Cancer Stem Cells in Glioma

By Haoyu Li

Cancer stem cells are first identified in a case of acute myeloid leukemia (AML), Bonnet and Dick (1997) were able to isolate a population of cells which induces AML in immunodeficiency mice. This population had similar expressions of markers that resembles haemopoietic stem cells, yet the AMLs it formed show homogeneous biological characteristics to the tumor the population was isolated from. Such a phenomena demonstrated the ability of the population to self-renew into fully developed cancer, thus, they were titled cancer stem cells. 

Glioma is one of the most common malignant tumors of the central nervous system and is characterized by extensive infiltrative growth, neovascularization, and resistance to various combined therapies (Ma et al, 2018). The most severe form, glioblastoma multiforme (GBM), has an average estimated patient survival time of less than 16 month (Ma et al, 2018). Much the same as other cancers, cancer stem cells have been found in gliomas. These glioma stem cells (GSCs) can form neurosphere-like structures. Such structures are often observed when neural stem cells (NSCs) are incubated in vitro (Brescia et al, 2013). Not long after, several GSCs related markers have been confirmed, the most prominent being CD133. Singh et al (2004) were able to demonstrate through experiments with immunodeficiency mice, the ability of CD133+ glioma cells to single-handedly induce a tumor. The tumor was almost identical in terms of morphology and marker expressions to the original tumor the cells were isolated from. Tumors were not developed by CD133cells in the same experiment. However, more recent research has suggested that, under specific conditions, CD133cells also possesses the potential to self-renew and induce tumor (Hua et al, 2011); hence a marker more discrete to GSCs is being seeked. Currently, markers used to identify GSCs include broad spectrum stem cell markers (CD133), NSC markers (A2B5, Nestin), embryotic stem cell markers (Sox, Nanog), and cell adhesion molecules (CD15, CD44, LICAM) (Hua et al, 2011). These markers are often highly expressed in GSCs, but from the names it is not hard to tell that their high expressions are not unique to GSCs. 

Debates about the origins of GSCs have been ongoing since their discovery. Multiple theories have been brought up, the most supported being that GSCs originate from NSCs. The reasons, according to Nakano et al (2015), are: 1. GSCs and NSCs are biologically similar. NSC isolation, incubation and differentiation induction techniques can be used directly on GSCs without adjustments and both group of cells form neurospheres in vitro. 2. NSCs being stem cells, is constantly and actively undergoing the cell cycle for self-renewal, making them more prone to genetic mutations compared to mature cells and progenitor cells. 3. Mutated NSCs have been shown to induce tumor in mice when overexpressing oncogenes, the tumor induced morphologically resembles a glioma. 4. Most gliomas develop close to the subventricular zone, the same region where NSCs are distributed. On the other hand, alternative theories suggest directed differentiation of progenitor cells and mature glial cells can induce glioma, revealing another possible origin for GSCs. Continuous transfection of HPV16 E6/E7(two oncoprotein sequences) into mature human astrocytes followed by Ras (a small GTP-binding protein that acts as a binary molecular switch) activation leads to astrocytoma, continue activation of Akt pathway forms GBM (Nakano et al, 2015). Activations of Ras and Akt pathways in Nestin+ progenitor cells also lead to GBM formation (Nakano et al, 2015). The above experiments in vitro demonstrates that both progenitor cells and mature cells can induce tumor when a significant quantity of mutations accumulate in the cells, but such a significant amount of mutations to the cells in vivo would rely heavily on low expressions of tumor suppressor genes, which does not occur quite so often to allow the accumulation. In contrast, NSCs being the most supported origin as described at the start of the paragraph, expectedly express less tumor suppressor genes due to their stem cell nature, and hence is more likely to be the true origin of GSCs. 

