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Glioblastoma is one of the deadliest malignancies and is virtually incurable. Accumulating evidence indicates that a small population of cells with a stem-like phenotype is the major culprit of tumor recurrence. Enhanced DNA repair capacity and expression of stemness marker genes are the main characteristics of these cells. Elimination of this population might delay or prevent tumor recurrence following radiochemotherapy. The aim of this study was to analyze whether interference with the Hedgehog signaling (Hh) pathway or combined Hh/Notch blockade using small-molecule inhibitors can efficiently target these cancer stem cells and sensitize them to therapy. Using tumor sphere lines and primary patient-derived glioma cultures we demonstrate that the Hh pathway inhibitor GANT61 (GANT) and the arsenic trioxide (ATO)-mediated Hh/Notch inhibition are capable to synergistically induce cell death in combination with the natural anticancer agent (−)-Gossypol (Gos). Only ATO in combination with Gos also strongly decreased stemness marker expression and prevented sphere formation and recovery. These synergistic effects were associated with distinct proteomic changes indicating diminished DNA repair and markedly reduced stemness. Finally, using an organotypic brain slice transplantation model, we show that combined ATO/Gos treatment elicits strong growth inhibition or even complete elimination of tumors. Collectively, our data show for the first time that ATO and Gos, two drugs that can be used in the clinic, represent a promising targeted therapy approach for the synergistic elimination of glioma stem-like cells.
Glioblastoma (GBM) still presents as one of the most aggressive tumours in the brain, which despite enormous research efforts, remains incurable today. As many theories evolve around the persistent recurrence of this malignancy, the assumption of a small population of cells with a stem-like phenotype remains a key driver of its infiltrative nature. In this article, we research Chordin-like 1 (CHRDL1), a secreted protein, as a potential key regulator of the glioma stem-like cell (GSC) phenotype. It has been shown that CHRDL1 antagonizes the function of bone morphogenic protein 4 (BMP4), which induces GSC differentiation and, hence, reduces tumorigenicity. We, therefore, employed two previously described GSCs spheroid cultures and depleted them of CHRDL1 using the stable transduction of a CHRDL1-targeting shRNA. We show with in vitro cell-based assays (MTT, limiting dilution, and sphere formation assays), Western blots, irradiation procedures, and quantitative real-time PCR that the depletion of the secreted BMP4 antagonist CHRDL1 prominently decreases functional and molecular stemness traits resulting in enhanced radiation sensitivity. As a result, we postulate CHRDL1 as an enforcer of stemness in GSCs and find additional evidence that high CHRDL1 expression might also serve as a marker protein to determine BMP4 susceptibility.
Glioblastoma is a very aggressive tumor and represents the most common primary brain malignancy. Key characteristics include its high resistance against conventional treatments, such as radio- and chemotherapy and its diffuse tissue infiltration, preventing complete surgical resection. The analysis of migration and invasion processes in a physiological microenvironment allows for enhanced understanding of these processes and can lead to improved therapeutic approaches. Here, we combine two state-of-the-art techniques, adult organotypic brain tissue slice culture (OTC) and light sheet fluorescence microscopy (LSFM) of cleared tissues in a combined method termed OTCxLSFM. Using this methodology, we can show that glioblastoma tissue infiltration can be effectively blocked through treatment with arsenic trioxide, as well as genetic depletion of the tetraspanin, transmembrane receptor CD9. With our analysis-pipeline we gain single-cell level, three-dimensional information, as well as insights into the morphological appearance of the tumor cells.