From neural stem cells (NSC) to Glioblastoma (GBM): a natural history of GBM recapitulated in vitro

Due to its aggressive and invasive nature glioblastoma (GBM), the most common and aggressive primary brain tumour in adults, remains almost invariably lethal. Significant advances in the last several years have elucidated much of the molecular and genetic complexities of GBM. However, GBM exhibits a vast genetic variation and a wide diversity of phenotypes that has complicated the development of effective therapeutic strategies. This complex pathogenesis makes it necessary the development of experimental models that could be used to further understand the disease, and also to provide a more realistic testing ground for potential therapies. In this report, we describe the process of transformation of primary mouse embryo astrocytes into immortalized cultures with neural stem cell characteristics, that are able to generate of GBM when injected in the brain of C57BL/6 mice, or heterotopic tumours when injected iv. Overall, our results show that oncogenic transformation is a fate for NSC if cultured for long periods in vitro. In addition, since no additional hit is necessary to induce the oncogenic transformation, our model may be used to investigate the pathogenesis of gliomagenesis and to test the effectiveness of different drugs throughout the natural history of GBM.


Introduction
Glioblastoma (GBM) is the most malignant and highly aggressive type of gliomas, which accounts for almost 50% of primary malignant brain tumours in adults. However, it still carries a poor prognosis, with a 5-year survival of 4.7%, due to its aggressive and invasive nature, and its remarkable heterogeneity [1][2][3][4][5]. These characteristics complicate the development of effective therapeutic strategies and compel the need for more trustworthy models to study the disease and test new drugs and therapies.
In vitro cultures are widely employed as suitable models to investigate both the pathogenesis and the therapeutics of GBM, although these models are imperfect for several reasons. Different lines of immortalized glioma cells, such as U87, U251, T98G, or CT-2A have been widely used for more than 30 years [6][7][8]. However, although the use of cell lines has provided a valuable knowledge about GBM, they undergo profound phenotypical changes when grown as monolayers, and fail to develop the defining morphological featur es of GBM tumours when injected in vivo. These cells also exhibit markedly different responses to cytotoxic treatments than those observed in patients, and controversy regarding their origins has recently appeared [8].
On the other side, the use of patient derived cells has become the gold standard of GBM preclinical studies, mainly to test personalized therapies, and a library of annotated and validated cell lines derived from surgical samples of GBM patients has been r ecently created [5,6]. It is expected that the use of validated cells, together with novel culture systems that better recapitulate the complex reality of brain tumours growing in situ [7] may provide a more suitable model for preclinical GBM research in the next future. However, despite their clear benefits over some current models, these models still have important limitations when investigating the pathogenesis of GBM, since they are established from already developed tumours, either spontaneously gener ated in a patient or experimentally induced in a cell line or laboratory animal. Therefore, it is very difficult to recapitulate the natural history of the oncogenic process and identify molecular targets involved in the early development of the disease.
In this study we describe a novel cellular model that allows to investigate the transformation of mouse neural stem cells (NSC) into aggressive GBM with metastatic capacity. Our results show that oncogenic transformation occurs in immortalized NSC because of long-term passaging in vitro, and without the need of any external hit. Furthermore, this transformation occurred with a 100% frequency, indicating that it is the fate of immortalized NSC in vitro, at least under our experimental conditions. Whether a similar process may occur in vivo, as a consequence of the dysregulation of the neurogenic niche, remains to be elucidated.

