Transcription factor-based gene therapy to treat glioblastoma through direct neuronal conversion

Objective: Glioblastoma (GBM) is the most prevalent and aggressive adult primary cancer in the central nervous system. Therapeutic approaches for GBM treatment are under intense investigation, including the use of emerging immunotherapies. Here, we propose an alternative approach to treat GBM through reprogramming proliferative GBM cells into non-proliferative neurons. Methods: Retroviruses were used to target highly proliferative human GBM cells through overexpression of neural transcription factors. Immunostaining, electrophysiological recording, and bulk RNA-seq were performed to investigate the mechanisms underlying the neuronal conversion of human GBM cells. An in vivo intracranial xenograft mouse model was used to examine the neuronal conversion of human GBM cells. Results: We report efficient neuronal conversion from human GBM cells by overexpressing single neural transcription factor Neurogenic differentiation 1 (NeuroD1), Neurogenin-2 (Neurog2), or Achaete-scute homolog 1 (Ascl1). Subtype characterization showed that the majority of Neurog2- and NeuroD1-converted neurons were glutamatergic, while Ascl1 favored GABAergic neuron generation. The GBM cell-converted neurons not only showed pan-neuronal markers but also exhibited neuron-specific electrophysiological activities. Transcriptome analyses revealed that neuronal genes were activated in glioma cells after overexpression of neural transcription factors, and different signaling pathways were activated by different neural transcription factors. Importantly, the neuronal conversion of GBM cells was accompanied by significant inhibition of GBM cell proliferation in both in vitro and in vivo models. Conclusions: These results suggest that GBM cells can be reprogrammed into different subtypes of neurons, leading to a potential alternative approach to treat brain tumors using in vivo cell conversion technology.


Immunocytochemistry
Cultured cells were fixed in 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) for 15 min at room temperature. The cells were washed 3 times using PBS and incubated in blocking buffer (5% normal donkey serum, 0.05% Triton X-100 in PBS) for 40 min. The cells were then incubated with primary antibodies in blocking buffer overnight at 4 °C. The next day, the cells were washed 3 times by 0.05% Triton X-100 in PBS and incubated with appropriate secondary antibodies conjugated to Alexa Fluor 488, Alexa Fluor 546, or Alexa Fluor 647 (1:1,000; Molecular Probes, Eugene, OR, USA) for 1 h at room temperature. After 3 washes with 0.05% Triton X-100 in PBS, the coverslips were mounted onto microscope slides (75 × 25 × 1 mm; VWR, Radnor, PA, USA) with a mounting solution containing 4′,6-diamidino-2-phenylindole (Invitrogen). The slides were first examined with a revolve microscope (Echo, Revolve R4; VWR) and further analyzed with a confocal microscope (LSM 800; Zeiss, Jena, Germany). The images were acquired and analyzed using Zeiss software (Zeiss). Antibody information was collected in Supplementary Table S1. RNA isolation, reverse transcription, and RT-PCR RNA isolations from cultured cells were performed at desired time points using a NucleoSpin® RNA kit (Macherey-Nagel) following the manufacturer's protocols. Reverse transcription was performed using 5× qScriptTM cDNA SuperMix (Quanta Biosciences, Beverly, MA, USA) from isolated RNA samples. PerfeCTaTM SYBR® Green SuperMix, ROXTM (Quanta Biosciences) was used for RT-PCR. Glyceraldehye 3-phosphate dehydrogenase was used as the internal control. Each sample had 3 replicates for each target. The sequences of all primers were listed in Supplementary Table S2.

Patch clamp recordings of cultured cells
For the converted neurons, whole-cell recordings were performed using a Multiclamp 700A patch clamp amplifier (Molecular Devices, Palo Alto, CA, USA) as previously described, 52 and the chamber was constantly perfused with a bath solution consisting of 128 mM NaCl, 30 mM glucose, 25 mM HEPES, 5 mM KCl, 2 mM CaCl 2 , and 1 mM MgCl 2 . The pH of the bath solution was adjusted to 7.3 with NaOH, and the osmolarity was at 315-325 mOsm/L. Patch pipettes were pulled from borosilicate glass (3-5 MΩ) and filled with a pipette solution consisting of 135 mM KCl, 5 mM Na-phosphocreatine, 10 mM HEPES, 2 mM EGTA, 4 mM MgATP, and 0.5 mM Na 2 GTP (pH 7.3, adjusted with KOH). The series resistance was typically 10-30 MΩ. For voltage-clamp experiments, the membrane potential was typically held at -70 or -80 mV. Data were acquired using pClamp 9 software (Molecular Devices), sampled at 10 kHz, and filtered at 1 kHz. The Na+ and K+ currents and action potentials were analyzed using pClamp 9 Clampfit software. Spontaneous synaptic events were analyzed using MiniAnalysis software (Synaptosoft, Decator, GA, USA). All experiments were conducted at room temperature.

Mitochondrial tracker incubation
MitoTracker™ Red CMXRos (Invitrogen) was used to show mitochondrial morphology and distribution. MitoTracker was diluted with culture medium to a final concentration of 500 nM. The cells were incubated with MitoTracker for 1 h and then fixed with 4% PFA. This was followed by the regular immunohistochemistry protocol.

BrdU labeling and cell proliferation assays
Cell proliferation was examined by BrdU incorporation. BrdU was added in cell culture medium (10 mM) at indicated durations. At the desired time points, the cells were fixed in 4% PFA for 15 min, and then treated with 2 M HCl for 1 h at room temperature, and washed by PBS, 3 times, with 5 min each time. This was followed by blocking and sequential incubations with anti-BrdU antibody (1:1,000; Accurate Chemical, Westbury, NY, USA) and corresponding secondary antibody.

Figure S11
Top upregulated differentially-expressed genes (DEGs) in response to Neurog2 or Ascl1 overexpression in human glioblastoma cells. A heat map with hierarchical clustering showing the top 25 upregulated DEGs (sorted by fold change, > 100 normalized read counts in at least one sample) in response to Ascl1 or Neurog2 overexpression in U251 human glioblastoma cells. The color was scaled within each row.
Normalized read count values are presented.  Figure S12 Investigation of the cell cycle gene set in response to Neurog2-induced neuronal conversion of human glioblastoma cells. Heat map illustrating the subsets of genes (read count > 1,000) during conversion corresponding to mitotic spindle hallmark in gene set enrichment analysis. Color scaled within each row. Red color indicates high expression level, while blue color indicates low expression level.