Resultados del análisis de costos de ciclo de vida

Stem cells possess two unique features: pluripotency and self-renewal, and it has been revealed that all tissues contain tissue-specific stem cells which continuously produce differentiated counterparts of the tissue to maintain its structure and function. The discovery of cancer stem cells that behaved in a similar manner to normal stem cells turned out to be a breakthrough in the area of cancer resistance and relapse (Dick et al., 2008). GBM was the first solid tumour from where self-renewing, multipotent, tumourigenic CSCs were isolated (Singh et al., 2003). It is clearly understood from various studies that GBM

Table 3.1 VCR (nM) PAC (nM) DOX (nM) TMZ (uM) U87 att 2.50 (0.76) 3.02 (1.41) 36.83 (7.7) 15.69 (1.81) U87 NS >50** _>50** _>2000** _>500** U87 Sus >50** _>50** _{892.3 (14.8)}** _>500** U251 att _{2.52 (0.74) 2.77 (1.27) 41.92 (7.99) 13.3 (0.64)} U251 NS _>50** _>50** _>2000** _>500** U251 Sus >50** _>50** _{958.72 (32.4)}** _>500** U373 Att 2.71 (0.43) _{2.04 (0.2) 46.51 (6.26) 40.45 (11.21)} U373 NS >50** >50** _>2000** _>500** U373 Sus >50** >50** _>2000** _>500**

131 CSCs are responsible for the invasive growth and GBM recurrence due to their chemoresistant nature. In an effort to study and target the therapeutic resistance of GBM CSCs the CSC culture system was used to isolate CSCs from patient tumour samples or short term cultures.

The GBM CSC culture system is adapted from the normal neural stem cell culture system. The growth factor-supplemented serum-free spheroid culture is widely accepted as a gold standard method to keep the CSCs in an undifferentiated state under in vitro conditions. It is also referred that serum free conditions will purge the differentiated progenies, and selectively enrich the CSC population (Lee et al., 2006). The major drawback of the serum-free enrichment system is that it is very expensive (approx. £150 for a 500mL bottle). Additionally this system does not exactly mimic the physiological conditions and there is no clear information on the origin/source of the CSCs growing in sphere culture. Another paradox for the NS system is the belief that CSCs once exposed to serum will differentiate irreversibly in to their progenies. So people supporting this selection hypothesis do not use established cell lines as a source for CSCs. They consider the cell lines to be fully differentiated cells grown for many years under serum containing culture conditions. On the contrary there are several reports on isolating CSCs from established cell lines. They show all CSC characteristics including initiation of tumour in animal models.

Our results also indicate that when the cells from established GBM cell lines such as U87, U251 and U373 are grown as NS in selective media they form

132 spheres and exhibit several CSC characteristics like CD133, ALDH, Sox2, Oct4, and Nanog. If this is the case it appears like most of the established cell lines have a residual population of CSCs that can be enriched and activated by this growth factor enriched medium and proper culture conditions. But on the other hand growing the cell lines as spheres in normal DMEM+10% serum conditions also induces better spheres and enhanced all the above mentioned CSC characteristics. Therefore from the results of our study and information from literature we can be definite that stemness in GBM CSCs is not determined by the culture medium. Also we can be sure that the serum free culture system is not mandatory for GBM CSC culture.

Therefore there must be another common factor in this culture method that induces and maintains the CSC phenotypes from established cell lines. The most important feature of this culture system is growing the cells in low adherence conditions that allow the cells to grow as spheres. It is well known that cells grown in suspension are more likely to aggregate with each other and form clumps. No matter how low the cell number is, unless they are grown as single cell suspension, the aggregation of the suspended cells is inevitable (Jensen et al., 2011). In our experiments we followed the standard NSC culture methods and therefore we used a cell density of 10,000 cells/mL. The cells aggregate together and form clumps which eventually become sphere-shaped in about 5-7 days after which the CSC populations become apparent. If the culture media does not induce GBM CSCs the other possible mechanism could be a stress- induced phenotype change that occurs in the spheres. If we have a closer look at the sphere cells they clearly have a hypoxic core and a necrotic

133 region surrounded by healthy cells. The cells in the centre of the sphere may have been subjected to severe hypoxia and nutrient deprivation that causes necrosis in the middle. This matches very closely with in vivo GBM tumour conditions where there is extensive hypoxia and necrosis due to lack of nutrients and oxygen in the centre of rapidly growing tumour mass (Jensen et al., 2011).