Generation of NSC with capacity to generate GBM in vivo
In keeping with our previous report [9], both LP-and HP-immortalized cultures obtained from mouse embryo brains retain their capacity to express specific markers for all neural lineages (data not shown). Also in concordance with our previous findings, neurosphere formation can be induced by placing the cells under NBE conditions (data not shown). Altogether, these characteristics indicate that both types of cultures can be considered as bona fide immortalized NSC. Table 1. Ge ne ration of GBM in mice afte r orthotopic inje ction of primary (1), LP-immortalize d NSC (2)  In addition, we also reported that HP-immortalized NSC show a sharp increase in their proliferative rate, as compared with either primary or LP-immortalized cells, together with the presence of numeral chromosomal abnormalities [10]. Since these phenotypical traits may indicate a potential for cellular transformation, in the present study, we first investigated their capacity to generate brain tumours in vivo. To this end, cell suspensions of either LP-or HP-immortalized NSC were injected into the cerebral parenchyma of C57BL/6 mice. As table 1 shows, development of orthotopic tumours was observed in all mice injected with HP-immortalized NSC (40,60 or 90 passages), as soon as 3.5 months of injection (see also Fig. 1A). In contrast, no tumours were observed in mice injected with either primary cultures or LP (15 passages) immortalized cells. Tumours showed many of the distinctive characteristics of GBM [2] including a marked cellular pleomorphism, and the presence regions of necrosis, together with extensive vascular hyperplasia, thrombus formation, invasion of vessel walls, and prominent Ki67 expression ( Fig. 1B and 1C). Demonstrati on that tumours are not spontaneously generated but arise from the injected cells was obtaining by detecting the expression of RFP in the tumour cells (Fig. 1D). Finally, as occurs with immortalized NSC, cells isolated from GBM can growth as a monolayer cultures, that can be induced to form neurospheres by placing them in NBE conditions (Fig. 1E). Furthermore, GBM-derived cells also retain their capacity to express specific markers from all the three neural lineages ( Fig. 1E and 1F), although a prominent decrease in GFAP expression exists. Immunohistochemical de tection of Ki67 shows incre ased e xpression in GBM (le ft), as compared with controls (right). (d) De monstration that GBM arise from inje cte d ce lls was obtaine d by immunohistoche mical de tection of the RFP prote in (le ft), while no RFP e xpre ssion was de tected in controls (right). (e ), (f). GBM-de rive d ce lls re tain NSC characteristics. Tumours we re dissociated and ce lls we re grown in DMEM (se e materials and me thods for furthe r de tails). Ne urosphere formation was induce d by placing the ce lls in NBE conditions. Expre ssion of spe cific line age marke r s for astrocytes (GFAP), oligode ndrocytes (MBP) and ne urons (ne stin) was de te cted by immunocytochemistry (e) (lowe r panels are the negative controls) or by semi-quantitative RT-PCR (f) (in this case, primary cells we re used as control).

Generation of NSC with capacity to generate heterotopic (metastatic) tumours in vivo
One of the more intriguing aspects of the biology of GBM is their ability to form metastasis, most likely because of the notion that extracranial/extraspinal metastases of human GBM are clinically rare [11][12][13][14]. Therefore, we took advantage of our cellular model to investigate the ability of either HPimmortalized NSC or GBM-derived cells to form metastatic (heterotopic) tumours in vivo after injection in the tail vein of C57BL/6 mice. As table 2 shows, macroscopic lung tumours were observed in all mice injected with HP-immortalized NSC (4 mice) after 6 months of injection. In contrast, no tumours were detected after 3 or 4 months of injection of HP-immortalized NSC. In addition, a macroscopic lung tumour was also detected in one of three mice injected with GBM-derived cells, although, in this case, the tumour developed as soon as 3 months after injection ( Table 2). Macroscopic examination of metastatic lungs showed the presence of singl e or multiple metastatic nodules with cannonball appearance and frequent haemorrhagic foci ( Fig. 2A). Haematoxylin and eosin staining of the lung metastasis showed the existence of distinct characteristics of GBM-derived cells, including the presence of marked cellular pleomorphism and necrosis (Fig. 2B), together with the existence of prominent Ki67 expression (Fig. 2C). As in the case of primary brain tumours, demonstration that metastatic (heterotopic) tumours arise from injected cells was obtained by investigating the existence of RFP expression (Fig. 2D). Furthermore, cells isolated from metastatic tumours can growth as monolayer cultures that can be induced to form neurospheres when placed in NBE conditions (Fig. 2E). As also occurs with GBM-derived cells, cells obtained from heterotopic tumours retained the expression of specific markers for all neural lineages ( Fig. 2E and 2F).  De monstration that GBM arise from inje cted cells was obtained by immunohistochemical de tection of the RFP prote in (le ft), while no RFP e xpre ssion was de tected in the control. (e ), (f). Ce lls from lung me tastatic tumours display NSC characteristics. Lung tumours we re dissociated, and ce lls were grown in DMEM (se e materials and methods for further de tails). Ne urosphere formation was induced by placing the ce lls in NBE conditions. Expre ssion of spe cific line a ge markers for astrocytes (GFAP), oligode ndrocytes (MBP) and ne urons (nestin) was de tected by immunocytochemistry in cells de rived from me tastatic lung tumours (e ). Lowe r pane ls are the ne gative controls. Notice the pre sence of ne urospheres attached to the culture dish in all cases. Expre ssion of specific line age marke rs was also de te cted by semi-quantitative RT-PCR (f) (in this case, primary cells we re used as control). Despite the low rate of extracranial metastasis that exists in GBM, recent reports have described the existence of circulating tumour cells (CTC) in the peripheral blood of patients with GBM [13,15]. Therefore, in order to further investigate the blood dissemination of injected cells, we performed cellular cultures of brain, lung, kidney, spleen, brain, bone marrow, testicles, and liver, obtained from mice injected with either HP-immortalized cells or GBM-derived cells. The presence of tumoral cells in these tissues, was demonstrated by growing primary cultures in DMEM, and inducing the formation of neurospheres by placing them in NBE conditions. Evidence of microscopic heterotopic tumours was found in kidney, liver and/or spleen in all but one mouse with macroscopic lung tumours induced by injection of HP-immortalized NSC (Table 2). Microscopic lung and liver metastasis were also observed in the two mice injected with GBM-derived cells that did not develop macroscopic tumours three months after injection ( Table 2). Formation of neurospheres was not observed, in any case, in primary cultures from bone marrow, testicle, or brain. As expected, neurospheres expressed specific markers for all neural lineages. (Fig. 3).

