A dangerous liaison: Leptin and sPLA2-IIA join forces to induce proliferation and migration of astrocytoma cells

Glioblastoma, the most aggressive type of primary brain tumour, shows worse prognosis linked to diabetes or obesity persistence. These pathologies are chronic inflammatory conditions characterized by altered profiles of inflammatory mediators, including leptin and secreted phospholipase A2-IIA (sPLA2-IIA). Both proteins, in turn, display diverse pro-cancer properties in different cell types, including astrocytes. Herein, to understand the underlying relationship between obesity and brain tumors, we investigated the effect of leptin, alone or in combination with sPLA2-IIA on astrocytoma cell functions. sPLA2-IIA induced up-regulation of leptin receptors in 1321N1 human astrocytoma cells. Leptin, as well as sPLA2-IIA, increased growth and migration in these cells, through activation/phosphorylation of key proteins of survival cascades. Leptin, at concentrations with minimal or no activating effects on astrocytoma cells, enhanced growth and migration promoted by low doses of sPLA2-IIA. sPLA2-IIA alone induced a transient phosphorylation pattern in the Src/ERK/Akt/mTOR/p70S6K/rS6 pathway through EGFR transactivation, and co-addition of leptin resulted in a sustained phosphorylation of these signaling regulators. Mechanistically, EGFR transactivation and tyrosine- and serine/threonine-protein phosphatases revealed a key role in this leptin-sPLA2-IIA cross-talk. This cooperative partnership between both proteins was also found in primary astrocytes. These findings thus indicate that the adipokine leptin, by increasing the susceptibility of cells to inflammatory mediators, could contribute to worsen the prognosis of tumoral and neurodegenerative processes, being a potential mediator of some obesity-related medical complications.


Introduction
Over the last years, many studies have stated a harmful synergy among cancer, obesity and diabetes: individuals with diabetes and elevated body mass index are more likely to develop cancer; and cancer patients, who also suffer from diabetes or obesity, show a higher risk of mortality than non-diabetic and non-obese ones [1][2][3]. According to this, a recent study has demonstrated that in high grade glioma patients, pre-existing diabetes and obesity are independent risk factors for early progression and death [4]. Glioblastoma is the most common a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Cell culture 1321N1 human astrocytoma cells, a gift from Dr. JH Brown (UCSD, San Diego, CA, USA), were cultured in DMEM supplemented with 100 U/ml penicillin/streptomycin, and 10% heatinactivated fetal calf serum (FCS) at 37˚C in 5% CO 2 . Primary mouse astrocytes were obtained as described previously [25]. More than 95% of cells were stained for astrocyte specific glial fibrillary acidic protein (Santa Cruz Biotechnology Inc).
Primary astrocytes and astrocytoma cells were incubated in serum-free medium 24 h before the experiments, and then stimulated as indicated, in the presence or absence of inhibitors.

Immunofluorescence studies
Serum-starved 1321N1 cells, on 18-mm cover glass coverslips, were fixed in cold 4% paraformaldehyde for 10 min, permeabilized in 0.3% Triton X-100 for 10 min and blocked with 3% BSA for 1h. Immunostaining with ObR antibody (H-300; Santa Cruz Biotechnology Inc) was performed at room temperature for 30 min, followed by incubation with FITC-conjugated secondary antibody. Cells were then analyzed under a Nikon Eclipse 90i (Nikon Instruments, Inc.).

Chemotaxis assay
Cell migration assays were performed using 24-well Transwell plates with 8μm-pore polycarbonate membrane filters, following the manufacturer's instructions. Lower wells were filled with medium containing vehicle, 50% FCS or leptin, as indicated. Cells (1x10 6 cells/ml) were added to the upper chamber. Before the migration assay was performed, and when indicated, cells were pretreated for 30 min with selective inhibitors, and then stimulated with sPLA 2 -IIA or leptin for 30 additional min. Migration was allowed for 4 h at 37˚C in 5% CO 2 . After incubation, nonmigrating cells were scraped off from the upper surface of the filter. Migrating cells were fixed with 4% paraformaldehyde and stained with 0.1% Crystal Violet in 20% methanol-PBS for 3 min. To quantify migration, the bound stain was solubilized and absorbance measured at 590 nm. Each experiment was repeated at least three times in triplicate. Chemotactic index was calculated from the number of cells migrating towards chemoattractant/number of cells migrating towards vehicle control.

