CD133 belongs to the Prominin family, and is also known as Prominin 1, with five transmembrane regions. Singh et al. (Singh et al., 2004) found that 100 CD133 positive cells is enough to induce tumorigenesis in the NOD/SCID mouse brain and whereas 100,000 CD133 negative cells were incapable of tumorigenesis. Subsequently, CD133 has been widely recognized as a biomarker of glioma stem cells.

Although many studies have demonstrated transplanted tumors using CD133+ cells, some researchers have reported on the limitations of CD133 as tumor stem cell marker. CD133+ cells serve as tumor stem cells in many organs, such as brain, lung and colon cancers, but expect for gastric or breast (Su et al., 2015). CD133+ cells only had tumor initiating effects in some glioma cells and were not found in other brain tumors, such as CD15+, CD133- medulloblastomas (Read et al., 2009). Different types of glioblastoma cells derived from different patients can produce CD133+ or CD133- tumor stem cells after serum-free culture in vitro, both of whom embrace stem cell features, tumorigenic characteristics and capability of re-generating CD133+ and CD133- cell populations. CD133+ glioma stem cells can differentiate into CD133- tumor cells; CD133- glioma cells injected into nude rats formed tumors containing CD133+ cells (Joo et al., 2008; Wang et al., 2008). Therefore, CD133+ cells are not the only cells with the characteristics of glioma stem cells, and CD133- cells exist as CSCs.

Recent studies have demonstrated that some glioma cell subpopulations highly express CD44, a distinctive cell adhesion molecule (Xu et al., 2010). CD44 is a glycoprotein commonly expressed in numerous malignancies (Bradshaw et al., 2016). CD44 knockdown in GBM xenograft models has inhibited tumor cell growth while improving the response to chemotherapy (Y. Xu et al., 2010). CD44 and CD133 are usually co-expressed in GBM spheres (Brown et al., 2015). Collectively, these data suggest that CD44 may be useful as a CSC marker.

Integrin-α6 is a member of the heterodimer integrin family and is a laminin member of the extracellular matrix protein family. Integrin-α6 can be used as a marker of neural stem cells and the expression of integrin-α6 can be used to detect the tumorigenic potential of normal neural stem cells (Corsini and Martin-Villalba, 2010). Integrin-α6 is highly expressed by the glioma stem cell population and can be used to isolate glioma stem cells (Lathia et al., 2010; Velpula et al., 2012). The function of integrin-α6 lies in self-renewal, proliferation, survival and growth of tumor cells in vitro, and so it can be used as a regulatory target of tumor growth, while its genetic knockout can reduce tumorigenesis (Lathia et al., 2010).

Also known as SSEA-1, it is a carbohydrate antigen on the cell surface. Read et al. (Read et al., 2009) found that tumor cells that are CD15+ and CD133- had the characteristics of tumor stem cells through mouse medulloblastoma experiments. The tumorigenicity of CD15+ cells is 100 times higher than that of CD15- cells in human glioblastoma, where all CD15+ cells were also found to be CD133+, while most CD133+ cells also expressed CD15, suggesting that CD15 is highly likely to be another surface marker of glioblastoma stem cells (Son et al., 2009).

L1 cell adhesion molecule (L1CAM) belongs to the nerve cell adhesion molecule category and to the type I transmembrane glycoprotein of immunoglobulin super family and is crucial in nervous system development. L1CAM supports the survival and proliferation of CD133+ glioma cells, both in vitro and in vivo, and can be targeted as CSC-specific marker for precise treatment in malignant gliomas (Bao et al., 2008). L1CAM activates some signaling pathways such as fibroblast growth factor receptor (FGFR) and focal adhesion kinase (FAK) through integrin, increasing the growth and motility of GBM cells in autocrine or/and paracrine manner. These effects can be intervened by using small-molecule inhibitors of FGFR, integrins and FAK (Anderson and Galileo, 2016).

Also known as Thy-1, CD90 is a member of the cell adhesion molecule immunoglobulin super family. CD90 has been found on the surfaces of nerve cells, thymocytes, fibroblast subsets, endothelial cells, mesangial cells, and hematopoietic stem cells, suggesting that CD90 is a surface marker in hematopoietic stem cells (Kumar et al., 2016), mesenchymal stem cells (Kimura et al., 2016) and hepatocellular stem cells (Yang et al., 2008). CD90 is overexpressed in GBM and is almost absent in low-grade gliomas or normal brain tissues. All CD133+ glioma cells expressing CD90, and CD90+/CD133+ and CD90+/CD133- cells have the same self-renewal ability, indicating that CD133+ glioma stem cells may be a subtype of CD90+ glioma cells (He et al., 2012). In addition, CD90+ cells were also found in glioma peritumoral vessels (Inoue et al., 2016). Therefore, CD90 can be used as a prognostic index of glioma, a marker of glioma stem cells and an indicator of glioma angiogenesis as well.

