**5. GSCs in the tumor: Target of therapies?**

The overlapping of tumor cell infiltration and edema remains a major problem in the brain adjacent to tumor (BAT), because of the difficulty of their distinction [98,99], even though somebody supports that white matter fibre tract infiltration can be recognized [97]. In exper‐ imental tumors transplanted into mice, it has been observed by superimposing immunohis‐ tochemistry to MRI that in edema districts around the tumor, reactive astrocytes and activated microglia increased Aquaporin-4 expression and invasive tumor cells coexist [100]. Aquapor‐ in-4 has been observed to correlate in peritumor tissue with edema and in the tumor with Hypoxia-Inducible Factor-1 (HIF-1), Vascular Endothelial Growth Factor (VEGF) and the grade of malignancy [101], whereas NAA seemed to be more suitable to detect low tumor infiltration in peritumor edema [102]. Of course, in the latter a damage to myelin sheaths takes

In the recognition of tumor cell infiltration in edematous areas by MRI, histological examina‐ tion of the surgical samples corresponding to the ROIs on rFSE or rFLAIR areas, is of great importance, in spite of the demonstration that removing T2 hyperintense non-enhancing areas and areas possibly containing ITCs, survival did not change [65]. It must be known that a T2 hyper-intense area may well correspond to a tumor (Figure 14B). A distinction would be possible, provided that there is no mismatch between the ROIs and sample removal. Usually, the cells composing edematous areas can be: tumor, normal or endothelial cells, macrophages or inflammatory cells and mainly reactive astrocytes. In our experience, the cell count is of paramount importance, especially when the number of non-tumor cells largely exceeds that of tumor cells, including in the former reactive astrocytes, microglia and endothelial cells. By comparing the number of cells in H&E stained sections and of GFAP+and CD68+cells with MRI variables, it has been found that normal cells, reactive astrocytes and microglia cells represent a rather stable quota of cells, so that variations of the total number of cells of a given area could be attributed to tumor cells. Reactive astrocytes, once no more proliferating, become fibrillogenetic and mature; usually, they do not exceed a certain number *per* field (Figure 12A). Therefore, they may influence the total number of cells only when tumor cell infiltration is mild. When the number of infiltrating cells is high, the astrocytic quota becomes insignificant. The same can be said for microglia/macrophages. Inside the tumor these cells are often found in perivascular or perinecrotic masses, but in peritumor tissue they are more regularly distributed and they too do not exceed a certain number *per* field (Figure 12B,C). CBV or Cho/ NAA values will be influenced by macrophages/microglia only when the total number of cells is very low, *i.e.* when tumor cell infiltration will be really mild, below a certain percentage of the total number of cells, taking into account that the number of reactive astrocytes plus that of microglia/macrophages usually corresponds to the half of that of normal cells (unpublished

ITCs can be detected only after a systemic study of the brain at autopsy, as in the whole mounting preparation technique (Figure 15); they cannot be detected in surgical material because this usually cannot include them [4]. ITCs remain as a sword of Damocles in regard

place and it is detectable by MRSI [103].

80 Tumors of the Central Nervous System – Primary and Secondary

data).

to tumor recurrence.

In the last decades, the aphorism is that the eradication of the tumor cannot be obtained by directing chemo-and radiotherapies to the entire tumor mass, composed of non-proliferating, differentiated and insensitive cells; on the contrary, such therapies would be successful if addressed to the cells responsible for growth, recurrence and resistance, *i.e.* GSCs. Therefore, the question is whether these cells can be *in vivo* detected by neuro-imaging and where are they located or generated in the tumor. To answer this question, a short discussion on the origin and nature of GSCs is necessary.

The hypothesis of GSCs is based on the concept that a rare subset of cells within GBM may have significant expansion capacity and the ability to generate new tumors. The remainder of tumor cells, which predominantly make up GBM, may represent partially differentiated cells with limited progenitor capacity or terminally differentiated cells that cannot form new tumors. Following the model of glioma origin from sub-ventricular zone (SVZ) after nitro‐ sourea derivatives [104], the most important hypothesis on gliomagenesis is today that GSCs derive by the transformation of Normal Stem Cells (NSCs) or progenitors, *i.e.* from B or C cells of the SVZ niche [105]. There is a great similarity between SVZ NSCs or progenitors and TICs and malignant gliomas most probably originate from the SVZ [106,107]. The concept is supported by the observation that GBM is almost always in contact with lateral ventricles [108]. This hypothesis cannot be applied to benign gliomas that should derive from mature glia. According to other hypotheses, also GBMs could derive from mature glial cells by acquiring stemness properties through a dedifferentiation process [109] or from stem cells of the white matter, NG2 cells. This origin would fit better with tumors far from the ventricles or with secondary GBM [110]. Also reactive astrocytes can be candidate for glioma origin [111,112], considering that they can acquire a stem-like phenotype [113].