Phosphatase assay
Cells were resuspended with PBS, subjected to three rounds of freeze/thaw and then sonicated for 10s. Serine/threonine and tyrosine phosphatase activities were measured using a commercially available specific phosphatase assay kits (Molecular Probes Inc). Five micrograms of protein were used in the assay. The generated fluorescent product was determined using a fluorometer (Ex 358 nm and Em 452 nm).

Data presentation
The results are presented as the mean ± SD or as representative results from more than three different batches of cells, unless otherwise stated. Statistical significance was assessed by using ANOVA followed by Bonferroni post-hoc testing, with Graph Pad Prism software, version 5 (GraphPad Software Inc).

Leptin stimulates EGFR-independent proliferation of 1321N1 cells
We first checked for the constitutive expression of the leptin receptor (ObR) on human 1321N1 astrocytoma cells ( Fig 1A). Then, we examined whether this expression could be altered during exposure to proliferative agents also present in the tumor microenvironment. As shown in Fig 1B, we found that cell stimulation increased constitutive ObR expression levels on the cell surface. After a 24 h-stimulation with sPLA 2 -IIA, EGF or PGDR the population of cells expressing high levels of OB-R increased from the 18% observed in control, to 42%, 45% and 66%, respectively.
We next analyzed leptin-induced proliferative responses in these cells, since cell growth is a well-known effect of this adipokine in tumoral cells. As shown in Fig 1C, leptin enhanced 1321N1 cell proliferation in a dose-dependent manner and reached a 2.1 ± 0.4-fold (p 0.001) increase when stimulated with 0.5 μM leptin for 24h.
Moreover, we analyzed whether leptin-induced proliferative responses in 1321N1 cells involved EGFR or PDGFR, important signaling mechanisms in cancer cells. We incubated cells for 30 min in the presence of different doses of the EGFR kinase inhibitor AG1478 or the PDGFR kinase inhibitor AG1296, before stimulation with 0.5 μM leptin or 0.4 μM EGF for 24h. The presence of specific inhibitors did not affect proliferation induced by leptin, whereas, as expected, pretreatment with the EGFR kinase inhibitor AG1478 inhibited the EGF-induced signalling and proliferation ( Fig 1D). Cross-Talk between leptin and sPLA2-IIA Leptin-induced Proliferation of 1321N1 cells requires the activity of classical ERK, Akt, pathways Next, to investigate whether the activation of Src/ERK/Akt/mTOR kinases were key elements of the leptin induced proliferation in 1321N1 astrocytoma cells, they were incubated in the absence or presence of an optimal concentration of human recombinant leptin (0.5 μM) at different times. Flow cytometry analysis revealed that resting 1321N1 cells constitutively expressed Tyr 416-phophorylated Src. Levels increased 4-fold over control by 3 min after leptin treatment, and declined to near baseline levels by 30 min (Fig 2A).
We also found that leptin treatment immediately increased the presence of phosphorylated extracellular signal regulated kinases (ERK, p-ERK), reaching a maximum level of phosphorylation around 10 min, decreasing afterwards to reach basal levels, approximately at 60 min ( Fig  2A, S1A Fig).
Similarly, we observed Akt phosphorylation on Ser473 with a time course that paralleled ERK activation/phosphorylation profile. In addition, the rapamycin-sensitive molecules P70 ribosomal protein S6 kinase (P70S6K) and S6 ribosomal protein (rS6) were also phosphorylated upon leptin exposure following a similar pattern, with a maximal effect at 15 min, which almost disappeared 60 min after incubation. 1321N1 cells stimulated with 0.5 μg/ml of sPLA 2 -IIA for 15 min were used as a positive control.
To identify the role of these signalling elements on leptin-induced astrocytoma cell growth, the mitogenic response of 1321N1 cells to 0.5 μM of leptin was tested in the presence or absence of several selective pharmacological inhibitors. As shown in Fig 2B, leptin-induced proliferation was almost abolished by inhibition of ERK with the MEK inhibitor PD98059. Inhibition of the PI3-kinase/Akt pathway with LY 294002, or with the specific mTOR inhibitor rapamycin, also diminished cell proliferation in a concentration-dependent manner ( Fig 2B). We also found that 20 μM of the Src inhibitor, PP2, blocked leptin-induced cell proliferation (not shown).