Despite similarities (which includes self-renewal, self-proliferation, multipotency, and stem cell homing behaviors) between GSCs and NSCs, Campos et al (2014) suggest that two key differences separates the two: 1. GSCs have stronger self-renewal capabilities. Neurosphere isolated from gliomas can survive approximately 4 months in vitro without significant proliferation, whereas NSCs can barely survive more than 1 month. 2. Differentiation progression differs. Cells derived from GSCs more often expresses markers from different marker families (e.g. derived cells are often β-tubulin-III+ GFAP+ double positive). Occurrence of such an expression pattern is seldomly seen in NSC derived cells.

Radiotherapy is a widely recognized way to treat cancer. GSCs are now thought to be the primary reason why malignant gliomas resist radiotherapy. Experiments have shown that following radioactive exposure, significantly larger tumors remain in CD133+ cell colonies compared to CD133 ones (Jackson et al, 2015). Further analysis reveals the former having increase activation of DNA damage repair mechanisms including prolongation of the cell cycle and expression of CHK kinase (an enzyme that coordinates the DNA damage response and cell cycle checkpoint response). Like radiotherapy, CD133+ colonies also possess higher chemotherapy resistances, concluded by the fact they have much higher levels of expression MDR1 & MDR2 (multidrug resistance proteins belonging to the ABC superfamily) (Jackson et al, 2015). In addition, immune escape is another strategy that allows GSCs to evade surveillance of the immune system and develop in the shades to resist therapy. This happens in one of three ways (Jackson et al, 2015): 1. Abnormally activating regulatory T (Treg) cells, whose usual role is to prevent a hyper immune response by triggering cell apoptosis among immunocytes attacking the tumor. 2. Expression of negative costimulatory molecules (e.g. MHC I, B7-H1, B7-H4), which are markers normally expressed on Treg cells. This way GSCs can mimic behavior of Treg cells and avoid triggering an immune response. 3. Secretion of immune-inhibitory cytokines (e.g. TGF-β, PGE2, CCL2, galectin-3). These cytokines bind to receptors on immunocytes and causes a cascade that either decrease the activity or leads to apoptosis. 

To sum up, research into GSCs was a big leap in our understanding of gliomas, but a few key questions remains unanswered: 1. Search for a surface marker unique to GSCs. GSCs shares many expression patterns with NSCs and progenitor cells, making them hard to target using antibodies, as these antibodies will inevitably damage surrounding healthy cells that share the same antigens as GSCs. A surface marker unique to GSCs will provide a more specific immune checkpoint target for immune therapy. 2. Origin of GSCs. Although evidence signifies the closeness between GSCs and NSCs, pathways involved in the mutation process remains ill-defined. 3. Mechanism which allows GSCs to resist and escape traditional combined radio and chemo therapies. Increased activation of DNA repair mechanism and overexpression of drug resistance protein were observed, but a signaling pathway that triggers these events is yet to be unraveled.

References:

Bonnet D. and Dick J. (1997). Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat Med. 1997 Jul;3(7):730-7. 

Brescia P. et al (2013). CD133 is essential for glioblastoma stem cell maintenance. Stem Cells. 31(5), pp. 857-869. 

Campos B. et al (2014). Aberrant self-renewal and quiescence contribute to the aggressiveness of glioblastoma. J Pathol. 234(1), pp. 23-33. 

Hua W. et al (2011). Progress in malignant glioma cancer stem cell markers. Chinese Journal of Surgery. 49(10), pp. 944-946. 

Jackson M. et al (2015). Glioblastoma stem-like cells: at the root of tumor recurrence and a therapeutic target. Carcinogenesis. 36(2), pp. 177-185. 

Ma Q. et al (2018). Cancer Stem Cells and Immunosuppressive Microenvironment in Glioma. Front Immunol. 9:2924. 

Nakano I. (2015). Stem cell signature in glioblastoma: therapeutic development for a moving target. J Neurosurg. 122(2), pp. 324-330. 

Singh S. et al (2004). Identification of human brain tumour initiating cells. Nature. 432(7015), pp. 396-401.

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