A2B5 is a ganglioside on the surface of the glial precursor cell membrane. Ogden et al. (Ogden et al., 2008) detected more A2B5+ cells than CD133+ cells in glioblastoma samples, and CD133+ cells were rarely detected. The cells were screened and sorted using flow cytometry, and sequential culture of A2B5+/CD133- and A2B5+/CD133+ cells showed stem cell proliferative activity while that of A2B5-/CD133- cells did not. Tchoghandjian et al. (Tchoghandjian et al., 2010) confirmed that A2B5+/CD133- and A2B5+/CD133+ cells could form tumor stem cell spheres, while A2B5-/CD133- cells could not. These studies also show that CD133- cell populations still contain cells with stem cell activity, and A2B5+ cells may be one type of such stem cells. CD133-/A2B5+ glioma-initiating cells possess a strong migratory and invasive capacity; these cells may be an important subpopulation with high invasive potential in GBM (Sun et al., 2015).

Recently, some typically expressed embryonic stem cells markers have been considered as the markers for tumor-initiating cells, such as c-Myc, SOX2, and OCT-4. These markers could be useful as a tool to identify and isolate CSCs (Ignatova et al., 2002). Moreover, Nestin, OCT-4, NANOG, SOX2, c-Myc, and KLF4 have been described as key players in the transcriptional regulation of glioblastoma CSCs (Ignatova et al., 2002; Yang et al., 2008; Guo et al., 2011; Zhu et al., 2014).

The transport of energy production molecules like glucose and lactate, nucleosides, and ions through the cell membrane by facilitated or active transport can be mediated by CMT. In addition, CMT aids the clearance of neurotoxic substances, metabolites of brain function, and neurotransmitters, through AETs (Ohtsuki and Terasaki, 2007; Sanchez-Covarrubias et al., 2014; Papademetriou and Porter, 2015).

The biochemical modification of small molecules enables changes in some parameters like solubility, stability, lipophilicity, and recognition by AETs. Redesign of drug aiming to improve the recognition by CMT and transportation through BBB can be achieved by coupling the drug to a regular CMT substrate. The molecular structure of the drug should mimic that of the endogenous CMT substrate (e.g., sugars, amino acids, nucleosides) with pharmacologic activity preserved, but preferably not affect CMT function to avoid possible side effects (Misra et al., 2003; Papademetriou and Porter, 2015).

In contrast to CMT, RMT promotes the permeability of some macromolecules into the brain, such as lipoproteins, hormones, nutrients and growth factors (Papademetriou and Porter, 2015). The RMT process is mediated by the binding of the molecule to a cell-surface receptor that presents in endothelial cells on the luminal surface, following endocytosis and transportation of vesicles to the destination, and sequential exocytosis of the vesicle to the extravascular space (Abbott et al., 2010; Georgieva et al., 2014; Papademetriou and Porter, 2015).

The approach targeting RMT requires the involvement of a specific ligand (e.g., an antibody or antibody fragment, synthetic peptide, natural ligand), which has affinity for an endocytic receptor expressed on the endothelial cell surface, to the chemotherapeutic drug or to a drug-loaded nanocarrier. Binding to the targeted receptor induces intracellular signaling cascades mediating invagination and formation of membrane-bound vesicles in the cell interior, and then intracellular vesicular trafficking transport to the abluminal endothelial plasma membrane (Abbott et al., 2010; Georgieva et al., 2014; Papademetriou and Porter, 2015).

Lipid-bilayer vesicles, namely liposomes, are popular drug systems for delivery due to their easy preparation, their encapsulation capability of a wide array of drugs, their biocompatibility, efficiency, non-immunogenicity, enhanced solubility of chemotherapeutic agents, and commercial availability. The clearance of liposomes by macrophages is relatively fast, so modifications of the liposome surface or size can extend their circulation time. Specificity for the nervous system is possible by coupling liposomes to aptamers or monoclonal antibodies against transferrin receptors (OX-26), glial fibrillary acidic proteins or the insulin receptor (Kanai et al., 2014). The use of liposomes for gene delivery has been demonstrated by injecting liposomes carrying a plasmid coding for the green fluorescence protein in rats. Also, in tumor therapy, liposomes carrying small interfering RNA (siRNA) have been deployed, while the diesteryl phosphoethanolamine poly carboxybetaine lipid which promotes endosomal/lysosomal escape was developed for systemic delivery of siRNA (Dai et al., 2014; Ozpolat et al., 2014).