However, the presence of different doses of the PKC inhibitor GF109203X did not markedly affect the response induced by leptin in 1321N1 cells (p>0.05) (Fig 2C and 2D, S1B Fig), while EGF induced cell proliferation, as well as ERK and Akt phosphorylation, was abrogated by pre-treatment of the cells with 5 μM of GF109203X ( ÃÃ p 0.001).
To understand and further characterize the activation sequence of leptin in 1321N1 cells, we incubated them in the presence or absence of selective inhibitors. We observed that ERK phosphorylation was abolished by pretreatment of the cells with the MEK inhibitor PD98059, which also abrogated activation of p70S6K and rS6, leaving Akt phosphorylation unaffected ( Fig 3A, S2A Fig). We also found that PP2, the Src inhibitor, blocked ERK phosphorylation (data not shown). LY294002, the PI3K inhibitor, significantly reduced the phosphorylation of Akt/p70S6K/rS6 signaling proteins, but it did not affect ERK ( Fig 3B, S2B Fig) or Src (data not shown) phosphorylation induced by leptin. In addition, rapamycin, the mTOR inhibitor, also reduced p70S6K and rS6 phosphorylation ( Fig 3C, S2C Fig).
Finally and as expected, the presence of the EGFR inhibitor, AG1478, did not affect leptinstimulated phosphorylation/activation of these signaling molecules, while it abolished EGFstimulated phosphorylation (Fig 3D, S2D Fig). The PDGFR kinase inhibitor AG1296 did not abolish the immediate phosphorylation of the studied kinases either (not shown).
Leptin synergizes with sPLA 2 -IIA to induce proliferation of 1321N1 cells Next, we compared the proliferative activity of leptin in 1321N1 cells with known mitogens implicated in tumor pathogenesis such as EGF or sPLA 2 -IIA (Fig 4). As demonstrated previously, 0.5 μg/ml of sPLA 2 -IIA induced a strong mitogenic response in a serum-free cell culture  [23,24]. The simultaneous presence of 0.5 μM leptin and sPLA 2 -IIA, either at optimal (0.5 μg/ ml) or suboptimal (0.2 μg/ml) concentrations did not yield any significant additive effect ( Fig  4A). Interestingly, although the addition of a suboptimal dose of leptin, 0.1 μM, to the optimal concentration of sPLA 2 -IIA (0.5 μg/ml) had no major effect on the proliferative response of the sPLA 2 -IIA, when it was combined with the lowest dose of the phospholipase, 0.2 μg/ml, the proliferative response induced was significantly higher than promoted by either agonist    (Fig 4B), suggesting the possibility that leptin synergizes with sPLA 2 -IIA to stimulate growth.
Although EGF also induced a dose-dependent mitogenic response in the serum-free culture, the addition of 0.1 μM of leptin had no further effect on the proliferative activity of EGF at any dose tested (Fig 4C), showing a specific link between sPLA 2 and leptin signaling.
Leptin modulates migration of untreated and sPLA 2 -II-treated 1321N1 cells We focussed on leptin and sPLA 2 -IIA, alone or combined, in the search for factors and mechanisms involved in the invasive behaviour of brain tumors.
Experiments using leptin as a chemoattractant revealed a dose-dependent chemotactic motility on these cells (Fig 5A). Besides, we also found that leptin increases their spontaneous and FCS-mediated migratory capabilities in a concentration dependent manner. Pretreatment of 1321N1 cells with the selective pharmacological inhibitors LY294002, PD98059 or rapamycin reduced the chemoattractant capacity of leptin, as well as leptin-induced migration, both baseline and FCS-mediated ( Fig 5B).
Before searching for a cooperative effect between leptin and sPLA 2 -IIA, we assessed the capacity of sPLA 2 -IIA to modulate 1321N1 migration. As shown in Table 1, sPLA 2 -IIA pretreatment stimulated both spontaneous and FCS-induced cell migration. Then, cells were stimulated with a combination of sPLA 2 -IIA and leptin, at concentrations eliciting none or minimal migratory responses, and we observed an increased cell mobilization compared to either stimulus alone (Fig 5C). Likewise, addition of leptin to the lower transwell chamber, at doses eliciting minor or no detectable chemoattractant effect in untreated cells, further increased the migration of sPLA 2 -IIA-treated 1321N1 cells (Fig 5D).