Figure 3.
Ide ntification of microscopic he terotopic tumours. Cell suspe nsions of 3x105 ce lls obtained from HP-immortalize d NSC (90 passages) or GBM-de rived cells we re injected in the tail ve in C57BL/6 mice and ce ll culture s from brain, lung, kidne y, sple e n, brain, bone marrow, te sticles, and live r were pe rformed to inve stigate the pre sence of micrometastasis. In all cases, cells we re grown in DMEM (se e materials and me thods for furthe r de tails)., and ne urosphere formation was induce d by placing the ce lls in NBE conditions. Re pre sentative image of a cell culture ge nerated from live r is shown. The pre se nce of ne urospheres attached to the culture can be appreciated. Expre ssion of specific lineage marke rs for astrocytes (GFAP), oligode ndrocytes (MBP) and ne urons (ne stin) was de te cted by immunocytochemistry. Lowe r panels are the negative controls.

Phenotypical characterization of immortal ized, tumoral and metastatic NSC
Altogether, our results demonstrate that continual passaging of cell cultures obtained from mouse embryo brain results in the generation of immortal cells with NSC characteristics that, finally, acquire the capacity to generate both primary GBM and heterot opic tumours in vivo. Therefore, to better understand the mechanisms underlying this transformation, we next investigated the presence of cellular aneuploidy, one of the hallmarks of the oncogenic process [16 -18]. As figure 4 depicts, more than 80% of primary cells have 40 chromosomes, but a sharp increase in the number of numerical chromosome abnormalities is observed in immortalized cells, even in LP cells. This finding is in concordance with our previous reports [19] and indicates that hyperploidy is an early event within the transformation process of NSC. Although the percent of cells with severe polyploidy is greater in HP-immortalized or in tumoral (either primary or heterotopic) cells, the most striking changes occur soon after immortalization is achieved.  for e ach cell type , showing a dramatic incre ase in the numbe r of hype rploid ce lls, alre ady pre sent in LP-immortalize d NSC (ce ll type 2), as compared with primary ce lls (cell type 1). An incre ase in the numbe r of highly hype rploid ce lls is also obse rved in HP-immortalize d NSC (ce ll type 3), as we ll as in the othe r ce ll type s. (b) Re presentative images of re sults pre sented in pane l A.
These changes in cellular aneuploidy correlate with other phenoty pical and biochemical modifications observed. When compared with primary cells, both LP-and HP-immortalized cells showed an increase in their proliferation rate ( Fig. 5A and 5B), a finding that is also in keeping with our previous results [19]. Interestingly, the increase in growth rate observed in HP-immortalized cells is similar to that observed in GBM-derived cells ( Fig. 5A and 5B), and contact inhibition was preserved in both cases (data not shown). However, the highest proliferative rate was observed in cells obtained from heterotopic tumours, either generated from HP-immortalized cells or GBMderived cells (Fig. 5A and 5B). These differences in cell proliferation sharply correlate with the changes observed in the expression of Ki67 and in the rate of BrdU incorporation. In fact, while less than 50% of either primary or LP-immortalized cells show immunoreactivity to Ki67, more than 90% of HP-immortalized or GBM-derived cells, and virtually all cells derived from heterotopic tumours are Ki67+ (Fig. 5C). In the case of BrdU incorporation, a significant increase was already observed in LP-immortalized cells when compared with control cells, but the most striking boost was found in HP-immortalized NSC. BrdU uptake remained similarly increased among all tumou r-derived cell types, either primary or metastatic (Fig. 5D). Also in keeping with our previous findings, continual cell passaging resulted in the appearance of dramatic morphological changes. Although flat, polygonal cells were still present in hastily gr owing cells, most of them displayed distinct    de rive d ce lls (ce ll type 4) or me tastasis -derived ce lls (ce ll type s 5 and 6) we re culture d, and ce ll numbe r was de te rmined by crystal-violet staining. A progre ssive increase in the growth rate was obse rved from 1 to 5-6. (b) Re pre sentative images of re sults pre sented in pane l A. (c) Ki67 staining in the diffe re nt ce ll type s. The pe rce nt of Ki67+ ce lls was de te rmined afte r scoring the m by immunocytochemistry. (d) BrdU incorporation was de te rmined by immunocytochemistry. In this case, e ach bar represents the me an+SEM of 3 e xpe rime nts in triplicate . *=p<0.05 vs primary ce lls.