Furthermore, trafficking cargo across the BBB is improved when using nanocarriers that target CMT. For example, liposomes targeting glucose transporter 1 (GLUT1) enhanced transport of daunorubicin (Ying et al., 2010), while doxorubicin delivery to the brain was 4.8-fold enhanced after equipped with liposomes that targeted glutathione transporters (2B3–101). This approach is particular suitable for small molecules delivery rather than that of large ones. In another study, 2B3–101 was reported as reaching clinical trials and this will be further detailed along this review (Birngruber et al., 2014; Papademetriou and Porter, 2015).

Nanoparticles (NPs) have also been widely studied, because of their high drug-loading capacity and protection against chemical and enzymatic degradation. NPs have enormous medical potential and have emerged as a major tool in nanomedicine, compared with conventional drug delivery methods. NPs are solid colloidal particles made of polymers ranging from 1 to 1000 nm, and are divided in two types, nanospheres and nanocapsules (Couvreur et al., 2002). An interesting application of NPs is the magnetic format of NPs that are made of a magnetic core of iron oxide or magnetite and a biocompatible covering shell of dextran or starch, to be distributed through an organism that is exposed to a localized magnetic field. In vivo GBM models have shown that magnetic NPs are promising. Detailed reviews concerning NP applications have already been published (Laquintana et al., 2009; Upadhyay, 2014).

Polymeric micelles range from 10 to 100 nm and have a core-shell architecture like NPs. They spontaneously self-assemble in aqueous solutions at concentrations higher than a threshold concentration termed the critical micelle concentration. The core is constructed mainly by hydrophobic polymer parts such as poly(caprolactone), poly (propylene glycol) (PPG), or poly(D,L-lactide), together with a hydrophilic shell made of poly(ethylene glycol) (PEG). Pluronic micelles (PEG-PPG-PEG) have emerged as good candidates for brain therapy, since they can easily cross the BBB and inhibit drug efflux. Micelles carrying paclitaxel were able to increase the toxicity of the chemotherapeutic drug in a LN18 human glioblastoma cell line (Liu et al., 2008; Laquintana et al., 2009).

Dendrimers are highly branched polymer molecules smaller than 12 nm. Conjugation to dendrimers confers enhanced delivery across the BBB, which in polyether-copolyester dendrimers loaded with methotrexate and D-glucosamine and tested against avascular glioma spheroids resulted in increased methotrexate potency. After a week, dendrimers do not affect the viability of neural cells nor induce local microglia activation even at submicromolar range of concentration. A better understanding of dendrimer distribution patterns may facilitate the design of nanomaterials for future clinical applications (Laquintana et al., 2009).

Metal particles have been studied extensively because it has been demonstrated that they enhance the susceptibility of tumor tissues to injury induced by radiation exposure, and are therefore a promising candidate for nanomedicine. Application of gold NPs prior to radiation produced distinctive DNA damage in tumors and improved the survival of tumor bearing animals (Joo et al., 2008; Bobyk et al., 2013). Previous research has suggested that the enhanced radiosensitization effects were led by low-energy electrons emission from gold particles and in a dose-dependent maner (Zheng et al., 2008). Similarly, the radiosensitization effect of silver NPs is also attributed to their interactions with the DNA repair system, which eventually leads to the arrest of DNA duplication and cell apoptosis (Xu et al., 2009). After irradiation, titanium dioxide (TiO2) induces tumor cell death by increasing the production of free radicals. The amount of reactive oxygen species generated is dose-dependent on the amount of TiO2 applied as a radiosensitizer when the cells are exposed in X-rays (Park et al., 2008).

Hyperthermic treatment strategies use a magnetic medium such as thermoseeds and magnetic NPs to apply moderate heating in a specific area of the organ where the tumor is located. The combination of carbon nanotubes (CNTs) with near-infrared radiation (NIR) was effective in debulking a tumor in rats, leading to tumor shrinkage without recurrence. Furthermore, this protocol could eliminate glioma CSCs, both drug-sensitive and drug-resistant glioma cells due to the broad-spectrum absorption of CNTs by gliomas. In contrast, normal cells were merely affected, demonstrating the lower uptake of CNTs (Santos et al., 2014). Hyperthermia in glioma treatment remains controversial because it is technically difficult to impose a lethal dose of heat to all cell populations within the glioma mass. The heterogeneous response to different grades of hyperthermia may change the biological nature of the surviving tumor cells. For example, following moderate thermal preconditioning human glioma cell lines demonstrate increased proliferation in vitro and aberrant aggressiveness in a xenograft model. The transient increase in growth of the CD133 subtype of gliomas after thermal preconditioning indicates that there might be a compensation for the loss of the thermal sensitive sub-population (Zeng et al., 2016).