Leptin modifies the temporal pattern of key signal-transduction elements activated by sPLA 2 -IIA on 1321N1 cells To elucidate the intracellular signaling mechanisms involved in the ability of leptin to potentiate sPLA 2 -IIA-induced biological responses, we investigated whether 0.1 μM leptin altered the time course of Src, ERK, Akt or p70S6 kinase activation induced by 0.2 μg/ml sPLA 2 -IIA. As shown in Fig 6 and S3 Fig, there was little or no increase in the magnitude of phosphorylation of the kinases studied after addition of a suboptimal dose of leptin (0.1 μM) to 1321N1 astrocytoma cells; however 0.2 μg/ml sPLA 2 -IIA led to a rapid transient activation of all the above mentioned kinases. Interestingly, incubation of the cells with leptin and sPLA 2 -IIA simultaneously led to an immediate and sustained increase in the phosphorylation of ERK, Akt and p70S6 kinase, up to 2h post stimulation. The effect on Src phosphorylation under these conditions was less pronounced.
These results may suggest that the ability of leptin to potentiate sPLA 2 -IIA-induced proliferation is due to the prolongation of sPLA 2 -IIA-induced kinases phosphorylation/activation.
Having previously established that sPLA 2 -IIA acts as a potent modulator of cell proliferation by inducing EGFR transactivation, we proposed to identify the potential role of EGFR in mediating the cooperative actions of leptin and sPLA 2 -IIA.
A significant and sustained increase in EGFR phosphorylation (Tyr 1176, Tyr 1068 and Tyr 845) was observed upon sPLA 2 -IIA stimulation, either alone or in combination with leptin, while leptin alone, as expected, showed no effect (Fig 7A). However, whereas in sPLA 2 -IIAstimulated cells EGFR phosphorylation level declined after 60 min of treatment, in cells treated with sPLA 2 -IIA+leptin the tyrosine phosphorylation status of EGFR was maintained, at least, 2h after stimulation. In keeping with these results, we showed that sPLA 2 -IIA-and sPLA 2 -IIA+leptin-mediated responses were sensitive to EGFR inhibition (Fig 7B and 7C, S4 Fig). We found that AG1478 was able to inhibit not only sPLA 2 -IIA-stimulated growth, but also the synergistic effect of leptin and sPLA 2 -IIA on 1321N1 cell proliferation. In addition, the persistent activation of the selected signaling proteins induced by the simultaneous addition of sPLA 2 -IIA and leptin was also dramatically reduced, particularly at later time points, where their phosphorylation was close to basal levels.

Effect of leptin on protein phosphatase activity
Since the homeostasis of protein phosphorylation events is governed by kinases and phosphatases, we looked for changes in the phosphatase activity, as a putative mechanism involved in the changes of the temporal phosphorylation pattern orchestrated by the presence of leptin. As shown in Fig 8, we found that leptin stimulation, both alone and in the simultaneous presence of sPLA 2 -IIA, decreased serine/threonine and tyrosine phosphatase activity in 1321N1 cells. In contrast, incubation of intact cells with sPLA 2 -IIA alone did not affect the phosphatase activity compared to non-stimulated cells. These findings suggest that sustained phosphorylation levels in MAPKs, Akt and EGFR promoted by co-stimulation of leptin and sPLA 2 -IIA in 1321N1 astrocytoma cells might be partly attributed to a reduced phosphatase activity.
Synergistic stimulatory effects of the combination of leptin and sPLA 2 -IIA on primary astrocyte cultures Next, we extended these observations related to the synergistic interactions between leptin and sPLA 2 -IIA to primary cultures of astrocytes. As shown in Fig 9A, cell proliferation was induced in serum-starved astrocytes upon treatment with leptin and sPLA 2 -IIA. Alike in 1321N1 astrocytoma cells, a synergistic effect on cell growth was found when astrocytes were stimulated with a suboptimal dose of leptin in combination with a suboptimal dose of sPLA 2 -IIA ( Fig  9C). Then, we analyzed the activation/phosphorylation of some representative intracellular signaling mediators associated with proliferation. Leptin and sPLA 2 -IIA increased ERK phosphorylation in a dose-dependent manner in primary cultures of astrocytes (Fig 9B). The simultaneous presence of both agonists at suboptimal doses also led to an immediate and sustained increase in the phosphorylation of the studied signaling proteins (Fig 9D, S5 Fig), as previously cells were treated as indicated for 30 min (loaded into top wells), and then allowed to migrate for 2 h to DMEM (C), or treated with medium or sPLA 2 -IIA (sPLA 2 ) for 30 min (loaded into top wells), and then allowed to migrate for 2 h to DMEM or leptin (loaded into the bottom wells) (D). ‡ p 0.001, n = 3.