Biochemical characterization of immortalized, tumoral and metastatic NSC
Finally, we investigated the biochemical changes underlying the timeline of the modifications that led to the transformation of primary cells into GBM-producing cells. Although GBM displays a heterogeneous profile, a common feature of all GBM types is an aberrant kinase signalling, and among the pathways implicated, the RAS -mitogen-activated protein kinase (MAPK), is one of the most frequently dysregulated in GBM cells [20 -22]. In keeping with this, undetectable levels of either Raf-1 or ERK1 phosphorylation were observed in both primary cultures and LP-immortalized cells (Fig. 6), while a clear rise was detected in HP-immortalized cells. Furthermore, this activation was progressively increasing throughout the transformation process, with the highest phosphorylation levels found in cells from heterotopic tumours. A similar pattern of activation was observed in the case the two leading members of the RSK (90 kDa ribosomal S6 kinase) family, RSK1 and RSK2, two downstream  effectors of the ERK pathway [23] (Fig. 6). RSK1 phosphorylation was undetectable in Figure 6. Transformation of immortalized NSC is associated with an incre ase in the activation of the ERK signalling pathway. Primary (ce ll type 1), LP-immortalize d NSC (ce ll type 2), HP-immortalize d NSC (ce ll type 3), GBM-de rive d ce lls (ce ll type 4) or me tastasis -derived cells (cell type s 5 and 6) were culture d and the phosphorylation le vels of proteins of the Ras-Raf-ERK pathway (Raf-1, ERK1, RSK1, RSK2), p27 and p53 we re inve stigated. A progre ssive increase in the activation of the pathway, was obse rved throughout the process, with the highe st lowe st phosphorylation le vels found in primary or LP ce lls (ce ll type 1 and 2, re spectively) and the highest le vels in ce lls obtained from he terotopic tumours (ce ll type s 5 and 6).
either primary or LP-immortalized cells, but progressively increased in the other cell types, with the highest phosphorylation levels detected in cells from heterotopic tumours. In the case of RSK2 no phosphorylation was detected in primary, LP-immortalized, and HP-immortalized cells, and phosphorylation levels were also unexpectedly low in cells from heterotopic tumours originated from HP-immortalized NSC. Interestingly, although, both RSK1 and RSK2 are associated with glioma malignity, RSK1 has been described as a potential progression marker and a therapeutic target for gliomas. In fact, RSK1 phosphorylation levels (Ser380), which more specifically reveals ER K activation, are higher in GBM [24]. Additionally, RSK1 phosphorylates p27 at Thr198, leading to accumulation of phosphorylated p27 in the cytoplasm [25]. Accordingly, p27 levels progressively increased with a similar pattern to that observed in RSK-1. These findings, together with the increased levels of p53 detected, may suggest that, as we previously shown [26], increased cell proliferation (and, eventually, cell transformation) may occur despite the existence of a normal activation of the cellular checkpoints involved in the induction of senescence. Surprisingly, a so clear pattern of activation was not observed when other signalling pathways were investigating. This is the case of the phosphatidylinositol 3-kinase (PI3K)-Akt pathway that is activated in almost 90% of all GBM [27][28][29]. However, we were unable to find any difference in the phosphorylation level of the leading members of this pathway, including PI3K, Akt, phosphatase and tensin homolog (PTEN) or mammalian Target of rapamycin (mTOR) (Suppl Fig. 1), among the different cells types. Overall, these results indicate that immortalization of NSC may be associated with a progressive activation of the Ras/Raf/ERK signalling pathway.  (4); while iv inje ction of HP-immortalize d NSC re sults in the formation of he te rotopic (me tastatic) tumours (5). Finally, formation of he te rotopic (me tastatic) tumours is also observed after iv inje ction of GBM-de rive d ce lls (6). As indicate d in the figure , the capacity to form ne urospheres was assessed in e ve ry cell type .

