Abstract
BACKGROUND
Glioblastoma (GBM) is the most aggressive primary brain tumor. The well-known cellular heterogeneity of this cancer, which includes glioma tumor-initiating cells with stem cell characteristics (GSCs), (co)influences therapy resistance and tumor recurrence. Bone Morphogenetic Protein-4 (BMP4) promotes differentiation of GSCs towards astroglial lineage while suppressing oligodendrocyte maturation. Treatment with BMP4 is associated with increased survival in mice. BMPs exert effects in cell-type and context dependent fashion, but also generate subtle heterogeneity in transcriptional response among similar cells. We studied first the cell viability of BMP4-treated primary GBM cultures followed by single-cell RNA-sequencing (scRNA-seq) on two differently responding cultures, and found correlation between their responses and therapy sensitivity.
MATERIAL AND METHODS
Cell viability, proliferation and apoptosis were assessed in 17 patient-derived BMP4-treated GBM cell cultures. We selected one culture in which this treatment induced high in vitro therapeutic efficacy, and one in which the treatment was ineffective, for analysis by scRNA-seq and then compared the results on the initial panel of 17 cultures.
RESULTS
After 7 days of treatment with BMP4, cell viability ranged from 28% (referred to as highest in vitro therapeutic efficacy) to 132% compared to untreated cells. scRNA-seq of the previously mentioned cultures in passage 7 showed that all neural cell types that are usually found in freshly resected GBM, were also present in our cultures. In the culture where BMP4 induced high in vitro therapeutic efficacy, BMP4 induces the formation of a large new cell population displaying decreased cell proliferation, increased migration and cell death, while the pro-inflammatory cells were depleted. RNA-velocity analysis revealed that, the cycling of cells was greatly diminished in the culture where therapy with BMP4 was efficacious, whereas this was enhanced in the GBM culture with the lowest cell viability after treatment. Astroglial differentiation was induced in all BMP4-treated cultures, while neuronal differentiation was reduced most in the cultures in which BMP4 induced lower or no in vitro therapeutic efficacy. In the culture without therapeutic efficacy of BMP4 cell cycle arrest was not induced anymore. In addition, OLIG1/2 mRNA and protein levels seemed predictive for BMP4-therapy efficacy, while activation of translation-associated genes (RPL27A, RPS27) was a suitable, immediate post-therapeutic marker for this.
CONCLUSION
scRNA-seq of in vitro GBM cultures provides advanced insights into the mechanism underlying therapy efficacy of BMP4. Neural differentiation status is distinctive for therapeutic efficacy of BMP4 in vitro before and after therapy.
Ureter single-cell and spatial mapping reveal cell types, architecture, and signaling networks