There are several recent evidence indicate that hypoxia can induce a pathway known as EMT which allows the fully differentiated cells to dedifferentiate to mesenchymal cells that shows CSC characteristics. If this is true, then it is very likely that all sphere cultures will eventually result in hypoxic core due to aggregation which will induce CSCs/mesenchymal cells through EMT program. This could be the possible reason why both serum and non serum GBM sphere cultures can show CSC phenotypes in them. Even if cells are grown as single cell suspension, the same scenario will apply to it after the cells divide and join together to form spheres. Under physiological conditions tumour cells under hypoxia reprogram to mesenchymal cells to facilitate migration, metastasis or to produce growth factors to initiate angiogenesis. Unlike the NSCs, the monolayer-cultured non-CSCs and sphere-cultured CSCs are interconvertible by oxygen concentrations. Hypoxia induces EMT and the cells can migrate out of the tumour and follow another program called MET where the mesenchymal cells become differentiated epithelial cells. This could be the reason why when grown as spheres, established cell lines get converted to mesenchymal phenotype and revert back to epithelial when they are adhered back to monolayer culture. We also had a question how some researchers isolated NS

134 CSCs from fresh samples or by growing them as single cell suspension. This is probably because they are enriching the mesenchymal or CSC phenotypes induced by physiological hypoxia in the primary tumour. A detailed analysis of how hypoxia can induce CSCs in GBM cell line derived spheres is given in chapter 4.

Despite optimal therapies GBM remains to be a fatal malignancy mainly due to their resistance towards conventional therapy and recurrence (Furnari et al., 2007). Studies from our group and many others have confirmed that GBM CSCs promote therapeutic resistance and relapse (Liu et al., 2012; Bao et al., 2006; Johannessen et al., 2009; Liu et al., 2006). In this study we also established that cell line-derived NS and SUS GBM CSCs show high level of chemoresistance compared to the ATT non-stem cell population. The exact mechanisms of GBM CSC chemoresistance are unknown, but probably due to a combination of slow cell cycle kinetics, resistance to oxidative damage, DNA repair mechanisms, avoiding apoptosis, hypoxia activated mechanisms and multidrug resistance proteins (Diehn et al., 2009; Bao et al., 2006; Liu et al., 2006). GBM CSCs probably remain in a quiescent state and may be more resistant to anticancer drugs that target rapidly dividing cells. It is found that cell cycle profiles of human breast CSCs had an extended G2 phase and hence more resistance to induction of apoptosis (Harper et al., 2010). The current standard chemotherapeutic agent known to improve survival in newly diagnosed GBM is TMZ, an oral methylating agent that induces DNA damage by methylated guanine adducts (Stupp et al., 2005). But invariable tumour recurrence after TMZ therapy point towards the presence of TMZ-resistant

135 subpopulation in GBM that express the repair enzyme MGMT which removes the DNA adduct. It is reported that MGMT is expressed in GBM sphere cultures isolated from patient samples so GBM CSCs also contribute to the chemoresistance to TMZ (Johannessen et al., 2009; Liu et al., 2006; Paranjpe et al., 2013). So we hypothesised that the expression of MGMT is elevated after growing the monolayer into spheres that results in resistance of NS and SUS to TMZ. But even after several experiments with positive controls we were not able to identify any MGMT expression in both NS and SUS GBM CSCs. This indicates that probably there are MGMT independent mechanisms present in GBM CSCs that renders the drug ineffective both in vitro and in vivo. This could be the possible reason why many MGMT negative GBM patients also develop resistance to TMZ and end up in tumour relapse.

GBM CSCs also express multidrug resistance proteins like P-gp, BCRP and ABCG2, whose drug efflux functions aid survival of GBM CSCs. When compared with autologous CD133− cells, GBM CSCs that are CD133+ are shown to express an array of anti-apoptotic proteins including FLIP, BCL-2 and BCL-XL that helps in survival after treatment (Liu et al., 2009). In addition to MGMT and other resistance proteins, GBM CSCs may have various other mechanisms like enhanced DNA repair, ALDH activity, oxidative stress balance, etc that can drive multidrug resistance to cytotoxics such as TMZ, BCNU, CCNU, carboplatin, Vincristine, Palitaxel, Docetaxel, Doxodubicin, VP16 and other drugs. Another mechanism of resistance in NS and SUS is thought to be hypoxia induced by cell aggregation and sphere formation. Hypoxia is widely acknowledged as a contributing factor in chemoresistance of solid tumours.

136 Some studies highlight that hypoxia induced EMT can drive chemoresistance (Harris, 2002). Other studies point out that the chemoresistant effects of CSCs are largely mediated by hypoxia inducible factors (HIFs) and other transcription factors highly expressed in GBM CSCs (Tanase et al., 2013). Our findings also indicate a means through which oxygen tension in NS and SUS culture systems could affect GBMs responsiveness to drugs. We have explained these facts in detail in the chapter 4.