Discussion
In the present study, we have demonstrated that continual passaging of immortalized NSC results in their transformation into cells that are able to produce either GBM or heterotopic tumours, when injected in adult mice. Although further studies are still necessary, our results support a role for NSC in the pathogenesis of GBM, at least for primary GBM, which accounts for more than 90% of this type of tumours [1]. This proposal is in keeping with other studies indicating that NSC may be the cells from which GBM originate, due to their self-renewal and proliferative capacities, and the relevance of the accumulation of somatic mutations in the process of gliomagenesis [29 -31].
The role of embryonic stem cells on cancer has been largely debated. Cancer initiating cells or cancer stem cells (CSC) are a subpopulation of cells that has the driving force of carcinogenesis [30,31] that can be identified in most types of human cancer [32][33][34][35]. Although not all CSC are originated from embryonic cells, specific criteria to define CSC include (1) the ability to self-renew; (2) the capacity to differentiate into different lineages (multipotency); and (3) the ability to initiate tumours in animal models, which recapitulate the original disease phenotype and heterogenicity [36 -39]. All these criteria are fulfilled by the HP-immortalized NSC used in this study and, therefore, they can be considered as truly CSC, thus supporting the hypothesis of NSC as the source of GBM. In this regard, it has been recently demonstrated that astrocyte-like NSC in the SVZ are the cell of origin that contains the driver mutations of human GBM [40]. Furthermore, astrocyte-like NSC that carry driver mutations migrate from the SVZ and lead to the development of high-grade malignant gliomas in distant brain regions [40].
Of special interest is the fact that no "external" hit is needed to induce the malignant transformation of NSC, other than their continuous passaging and the stress induced to overcome replicative senescence [19]. In fact, in contrast with the results reported in MEF placed on a 3T3 protocol, in which immortal cells emerge at low frequency [10], we were able to generate immortal NSC with a 100% frequency, and all HP-immortalized NSC were able to generate GBM when orthotopically injected in mice. Therefore, it is tempting to speculate that the mechanisms that allow primary cells to escape from replicative senescence are those leading to malignant transformation. In this context, it is interesting to note that those NSC that can overcome replicative senescence show increased activation of both p27 and p53 [19, and the present work]. This result, together with the early occurrence of hyperploidy in immortalized NSC may suggest the existence of an uncoupled response to the activation of the DNA damage response (DDR). Interestingly, similar results have been previously reported by our group in response to oncogene-induced senescence (OIS) in primary MEA [26]. In these cells, activation of Ras and elimination of RB does not induce cellular senescence, despite the activation of the cellular checkpoints related to DDR. It must be emphasized that MEA are the cells that give rise to immortal NSC after overcoming replicative senescence [9, 19 and the present study]. Therefore, the ability of MEA to escape cellular senescence, either replicative or OIS, could underlie an important role in gliomagenesis.
Although we do not presently know the mechanisms underlying this defective response, it is interesting to notice that the transition from primary to immortalized cells is associated with an almost total loss of euploid cells. Aneuploidy is one of the hallmarks of most human cancers, with around 90% of all solid tumours b eing aneuploid, mostly hyperdiploid [41][42][43]. Although both the mechanisms leading to aneuploidy and their consequences on tumorigenesis are still controversial, it has been proposed that tumour cells with significantly elevated genomic content (polyploid t umour cells) facilitate rapid tumour evolution. Furthermore, aneuploidy has been shown to precede transformation in a variety of cancers [44][45][46][47], and it has been proposed a role of aneuploidy during tumour initiation [16,17]. In keeping with this, severe polyploidy was also an early event in the natural history of the transformation process described in the study, so it can be hypothesized that it may be involved in the appearance of the other biochemical and phenotypical changes observed, including the increased activation of ERK signalling [48,49].
It remains to be elucidated whether similar mechanisms may account for the oncogenic transformation of NSC in vivo. Under physiological conditions, NSC are regulated by the orchestration of intrinsic and ext rinsic signals that provide the complex regulatory architectures present in the neurogenic niche. The special microenvironment defined within the neurogenic niche permits the maintenance of the NSC pool and prevents terminal differentiation, thus allowing the balance between cell loss and cell replacement in the central nervous system [50 -52]. Under our experimental conditions, cells are grown in the presence of FBS and, therefore, exposed, for a long time, to a wealth of growth factors that are not present within the neurogenic niche. Therefore, it is tempting to speculate that disturbance of the microenvironment within the neurogenic niche may result in a dysregulated proliferation of NSC that, eventually, may cause the appearance of a GBM. In this respect, it has been shown that NSC niches of the human brain may be a mutational source for brain somatic mutations [42].
Another interesting conclusion of our study is the capacity of immortalized NSC to disseminate and form heterotopic tumours when injected in mice. Extracranial/extraspinal metastasis is a very uncommon entity in GBM, and only a low number of patients develop extracranial metastasis [11-Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 28 August 2020 doi:10.20944/preprints202008.0637.v1 14]. Although historically, GBM were not believed to metastasize because of the presence of the blood-brain barrier, improvements in the treatment of primary GBM has led to an increased in reported metastasis, mostly in the lungs, but also in other organs such as lymphatic nodes, bone, or liver [11][12][13][14]. In keeping with this, we found that lungs were the organs th at more frequently presented heterotopic tumours (macroscopic and microscopic), but a widespread hematogenic dissemination was observed. This capacity for hematogenic dissemination is in concordance with recent studies demonstrating the existence of circulating tumour cells in the blood of 20%-39% of patients with GBM [53,54]. Since occurrence of extracranial/extraspinal metastasis is expected to increase as GBM treatment becomes more efficient, our results support the validity of our model to mimic in vitro the natural course of the disease in vivo. It is also quite remarkable that HPimmortalized NSC, have the same capacity to be highly metastatic than cells obtained from primary GBM. This indicates that metastatic genotype is acquired early in tumour progression as has been already proposed [55,56].
In summary, we have developed an experimental model that allows to recapitulate the process of transformation of mouse embryo NSC into GBM-producing cells in vitro. Since most of the cells enter in senescence when submitted to the 3T3 protocol [19 and the present study], we do not presently know whether those cells that are able to overcome senescence, are the NSC present in the culture or just a fraction of them. In other words, it remains to be elucidated wh ether all NSC have the capacity of immortalization, or just some of them, as a consequence of a transformation process. In any case, we have found that the transformation process occurs with a 100% frequency, thus suggesting that oncogenic transformation is the unavoidable fate for, at least, a population of NSC, when undergoing long-term proliferation in vitro. Furthermore, the transformation likely occurs because of the presence of serum factors in the culture medium, thus highlighting the importance of maintaining the NSC in an adequate environment. While remains to be elucidated whether a similar mechanism may be involved in the pathogenesis of GBM in vivo, a warning should be made about the transplantation of NSC as a tool for neural repair [51].
Finally, we also describe a novel model of gliomagenesis that recapitulates in vitro the transformation of primary MEA into cells that are able to generate both GBM and heterotopic tumours in vivo. This model has two important advantages over current systems. First, it can be used to recapitulate the pathogenesis of GBM and, therefore, to elucidate the timeline of the mutations that accounts for the generation of GBM. Second, it provides with a valuable tool to test the effectiveness of therapeutics at different times throughout the natural history of the disease. This characteristic would help to the development of personalized treatment strategies by tailoring drug effectiveness with mutational profiles. Finally, given the ability of all cell types (including those derived from GBM or heterotopic tumours) to form neurospheres when placed on LBE conditions, they can be used to generate 3D organoid culture systems [57,58] to capture the phenotypic and molecular particularities of GBM, as we have recently shown [59].