GSCs develop in niches that can be perivascular or perinecrotic [114]. In perivascular niches there is a close contact between endothelial cells and Nestin+and CD133+cells [115]; the former would favor the self-renewal of the latter, mainly by Notch, and the opposite would happen for angiogenesis through VEGF and hypoxia/HIF-1 [115-120]. In perinecrotic niches, GSCs are generated by hypoxia through HIF-1. Really, in the niches there can be a complicated rela‐ tionship among different cell types, such as macrophages, pericytes, astrocytes, *etc.* with a multiple signalling [54,121,122]. In our experience, perinecrotic GSCs could be the remnants of GSCs that populated hyper-proliferating areas before the development of circumscribed necroses within them; this would take place because of the imbalance between the high proliferation capacity of tumor cells and the low one of endothelial cells [2,123]. GSCs, either as NS or AC, are heterogeneous as for stemness properties, clonogenicity and tumorigenicity and they have been regarded as at the top of a cell hierarchy for some molecular signs [49,50]. Stemness among tumor cells could be distributed in a spectrum with a *crescendo* from quiescent highly differentiated cells, where it is nil, to those in which it reaches the highest degree of expression. Stemness would be regulated by the microenvironment [53] and it could be the feature of a functional status rather than of a subset of cells [124,125]. As it is lost during differentiation of normal cytogenesis beyond the stage of progenitors, in the opposite way it is gained by dedifferentiating tumor cells when they reach the stage of progenitors.

Using intravital microscopy, labelled GSCs could be followed in their propagation and responsibility in producing glioma heterogeneity [41], but data are not available by MRI techniques. Bone marrow-derived endothelial precursors, labelled by super-paramagnetic iron oxide nanoparticles, could be demonstrated in glioma-bearing immunodeficient SCID mice by MRI [128], but no similar procedure has been adopted for GSCs. The only possibility is to use the spatial relationship between MRI variables and tumor phenotypes [33] including

Spatial Relationships of MR Imaging and Positron Emission Tomography with Phenotype, Genotype and...

This work was supported by Grant n. 4011 SD/cv 2011-0438 from Compagnia di San Paolo,

, Antonio Melcarne3

, Laura Annovazzi1

and Angelina Cistaro6,7

, Tetyana Denysenko1,3, Carola Junemann3

1 Neuro-bio-oncology Center of Policlinico di Monza Foundation, Vercelli, Italy

6 Positron Emission Tomography Center IRMET S.p.A, Euromedic inc., Turin, Italy

7 Institute of Cognitive Sciences and Technologies, National Research Council, Rome, Italy

[1] Coons SW, Johnson PC, Scheithauer BW, Ytes AJ, Pearl DK. Improving diagnostic ac‐ curacy and interobserver concordance in the classification and grading of primary

, Marta Mellai1

, Angela Piazzi1

, Cristina Casalone4

, Elena Prodi2

http://dx.doi.org/10.5772/58391

83

,

, Paola Cassoni5

,

,

into the phenotype the expression of GSC stemness status.

, Consuelo Valentini2

, Valentina Caldera1

, Piercarlo Fania6

2 Neuroradiology Unit, CTO Hospital, Turin, Italy

3 Neurosurgery Unit, CTO Hospital, Turin, Italy

gliomas. Cancer 1997; 79(7): 1381-1393.

5 Department Medical Sciences, University of Turin, Turin, Italy

4 Istituto Zooprofilattico, Turin, Italy

**Acknowledgements**

Turin, Italy.

**Author details**

Davide Schiffer1

Giovanna Carrara2

Cristiano Corona4

Rebecca Senetta5

**References**

The heterogeneity of GBM, before discussed, conditions different genetic assets of the cells in the different clones; going from the samples of the most malignant areas of the tumor to those of tumor periphery, the potential of generating NS or AC decreases. The conclusion is that stem cells are kept as such by microenvironments and these are realized in the most malignant sites of the tumor [51,55].
