**1. Introduction**

76 Advances in the Biology, Imaging and Therapies for Glioblastoma

Stupp, R., Mason, W. P., van den Bent, M. J., Weller, M., Fisher, B., Taphoorn, M. J.,

(March 2005), pp987-996, ISSN 0028-4793

Belanger, K., Brandes, A. A., Marosi, C., Bogdahn, U., Curschmann, J., Janzer, R. C., Ludwin, S. K., Gorlia, T., Allgeier, A., Lacombe, D., Cairncross, J. G., Eisenhauer, E. & Mirimanoff, R. O. (2005). Radiotherapy Plus Concomitant and Adjuvant Temozolomide for Glioblastoma. *New England Journal of Medicine,* Vol.352, No.10

> Gliomas are the most common form of brain tumors, contributing to more than half of the incidence of brain tumors. They are derived from three basic types of glial cells: astrocytes, oligodendrocytes and ependymal cells. The most frequent are diffusely infiltrating astrocytomas, further classified into astroytomas (A), anaplastic astrocytomas (AA) and glioblastoma, equivalent to World Health Organization (WHO) grade II, II and IV, respectively (Kleihues & Cavenee, 2000). The term glioblastoma is used synonymously with glioblastoma multiforme (GBM), which suggests, the histopathology of this tumor is extremely variable (Kleihues & Cavenee, 2000). GBM is the most common and lethal type of astrocyte-derived tumor, corresponding to 50% of adult primary brain tumor cases, followed by anaplastic astrocytoma (30%) and astrocytoma (20%) (Greenberg, 2010). GBM may develop from astrocytoma or anaplastic astrocytoma (secondary GBM), but more frequently they manifest after a short clinical history *de novo*, without any evidence of a less malignant precursor lesion (primary GBM) (Farhadi & Rutka, 2008). Although primary brain tumors are relatively rare compared with carcinomas, they are characterized by higher mortality rates and increased disability. The overall annual incidence rate of primary malignant and benign brain tumors in developed countries is approximately 15 per 100,000 individuals, and for primary malignant brain tumors it is 7 per 100,000 (Minn et al., 2008). Brain tumor incidence and mortality have increased by up to 300% over the past 3 decades primarily in people aged over 75 years (Davis et al., 1996 as cited in Minn et al., 2008; Wrensch et al, 1993 as cited in Minn et al., 2008).

> Malignant gliomas are among the most challenging of all cancers to treat successfully. The tumor cells vigorously invade surrounding tissue, which renders complete surgical resection difficult and contributes to the high incidence of the recurrence (Merzak et al., 1995). Invasion of glioma cells into adjacent brain tissue is dependent on their interaction with the extracellular matrix (ECM) and possible destruction of matrix barriers (Pilkington, 1994). Tumor cells at the invasive front have to detach from the primary tumor mass and reattach to ECM components or to surrounding tissue elements. In general, invasiveness may result in deformation and destruction of the brain architecture which leads to the fatal outcome for the patient. Proteolytic modification of ECM components, such as laminin and

Prognostic Significance of Immunohistochemical Markers in Glioma Patients 79

Previously, either commercial cell lines derived from human GBM or multicellular tumor spheroids from human gliomas have been used as a model for the study of brain tumor invasion (Bjerkvig et al., 1990; Engebraaten et al., 1999). Since all these cell types can grow in vitro, the cultures must be characterized to ascertain the cell subpopulations which have been selected through the culture conditions. Morphological characterization is not sufficient and a panel of markers may be required to define the populations present. However, as the cells adapt to the tissue culture conditions, they may lose the ability to express one or more of these markers. The need for better and more relevant brain tumor

**2.1.1 U87 human glioblastoma cell xenografts in rat brains and on the chicken** 

The objective of our experimental work was to develop a simple and cheap animal model, with high tumor take rate, for brain tumor progression studies (Strojnik et al., 2006; Strojnik et al., 2010). A tumorigenesis model was presented, originating from tumor spheroids prepared from U87 human glioblastoma cell line, in the brain of rats and on the chick chorioallantoic membrane (CAM). U87 cells are considered to be a rapidly proliferating cell line, which can be grown in culture as monolayers and tumor spheroids. The U87 cell suspension, or precultured U87 tumor spheroids, was inoculated into the brain of 4-week-

Fig. 1. Coronal rat brain tissue sections demonstrated solid, well demarcated, grayish tumor

models is generally acknowledged.

old rats and on CAM on embryonic day seven.

with an area of hemorrhage (arrow).

**chorioallantoic membrane** 

fibronection, is believed to facilitate the invasive spread of tumor cells (Gladson, 1999; Goldbrunner et al., 1999). Lysosomal cysteine cathepsins have been implicated in tumor progression. Proteolytic enzymes, including cahtepsins (Cats), mediate the invasion process, either acting alone or participating in proteolytic cascades (Schmitt et al., 1992; Sloane et al., 1994). Increased activity of proteolytic enzymes is observed during brain tumor progression (Frosch & Sloane, 1998; Levičar et al., 2003). Cathepsins are responsible for intracellular protein turnover and are vitally important for normal cell and organ development. The activity of the cysteine cathepsins can be regulated at various levels, ultimately by their endogenous inhibitors (Lah & Kos, 1998; Calkins & Sloane, 1995). In brain tumors, downregulation of the total inhibitory activity of cystatins has been observed, presumably contributing to tumor malignancy (Sivaparvathi et al., 1996a).

In addition, tumor growth is critically dependent on blood supply and the development of new capillaries. In the case of tumor cell-induced angiogenesis, endothelial cells invade surrounding tissue in a process similar to that observed for tumor cells (Paku, 1998).

Human GBM also contains a various amounts of brain tumor stem-like precursor cells (BTSC) (Singh et al., 2003), which indicates a hierarchical model of tumorigenesis. The BTSCs display self-renewal potential, ex vivo multipotency and, most importantly, the ability to establish and expand the tumor in vivo. Moreover, genetic analyses of the BTSC in various patients have revealed distinct patterns of up-regulated genes, including patientspecific genes expression (Galli et al., 2004).

Despite recent advances in neuro-imaging, neurosurgical resection techniques and the development of novel adjuvant therapies, the long-term survival of patients suffering from malignant glioma remains low. Although treatment with temozolomide and radiotherapy improved median survival after diagnosis of GBM from 12 months to 14 months (Kalkanis & Rosenblum, 2008), the survival rate still ranges from a few months to several years, which, together with the poor prognosis, points the need for new, independent prognostic factors that may enable individualized treatment modalities, including molecular based therapies, of patients with unfavourable prognosis. Our studies are aimed to reveal differential expression and compare the prognostic significance of potential biological markers in glioma patients.