Discussion
This study provides possible mechanisms to better understand how obesity-related inflammation may affect risk and prognosis of multiple diseases. We focused on cancer and glial tumors in particular, since a recent study shows that obesity worsens the prognosis of glioma patients [4]. To date, the pathophysiology and molecular mechanisms underlying these associations are not fully understood, although several hormonal systems-have been studied to some extent, and a leptin-cytokine cross talk has been observed in breast cancer [26] The role of leptin and sPLA 2 -IIA in the initiation and metastasis of tumors has been documented and reviewed, and here we pay attention to both proteins as emerging candidate molecules linking obesity and inflammation in glioma tumors [6,8,21]. This study illustrates key signaling molecules involved in leptin-induced proliferation and migration of human astrocytoma cells, and demonstrates a cross-talk between leptin and the inflammatory sPLA 2 -IIA. We observed that leptin increases the proliferative and migratory effects of sPLA 2 -IIA on both astrocytoma cells and primary astrocytes. The synergy between them is found at doses that individually show no activation, or activation below the reported maximal effects. Synergy between inflammation-related factors has already been pointed as an additional mechanism of the inflammatory system to sustain or amplify inflammatory responses when suboptimal cytokine/chemokine concentrations are present [27][28][29].
Effects of leptin on different glioblastoma cell lines had been previously examined in vitro, and leptin has been found: i) to induce migration and invasion of C6 rat cells linked to the activation of p38MAP kinase and NF-κB pathway, ii) to promote human U87 cells proliferation mainly through Janus-Activated Kinase (JAK)/Signal Transducer and Activator of Transcription 3 (STAT3) signaling pathway and iii) to enhance growth and migration of human LN18 and LN229 cells along with the activation of the PI-3K/Akt and STAT3 pathways [30][31][32]. The present study reveals that leptin-induced human 1321N1 astrocytoma cells growth and mobility requires the activation of key intracellular signal transduction elements from the major signalling cascades: MEK/ERK and PI3K/Akt/mTOR/P70S6K pathways, but not PKC activation as required by other proliferative molecules, such as EGF or sPLA 2 -IIA itself [24] Our data also suggest that both cascades converge to regulate pro-tumoral events, since inhibition of any of those pathways significantly diminishes leptin effects. Interestingly, although in some cell types leptin signaling is dependent on EGFR transactivation, in 1321N1 astrocytoma cells the presence of the specific EGFR inhibitor, AG1478, does not abrogate the mitogenic effects induced by leptin alone [33].
The directed chemotactic migration of tumoral cells is an essential component of tumor dissemination during progression and invasion. Here, we show that leptin not only behaves as a chemoattractant for 1321N1 cells, but it also improves FCS-induced chemotaxis. These results are inline with previous studies demonstrating cooperative chemotactic responses between leptin and some mediators from the molecular network by which obesity impacts/ modifies? the pathological manifestation of carcinogenesis, such as IGF-I [19]. In addition, we found that leptin-promoted chemotaxis of 1321N1 cells is dependent on activation/phosphorylation of similar crucial kinases, hence signaling cascades, to leptin-induced mitogenesis.
