Direct Reprogramming of Glioma Cells to Neurons via Small Molecules and a Novel Protein Delivery System

Lee, Christopher (University of Victoria)
Willerth, Stephanie M. (University of Victoria)

Introduction

Glioblastoma is one of the most aggressive forms of cancer. It is the most common of all malignant central nervous system tumours and the deadliest primary brain cancer. (Ostrom et al., 2016). Aggressive treatments of surgery followed by radio- and chemotherapy can minimally increase life expectancy, but have found little success in producing a significantly improved prognosis.

One of the methods for alternative treatment of glioblastoma is the forced conversion of the cells to a terminally differentiated, non-proliferating neuronal state. Direct reprogramming refers to the conversion of one specific cell type to another. The most common method for reprogramming a given cell into a neuron involves virally expressing neuronal transcription factors, such as Ascl1. Viral expression has long been cemented as the most population reprogramming method. However, issues can arise with the use viral expression system, including difficulty controlling the level of protein expression and oncogenesis due to the lack of control over viral integration into the host cell genome (Hu, 2014).

One alternative to viral expression of a protein is direct supplementation of the protein product. Recently, the company iProgen Biotech has developed a unique recombinant protein delivery system that allows for high uptake of the target protein. In the case of Ascl1, the protein is functionalized with intracellular protein delivery technology (IPTD), which allows for a high level of transduction. This recombinant protein is referred to as Ascl1-IPTD, and has been shown to effectively reprogram human induced pluripotent stem cells into neuronal-like cells with a much higher level of control when compared to viral reprogramming methods (Robinson et al., 2016).

Another direct reprogramming method that has garnered increasing interest is the treatment of target cells with small molecule cocktails. These cocktails contain a variety of chemicals that affect the epigenetics of the cell, and drive differentiation free of exogenous transcription factors.

Whether Ascl1-IPTD and a cocktail of small molecules, alone or in conjunction are able to directly convert glioblastoma cells to functional neurons has not been investigated up to this point. The objective of this study is to investigate and quantify the reprogramming ability of Ascl1-IPTD and a small molecule cocktail on glioblastoma cells. It is hypothesized that the treatment should be able to drive differentiation of glioblastoma cells into neuronal-like cells. These cells would show an increase in the physical aspects of neurons, be able to be identified with common neuronal markers, and show a marked decrease in proliferative ability.

Materials and Methods

Cells were split into 4 groups: 1) cells to receive only ASCL1-IPTD supplementation; 2) cells to receive ASCL1-IPTD and small molecule supplementation - ASCL1 and chemically induced neurons (ACiNs); 3) cells to receive only small molecule supplementation - chemically induced neurons (CiNs); and 4) a control group that received no supplementation.

Human U87 glioblastoma cells were seeded and grown on astrocyte media (AM) for 3 days, and then switched to neuronal media (NM). At day 0, media was switched to NM media containing small molecules (NM+SM) for ACiN and CiN groups. On days 1 and 2 protein was supplemented at 5 um/mL for PO and ACiN groups. On day 7 ICC was performed on a portion of the cells for neuronal cytoskeletal protein beta tubulin (TUJ1), remaining cells were switched to maturation media (MM) and were allowed to continue growth.

Results

Cells treated with only ASCL1-iPTD showed a marked decrease in confluence in the days immediately following treatment, but failed to show significant neuronal morphology. ICC at day 7 revealed insignificant TUJ1 expression. Neuronal morphology and decreased confluence was significantly observed by day 6 for both ACiN and CiN populations, with ACiNs showing a more marked decrease in confluence. ICC imaging for TUJ1 on day 7 showed significant neuronal conversion for both ACiNs and CiNs. (Fig. 1)

N=12 images from 3 wells each were taken and TUJ1 expression was calculated to be <1% for the controls, 63.6% for CiNs, and 58.4% for ACiNs. Neurite length was hand-measured for controls, ACiNs, and CiNs. (Table 1)

At 13 days, CiNs showed signs of complex morphologies, showing morphology similar to terminally differentiated neurons and astrocytes. Too few ACiNs survived to day 13 to identify significant signs of complex morphologies. (Fig. 2)

Discussion and Conclusion

It was found that ASCL1-iPTD alone did not show significant neuronal conversion, but was found to be extremely potent in decreasing confluence of glioblastoma cells. This was not expected, as we have not seen a result such as this for ASCL1-IPTD treated human astrocytes, iPSCs, or fibroblasts. The sharp decrease in cell number limited the amount of ASCL1-IPTD supplemented to the cells, possibly limiting Ascl1-IPTDs reprogramming effectiveness.

ACiNs displayed significant neurite growth and TUJ1 expression at day 7, however this length was lower than for the CiNs. Originally it was hypothesized that ASCL1’s role as a pioneer neural reprogramming factor would facilitate neural reprogramming. However, it was seen that there was no significant difference between the % positive expression of TUJ1 between ACiNs and CiNs; furthermore neurite length was lower for ACiNs when compared to CiNs.

This work has provided initial evidence that ASCL1-iPTD is a strong inhibitor of U87 glioblastoma cell growth, and that a cocktail of small molecules is able to reprogram glioblastoma cells alone, or in the presence of ASCL1-iPTD. However, no evidence has been found that ASCL1-iPTD contributes to neuronal reprogramming. Future work aims to optimize the small molecule conversion protocol to produce more mature neurons, and to investigate whether withdrawing specific chemicals from the small molecule cocktail will improve ACiN conversion.


Figure 1. Reprogrammed U87 glioblastoma cells stained exhibiting the neuronal cytoskeletal protein beta tubulin (TUJ1) on day 7 with phase microscopy contrast for day 6. (A) TUJ1 Control, (B) TUJ1 ACiN, (C) TUJ1 CiN, (D) phase control, (E) phase ACiN, (F) phase CiN.

Table 1. Neurite length of reprogrammed U87 glioblastoma cells.

Figure 2. Complex morphologies displayed by reprogrammed U87 glioblastoma cells that had been treated with a cocktail of small molecules.

Acknowledgements

The authors would like to acknowledge iProgen Biotech inc. for their support in this research.

References

Hu, K. (2014). Vectorology and factor delivery in induced pluripotent stem cell reprogramming. Stem Cells Dev. 23, 1301–1315.

Ostrom, Q.T., Gittleman, H., Xu, J., Kromer, C., Wolinsky, Y., Kruchko, C., and Barnholtz-Sloan, J.S. (2016). CBTRUS Statistical Report: Primary Brain and Other Central Nervous System Tumors Diagnosed in the United States in 2009–2013. Neuro-Oncology 18, v1–v75.

Robinson, M., Chapani, P., Styan, T., Vaidyanathan, R., and Willerth, S.M. (2016). Functionalizing Ascl1 with Novel Intracellular Protein Delivery Technology for Promoting Neuronal Differentiation of Human Induced Pluripotent Stem Cells. Stem Cell Rev and Rep 12, 476–483.

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