100 bp paired end reads to allow for isoform and splice junction reconstruction (96 samples, annotated MGH30L). Cells were also cultured in serum free conditions to generate gliomasphere cell lines for MGH26, MGH28, and MGH31 (CSC) which were then differentiated using 10% serum (FCS). Population RNA-seq was performed on these samples (3 CSC, 3 FCS, 6 total). The initial dataset included 875 RNA-seq libraries (576 single glioblastoma cells, 96 resequenced MGH30L, 192 single gliomasphere cells, 5 tumor population controls, 6 population libraries from CSC and FCS samples). Data was processed as described below using RSEM for quantification of gene expression. 5,948 genes with the highest composite expression either across all single cells combined (average log2(TPM)>4.5) or within a single tumor (average log2(TPM)>6 in at least one tumor) were included. Cells expressing less than 2,000 of these 5,948 genes were excluded. The final processed dataset then included 430 primary single cell glioblastoma transcriptomes, 102 single cell transcriptomes from cell lines(GBM6,GBM8), 5 population controls (1 for each tumor), and 6 population libraries from cell lines derived from the tumors (CSC and FCS for MGH26, MGH28 and MGH31). The final matrix (GBM_data_matrix.txt) therefore contains 5948 rows (genes) quantified in 543 samples (columns). Please note that the samples which are not included in the data processing are indicated in the sample description field.

Project description:Cerebral organoids â three-dimensional cultures of human cerebral tissue derived from pluripotent stem cells â have emerged as models of human cortical development. However, the extent to which in vitro organoid systems recapitulate neural progenitor cell proliferation and neuronal differentiation programs observed in vivo remains unclear. Here we use single-cell RNA sequencing (scRNA-seq) to dissect and compare cell composition and progenitor-to-neuron lineage relationships in human cerebral organoids and fetal neocortex. Covariation network analysis using the fetal neocortex data reveals known and novel interactions among genes central to neural progenitor proliferation and neuronal differentiation. In the organoid, we detect diverse progenitors and differentiated cell types of neuronal and mesenchymal lineages, and identify cells that derived from regions resembling the fetal neocortex. We find that these organoid cortical cells use gene expression programs remarkably similar to those of the fetal tissue in order to organize into cerebral cortex-like regions. Our comparison of in vivo and in vitro cortical single cell transcriptomes illuminates the genetic features underlying human cortical development that can be studied in organoid cultures. 734 single-cell transcriptomes from human fetal neocortex or human cerebral organoids from multiple time points were analyzed in this study. All single cell samples were processed on the microfluidic Fluidigm C1 platform and contain 92 external RNA spike-ins. Fetal neocortex data were generated at 12 weeks post conception (chip 1: 81 cells; chip 2: 83 cells) and 13 weeks post conception (62 cells). Cerebral organoid data were generated from dissociated whole organoids derived from induced pluripotent stem cell line 409B2 (iPSC 409B2) at 33 days (40 cells), 35 days (68 cells), 37 days (71 cells), 41 days (74 cells), and 65 days (80 cells) after the start of embryoid body culture. Cerebral organoid data were also generated from microdissected cortical-like regions from H9 embryonic stem cell derived organoids at 53 days (region 1, 48 cells; region 2, 48 cells) or from iPSC 409B2 organoids at 58 days (region 3, 43 cells; region 4, 36 cells).

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Glioblastoma-cortical organoids recapitulate cell state heterogeneity and intercellular transfer

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