Cell cultures
Immortalized NSC were generated from 13.5 dpc (day post coitum) C57BL/6 mice embryos, as previously described [19,26]. Briefly, pregnant C57BL/6 mice were sacrificed at 13.5 dpc and uterine horns were then dissected. Each embryo was separated from its placenta and embryonic sac and transferred to an individual petri dish with Dulbecco's modified Eagle's medium (DMEM, Sigma -Aldrich, Madrid, Spain). The head of each embryo was individually transferred to fresh petri dishes, and brains were then isolated, finely minced, and dissociated by mechanical shearing, followed by filtration through a 40-micron mesh. Cells were plated in Falcon polystyrene culture dishes (BD Biosciences, Madrid, Spain) and grown in DMEM supplemented with 10% foetal bovine serum (Thermo Fisher Scientific, Madrid, Spain), 2 mM glutamine, 2.5 U/mL penicillin, and 2.5 mg/mL Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 28 August 2020 doi:10.20944/preprints202008.0637.v1 streptomycin (all from Thermo Fisher Scientific). Cultures were maintained at 37ºC in a humidified atmosphere of 5% CO2. This method has been proved to consistently yield more than 95% of GFAP+ primary mouse embryo astrocytes (MEA) [9,19,26]. Immortalization of primary MEA was achieved by using the 3T3 protocol [10], with appropriate modifications [19]. Cells were tra nsferred to new dishes with fresh medium every 3 days, with the same cell density (3·10 3 /cm 2 ) seeded in every transfer. Continual cell passaging was used to generate long-term cultures of low passage (LP)immortalized cells (below 20 passages) or high pass age (HP)-immortalized cells (40-100 passages) (see Fig. 1 for further details). Both the immortalization protocol and the long-term maintenance of immortalized cells were carried out in DMEM. Formation of neurospheres was induced by using a modification of the neurosphere assay [9,60]. In this case, cells growing in DMEM, were trypsinized and transferred to fresh polystyrene culture dishes (approximately 3×10 5 /dish) with semi-synthetic (neurobasal) medium supplemented with 100 U/mL penicillin, 100 μg/mL st reptomycin, 1% Gibco B27 supplement, 10 ng/mL bFGF, and 10 ng/mL EGF (NBE conditions) [61]. Since immortalization occurred with a 100% frequency, and no differences were observed in the outcome of the cells, results obtained with different batches of immor talized NSC are presented throughout this study.
To ensure the detection of the immortalized cells after injection in mice, and to rule out a spontaneous origin of the generated tumours, luminescence and fluorescence reporter systems were developed.
To create the luminescence reporter, the pBABE-puro-Luc vector was transfected into Phoenix ecotropic retroviral packaging cells (a gift from C. Watzl) [62] by using JetPEI polyplus transfection system (Illkirch, France). Retroviruses-containing supernatant was collected 48 hours after transfection and used to infect immortalized cell cultures, using polybrene (5 g/mL) as transduction enhancer. Cells were then selected for 48 hours with 2.5 g/mL puromycin, and infection efficiency was measured by quantifying the luciferase activity using an optical in vivo imaging system (IVIS Spectrum, Perkin-Elmer, Waltham, MA). A red fluorescence reporter gene was created by replacing the sequence coding for GFP in a pMSCV PIG vector (Puro IRES GFP empty vector) with the sequence encoding the monomeric far-red fluorescent protein TagFP635 from a pTagFP635-C vector. The new vector was transfected into Phoenix ecotropic retroviral packaging cells and the supernatant was used to infect immortalized cells, as indicated before.