To further increase selectivity for CSCs, antibodies against the CD133 surface marker can be employed as a targeting moiety. Photothermal therapy using single-walled carbon nanotubes (SWNTs) conjugated with anti-CD133 antibodies (CDSWNTs) produced a targeted lysis of CD133+ GBM CSCs, while CD133- GBM cells remained intact in vitro. A discernible shrinkage of tumor after subcutaneous NIR laser irradiation following CDSWNT administration in this particular ectopic GBM tumor model (Wang et al., 2011). NIR photoimmunotherapy, employing a monoclonal CD133 antibody (mAb) conjugated to an IR700 phototoxic phthalocyanine dye, permitted a spatiotemporally controlled elimination of tumor cells through specific image guidance. Rapid cell death was observed after CD133 mAb intravenous administration followed by harmless NIR light applied through the intact skull. This proof of principle study offers a promising theranostic agent that can be applied in intraoperative imaging or histopathological evaluation to define the tumor borders, as well as eradication of CSCs specifically and efficiently (Jing et al., 2016).

Antitumor antibiotics are a form of chemotherapeutic that interferes with DNA and slows or stops cancer cells from multiplying. Antitumor antibiotics demonstrate promise in treating gliomas. For example, doxorubicin (trade name: Adriamycin^®), daunorubicin (trade name: Cerubidine^®), and bleomycin (trade name: Blenoxane^®), show powerful anticancer activity against gliomas cells in vitro. Their efficacy in vivo was reported to be poor, which was largely attributed to their inability to penetrate the BBB (von Holst et al., 1990). However, once these antibiotics are encapsulated in PEGylated liposomes (for example, Doxil^® is a PEGylated form of liposomal doxorubicin), the prolonged survival of treated animals is observed following an enhanced local antitumor effect (Sharma et al., 1997). Overall, the antitumor effects of liposomal doxorubicin, daunorubicin, or bleomycin have been unsatisfactory against glioma in patients (Fabel et al., 2001; Fiorillo et al., 2004). Further, to promote the efficacy of liposomal formulations against brain tumors, more effective drug-delivery strategies are clearly in need. For example, the combination of ultrasound-induced microbubbles, which create transient local BBB permeability, with liposomal doxorubicin has been reported to have a significant antitumor effect (Aryal et al., 2013, 2015).

Recently, the advance of new technologies that facilitate the engineering of the cell genome, like clustered regularly interspaced short palindromic repeats (CRISPR)/Cas 9 and silencing RNA, has provided new methods to deliver nucleic acids to the brain, and in particular for glioma treatment. For this purpose, positively charged and degradable polymers, including chitosan, poly(beta-amino esters), poly(amidoamines), and many other cationic polymers have been used, because of their cationic nature, which allows complexation with negatively charged molecules like DNA or RNA. Inorganic NPs are better applied for imaging and drug delivery purposes, because their synthesis is easily tunable and reproducible (Cardoso et al., 2007; Tzeng and Green, 2013). Some examples are injectable superparamagnetic iron oxide NPs, which are used as contrast agents for magnetic resonance imaging, and gold NPs that are used to carry a conjugated drug. Coated spherical gold NPs carrying a highly oriented layer of siRNA are well protected from nuclease degradation and provide highly efficient knockdown.

Liposomes have also been used to deliver the IFN-β gene in mouse models of glioma, resulting in immune response induction and reduced tumor growth. Five malignant glioma patients were treated using liposomes carrying the IFN-β gene in a pilot clinical trial and four patients showed > 50% tumor reduction or stable disease (Yoshida et al., 2004). Moreover, since Apo2L/tumor necrosis factor-related apoptosis inducing ligand (TRAIL) is fairly specific for cancer cells, a TRAIL plasmid encapsulated in PEG-conjugated PLA NPs (<120 nm) was injected intravenously and caused an increased median survival time (Hawkins, 2004; Lu et al., 2006).

To avoid GBM recurrence, the protein product of the delivered gene should be designed to be active in CSCs. In addition, the construct can be under the control of a cancer-specific promoter, such as survivin or PEG3, to ensure that healthy cells are not transfected or transduced (Su et al., 2005; Van Houdt et al., 2006). The delivery of miRNAs, such as miR-124 and miR-137, can induce terminal differentiation and cell death in murine CSCs in vitro (Silber et al., 2008). Moreover, Gangemi et al. used an shRNA-expressing plasmid in a retroviral vector for in vitro knockdown of SOX2, leading to inhibited CSC proliferation, self-renewal, and tumor-initiating capacity (Gangemi et al., 2009).

Recently, modified siRNAs have been developed that are protected from nuclease degradation and can be readily taken up into cells. This type of modification allows researchers to focus on developing engineered NPs with a prolonged circulation time and site-specific delivery, instead of siRNA protection, thus accelerating clinical translation.