Our study also found out that surface leptin receptor is enhanced by pro-inflammatory sPLA 2 -IIA and growth factors, such as EGF and PDGF. Since inflammation and over- expression of growth factors in glioma tumors involves pro-inflammatory proteins production, including sPLA 2 -IIA; and these factors induce upregulation of the leptin receptor, it is conceivable that leptin has a cooperative role. Leptin enhances sPLA 2 -IIA-mediated effects in 1321N1 astrocytoma cells, and sPLA 2 -IIA enhances leptin capacity to signal by inducing overexpression of its receptor, thereby possibly contributing to a more aggressive behaviour of the basal tumor phenotype. sPLA 2 -IIA has been studied in different types of human malignancies, and it has been shown to exert diverse, and even contradictory, biological effects, probably due to tissue-or cell-specific functions [20,23,34]. We have previously reported that sPLA 2 -IIA mediates 1321N1 cell proliferation, and here we demonstrate that it also modulates astrocytoma cell mobility. Additionally, we found that the rapid and transient activation of crucial kinasessuch as Src, ERK, Akt, P70S6K -induced by sPLA 2 -IIA are prolonged in time in the simultaneous presence of leptin. These amplifying effects of leptin in the phosphorylation state of those signaling elements might be the cause of its synergistic effect, since a long-lasting phosphorylation could enhance the activity of the intracellular pathways involved in the proliferative and migratory responses to the phospholipase in 1321N1 cells.
Synergistic interactions between leptin and several factors have been previously described in the regulation of diverse cell functions. Leptin interacts with cholecystokinin at the level of vagal afferent neurons to control the function of the early growth response protein-1 (EGR1), and synergistically increase phosphorylation of STAT3 to enhance neural excitability on the nodose ganglia neurons [35,36]. Leptin also synergizes with stem cell factor in the stimulation of murine hematopoietic progenitor cells proliferation, and with thyroid hormone to promote growth plate chondrocyte proliferation and terminal differentiation [37,38]. A similar synergism on the activation of intracellular kinases has been reported between leptin/IL-1 and leptin/IFNγ to activate NOS type II in chondrocytes, and a synergistic activation of ERK and EGFR has been associated with cell growth in oesophageal adenocarcinoma cells exposed to leptin together with acid [39][40][41].
In 1321N1 cells, we identified EGFR phosphorylation upon sPLA 2 -IIA stimulation, and we observed that the simultaneous presence of leptin prolonged its phosphorylation time-course, although a direct action of leptin on EGFR phosphorylation was excluded analyzing results from cells treated with leptin alone. Activation of the EGFR, by extracellular stimuli in addition to EGF-like ligands, is widely recognized as a potent signal in the regulation of diverse biological processes including cell proliferation, survival and motility, depending on the cellular system, the stimulus and the repertoire of signaling molecules recruited [42]. Therefore, it could be hypothesized that the sustained phosphorylation promoted by leptin on sPLA 2 -IIA-EGFR transactivation is an important step in the cooperative effects of both proteins.
This prolonged phosphorylated status promoted by leptin when sPLA 2 -IIA is present, would also be consistent with leptin acting through the regulation of the activity of serine/threonine phosphatases and/or tyrosine phosphatases. It has been demonstrated that leptin reduces the enzymatic activity of the serine/threonine phosphatase PP-1 in INS-1 cells, and inhibits the phosphatase activity of the tyrosine phosphatase PTEN in mouse hypothalamic cells and pancreatic β-cells [43,44]. Furthermore, it has been reported that inhibition of the dual phosphatase MKP-1 increases ERK phosphorylation, and a prolonged EGFR phosphorylation is triggered upon inhibition of the dual protein phosphatase cdc25A, and the tyrosine phosphatases PTP1B and SHP-2 [45][46][47][48]. In this study we find that in the presence leptin, the activity of protein phosphatases is notably diminished. However, further studies should be performed to elucidate whether leptin is either indirectly regulating protein phosphatase inhibitors, or directly affecting the activity of protein phosphatases, and, if so, to identify the phosphatase(s) responsible involved. This synergic interaction between leptin and sPLA 2 -IIA was also observed in primary astrocytes. Since sPLA 2 -IIA has been found upregulated in diseases that affect the nervous system, such as stroke and Alzheimer's disease, and these disorders are considered obesity-related diseases, our results might also have relevance and contribute to explain some of the obesityrelated medical complications also in non-tumoral pathologies of the nervous system [49][50][51].
In conclusion, the synergic interaction between obesity-and inflammatory-related mediators provides an additional mechanism to sustain a harmful response in the presence of suboptimal concentrations of either molecule. As a consequence, low concentrations of inflammatory mediators in the tumor microenvironment, or in damaged brain areas, could be stimulating cell growth and dissemination in overweight or obese subjects, thus being a mechanism by which obesity accelerates development and malignancy of cancer and other CNS pathologies.