Generation of orthotopic tumours
Orthotopic tumours were generated by stereotaxic injection of immortalized NSC. Six -week-old C57BL/6 male mice were anesthetized by intraperitoneal injection of a ketamine (80 -100 mg/kg) and xylazine (16-20 mg/kg) mixture and returned to a holding cage briefly before being placed on a stereotaxic frame (David Kopf Instruments, Tujunga, CA). The surgical area was carefully scrubbed, and a sagittal incision was made through the scalp. The periosteum was then retracted b y gently rubbing and microinjections were made in the brain parenchyma according to a topographical map [63] (coordinates: anterior −0.2 mm, lateral 1 mm from bregma and depth 2.2 mm from the skull surface) by using a 25-gauge needle (Hamilton 7001 syringe, Hamilton Company, Reno, Nevada). The needle was gently inserted into the skull opening, retracted to accommodate the injection volume (5 L) and the cell suspension (1x10 5 cells) was slowly injected. Once the micro syringe removed, the head skin was sutured with surgical silk. Mice were monitored for anaesthetic recovery and postsurgical pain. To obtain GBM-derived cell cultures, tumours were finely minced and dissociated by mechanical shearing as indicated above. Cell cultures were grown in DMEM as descr ibed and neurosphere generation was induced by placing the cells in NBE conditions.

