Distancia mínima entre filas de captación

4.1.5.1 CD44:

CD44 is a principal transmembrane cell surface glycoprotein that acts as a receptor for hyaluronan (HA). CD44 plays a vital role in alteration and degradation of HA which leads to migration and invasion of cancer cells and consequently metastasis (Rodgers, 2006; Nagano and Saya, 2004). Under hypoxic conditions HA is degraded into smaller subunits by enzymes called hyaluronidases. HA fragments promote angiogenesis and also can bind with CD44. The binding of HA-CD44 activates the cytoplasmic tail of CD44 and interact with actin cytoskeleton through a group of linked proteins called ezrin- radixin-moesin (ERM) proteins. Interactions between CD44 and cytoskeletal components modulate the adhesion of cancer cells to each other as well as to endothelial cells and increase the migratory potential of the cells (Mori et al., 2008). CD44-HA binding is also known to activate EMT program through TGF- pathway and by activation of Rho-GTPases and protein kinase C and subsequently various CSC genes like Nanog, OCT4, Wnt and -catenin. CD44 also interacts with MMPs and increases the activity of MMP-dependant degradation of ECM thereby promoting invasion and metastasis (Chetty et al., 2012). The expression of CD44 has been associated with more metastatic and resistant phenotypes of hepatocellular carcinoma, breast cancer, colorectal cancer, pancreatic cancer, head and neck cancer and ovarian cancer. The significance of CD44 in GBM CSCs has not been studied clearly but it is thought that EMT phenotypes expressing high levels of CD44 could be the

147 potential reason behind aggressive nature of GBM. Thus CD44 has been suggested as an important differentiator of epithelial versus mesenchymal characteristics (Mani et al., 2008).

4.1.5.2 Cadherin Switch

Cadherins are a superfamily of adhesion molecules that play vital roles in embryogenesis, tissue recognition and maintaining epithelial structure and tissue architecture. E-cadherin is a calcium-dependent cell adhesion molecule that mediates cell-cell adhesion, one of the hallmarks of epithelial cells. Another well-characterized cadherin is N-Cadherin which is expressed in many cells and predominantly in mesenchymal cells. The interaction between cadherins results in stable and strong adhesive forces between cells and is thought to be regulated by environmental signals, growth factors and cytokines. Accumulating evidence from many epithelial cancers suggests that multiple changes in cadherin expression occur during tumour growth that results in tumour progression. Particularly a phenomenon known as “cadherin switching” play a critical role during the process of EMT where E-cadherin is switched off and N- cadherin is switched on resulting in the conversion of nascent tumour cells from an epithelial to a mesenchymal phenotype. The loss of E-cadherin has been proved to be an important reason for the disruption of tight cell-cell contact during EMT leading to an invasive/ metastatic state (Maeda, 2005; Baum, Settleman and Quinlan, 2008; Nieman, 1999). In addition, the mesenchymal N- cadherin becomes upregulated that promotes a motile state in cancer cells allowing the dissociation of individual cells from primary tumour and their

148 interaction with endothelial cells. This enhances migration and extravasation of tumour cells to a distant metastatic site through circulation. Therefore the switch between the classic E and N cadherins is used as a hallmark of EMT and the acquisition of mesenchymal phenotype (Hazan et al., 2004).

4.1.5.3 Vimentin

Vimentin is a major constituent of intermediate filaments that comprise the cytoskeletal proteins along with tubulin and actin microtubules. Vimentin is predominantly expressed in mesenchymal cells and are often used as a marker of cells undergoing EMT or cells derived from the mesenchymal origin (Satelli and Li, 2011). It is a major cytoskeletal component of mesenchymal cells because of its dynamic nature and its ability to offer flexibility in shape of cells thereby increasing their mobility (Satelli and Li, 2011). Moderate to strong vimentin expression was found in normal human brain especially in the ependymal cells, meninges and choroid plexus (Yamada et al., 1992). Expression of vimentin in GBM cells was shown to be dependent on the cellular density and chemo/radio treatment and is shown to mediate integrin trafficking which is vital for propelling GBM malignancy (Fortin et al., 2010). In other CNS tumours like meningiomas, schwannomas and neurofibromas vimentin expression was associated with the migration potential and infiltrative/invasive nature of these tumours (Bouamrani et al., 2010; Kawahara et al., 1988).

149

4.1.6 Rationale and aims of this study

The conventional sphere culture system is based on the above mentioned fact that GBM CSCs are preserved in the tumour microenvironment by hypoxia and hence can be isolated, enriched and studied using suitable serum-free selective medium. But we believe that when the cells grown in sphere culture system the aggregation of cells will first induce hypoxia and activate hypoxia mediated EMT in vitro that results in mesenchymal-CSC phenotypes. These cells are further maintained and protected by self renewal and embryonic signalling pathways activated under hypoxic environment. The main goal of this study is to show that CSC phenotypes induced in NS and SUS cultures of GBM cell lines are due to hypoxia induced EMT. Although the spheres are grown under normal O2 conditions, spheroids of larger size become hypoxic due to diffusion gradient (Lee et al., 2006). We aim to prove that any established cell lines grown under sphere culture method will have the same effect and result in CSCs which are mesenchymal cells. By this way we would like to implicate that the GBM CSCs that are chemoresistant in actual primary tumour could possibly be mesenchymal cells and also establish the fact that hypoxia is the key target.

If hypoxia is the problem, we would like to know whether hypoxia can induce EMT and CSCs while the cells are grown as attached monolayer. We aim to do this by culturing monolayer of GBM cell lines under normoxic and hypoxic conditions and determine whether there are any signs of EMT or CSC characteristics under hypoxic conditions. If there are mesenchymal changes observed we also would like to examine whether these cells are resistant to

150

anticancer drugs. By this way we can be sure whether or not the hypoxia induced mesenchymal cells are the true culprits in GBM chemoresistance. In this chapter we will discuss the possible mechanisms of how hypoxia can drive EMT and analyse the possibilities of these mechanisms in our NS and SUS CSCs from GBM cell lines.

4.2 Experimental design

Detailed information on materials, products, manufacturers and methodologies used for the entire study has been described in chapter 2. The following are specific experimental designs and methods used for this part of the study.