Generation of heterotopic (metastatic) tumours
To investigate the hematogenous dissemination of either HP-immortalized cells or GBM-derived cells, 3-week-old male mice were anesthetized with isoflurane (3% v/v), placed in a restrainer and injected with the corresponding cell suspension (3x10 5 cells diluted in 500 µL of saline solution). Cells were injected in the caudal vein with a 25-gauge needle, and animals were monitored for 5-10 minutes to ensure haemostasis. Cell cultures from macroscopic metastatic lung tumours were performed as described. After carefully dissenting the tumours from the surrounding tissue, they were finely minced and dissociated by mechanical shea ring. Cell cultures were grown in DMEM and neurosphere generation was induced by placing the cells in NBE conditions.
In order to test the presence of micrometastasis in several organs, cell cultures from lung, kidney, spleen, brain, bone marrow, testicles and liver were performed. Except for bone marrow, tissues were finely minced and dissociated as described. Form bone marrow cultures, femora and tibiae of the mice were harvested and flushed using phosphate-buffered saline (PBS) in a 1-ml syringe with a 25gauge needle. Cells were collected by centrifugation and seeded. In all cases, cultures were grown in DMEM and neurosphere generation was induced by placing the cells in NBE conditions.

In vivo imaging
For in vivo monitoring of tumoral growth, luciferin was injected intraperitoneally at a dose of 10 μl/g body weight. Mice were then anesthetized with isoflurane (3% v/v) and placed on the imaging stage of an optical imaging system (IVIS Spectrum). Images were collected every minute from 15 to 30 min after luciferin injection, and photon emission was quantified using Living Image Software (Living Image 3.1, PerkinElmer).

Immunocytochemistry
For immunocytochemical analysis, approximately 5×10 3 cells were seeded in glass coverslips, placed in 24-multiwell plates, and incubated for 24h, as described above.

Metaphase chromosome preparation
Metaphase spreads were prepared after treating the cells with colcemid (Sigma -Aldrich, 0.1 µg/mL) for 7 hr. Cells were then incubated in hypotonic buffer (0.05 M KCl, 0.0034 M trisodium citrate) for 20 min at 37°C and fixed in 75% methanol, 25% acetic acid. Cells were then spotted onto microscope slides and stained with 2% Wright stain (Sigma -Aldrich, 0.1 µg/mL) in Gurr buffer, pH 7.0 (Invitrogen). Metaphase chromosomes were scored using a Leica 2005 microscope under a 100X oil objective lens. At least 50 metaphases were analysed from three independent experiments.

Cell proliferation assay
Cells were seeded at 10x10 3 (primary), 5x10 3 (immortalized low passage) or 2,5x10 3 (immortalized high passage, GBM-derived and metastasis-derived) and grown in DMEM for 1, 3, 5 or 7 days. At these times, cells were fixed and stained with 0.1% violet crystal (Sigma) for 30 minutes and then washed with PBS to remove the dye excess. Dye content was quantified in a spectrophotometer. To determine the rate of BrdU incorporation, 10 6 cells were seeded in poly-L-lysine-coated slides (1 mL/slide) and maintained for 24h. Cells were grown in the presence of BrdU (10 mM, Sigma -Aldrich) for 2.5h, and BrdU+ cells were detected by immunocytochemistry using a monoclonal antibody (BD Biosciences, dilution 1:2000). Representative images were captured with an Olympus DP72 camera (Olympus Optical Co., Tokyo, Japan), and the number of BrdU+ cells was quantified in 10 random fields per slide.

Dot blot assay
A multiplexed protein detection kit (C-Series Human/Mouse AKT Pathway Phosphorylation Array C1, RayBiotech, Norcross, GA) was used to investigate the existence of changes in the signalling machinery among the different cell types generated. All procedures were performed according to the manufacturer's instructions Briefly, cells were washed with ice-cold PBS and solubilized in lysis buffer. Cell lysates were then centrifuged (14,000 rpm, 5 minutes, 4 ° C) and protein concentration was determined using the Bradford assay (Bio-Rad Laboratories). Membranes were blocked for 30 min at room temperature and incubated overnight at 4ºC with 200 g of each sample, before incubation with the detection antibody cocktail (2h at room temperature). Immunoreactivity was detected by chemiluminescence using X-ray film (Fuji Medical, Japan) to visualize the dots. Images were analysed with the ImageJ open source image software (https://imagej.nih.gov/ij/). Data were corrected for the local background and normalized for positive and negative internal controls.