This is the first time that the CSF protein profile has been examined prior to either the imaging or symptomatic detection of brain tumors. It is made possible by the availability of an experimental rodent model in which brain tumors invariably develop several weeks after a prenatal exposure to a neurocarcinogen. Investigations in several laboratories, including ours, have established predictable pathological landmarks including the appearance at P30 of abnormal cell and cell clusters expressing the neuroepithelial marker nestin without hyperplasia that become associated with cellular hyperplasia by P90 
CSF is produced mainly by the choroid plexus, an intraventricular structure formed by the invagination of the pia mater. The choroid plexus regulates chemicals in the brain not only by selectively limiting the access of blood-borne substances to the CNS, but also by serving as a unique source of essential molecules to the cerebral compartment including VEGF, IGFII transferrin, PGD2S and transthyretin 
. In addition, up to 30% of CSF is composed of bulk flow drainage of the interstitial liquid of the nervous tissues (which lack significant lymphoid structure) into the ventricles and subarachnoid space 
. The CSF thus represents an optimal “reporter” of the physiological and pathological status of the CNS, since both its much lower volume compared to blood, and insulation from exposure to the systemic circulation, favors the relative over-representation of brain proteins in CSF.
A major difficulty with interpreting results of CSF protein analysis, however, is that protein composition is dependent on subject age, the site where CSF is accessed (there being a two fold difference in albumin concentration between lumbar and cisternal sampling 
), the extent of blood contamination and the effects of a space occupying lesion. Thus, although there have been a number of clinical studies that have screened CSF obtained from brain tumor patients for differences in protein composition 
, it has been difficult to adequately control for these differences. Our study is therefore unique in that it is carefully balanced for subject age, site and method of CSF acquisition, as well as controlling for presence of contaminating blood (which even in minimal quantities can impact protein composition 
(see also Supporting Information S1
, Figure S1
We chose the ages to be studied based on the staggered appearance of nestin-expressing cell clusters (which start at P30) and the association of these cells with surrounding cellular hyperplasia (which can be detected as early as P60) 
. In this series, our pathological analysis confirmed the presence of nestin+
cell clusters in every rat examined at all three ages (n
63), while hyperplasias were detected in only 18% of brains examined at P60 and then 67% at P90. In association with this, we found that the global protein fingerprint was not significantly different between ENU-exposed and control subjects at P30 based on FDR analysis, after which increasing differences were noted at P60 and P90. No rat was found to have a macroscopic tumor at any of the times studied. Based on this, we conclude that CSF protein alterations noted in this study are more reflective of the cellular hyperplasia stage of this process.
To develop further insights as to the types of proteins that were altered and whether they pointed to a particular biologic process, we next identified those peaks that were most significantly different between ENU-exposed rats and controls. Several of the peaks represented albumin, which was consistently elevated in ENU-exposed rats after P60, a finding confirmed by Western blotting. Albumin levels in CSF have been noted to be elevated in patients with glioblastoma 
and has been attributed to either disturbance of the blood brain barrier or release from tumor. Considering the microscopic size of ENU-induced hyperplasia at this age, it seems unlikely that the increased albumin reflects either albumin release from tumor cells or the impact of a space occupying lesion. Therefore, our finding would thus support Schuhmann et al
’s conclusions that this reflects a blood brain barrier disturbance that is present long before imaging changes are apparent.
Another peak with a mass of 22.9 kD was decreased in ENU exposed rats and identified as prostaglandin D2 synthase, also known at β-trace. PGD2S is exclusively brain derived and is one of the most abundant proteins in the CSF 
, representing approximately 3% of the total protein in the CSF. It is expressed in the arachnoid membrane of the leptomeninges and choroid plexus 
, in oligodendrocytes and astrocytes but not mature neurons 
. The level of PGD2S in CSF is approximately 35 × that measured in plasma 
. Loss of PGD2S has been postulated to represent an important event in glioma progression 
and its levels in CSF have been reported to be decreased in medulloblastoma 
Our subsequent studies however indicate that changes noted in PGD2S peak intensities probably reflect an artifact caused by the effect of albumin concentration on the intensity of the PGD2S peak, as supported by the observations of almost a perfect inverse correlation between the PGD2S and albumin peak intensities () and an inability to confirm a decrease in PGD2S CSF protein levels with immuno-slot blot assays. Therefore, we conclude that PGD2S is not significantly altered early in our model of brain tumor development, at a time when no macroscopic tumors are found.
The identification of an increased concentration of a cleaved fragment of α1-macroglobulin is a unique finding that points to the advantages of using a top down MS approach, since it would have not been detected using a bottom up MS/MS approach that starts with proteolytic digestion of the sample. This macroglobulin is synthesized as a single polypeptide chain that is subsequently cleaved to form heavy and light chains. The novel fragment reported here is near the C-terminus of the heavy chain that is created during processing of the full-length protein. The m/z 3493 peptide is missing the C-terminal Arg residue of the heavy chain, and no evidence of a peak representing the 3493 peptide with the additional C-terminal Arg (a theoretical m/z 3650) was found in either unfractionated or anion exchange-fractionated CSF. This macroglobulin is a broad spectrum protease inhibitor that is constitutively active (in contrast with other rat α macroglobulins, which are more acute phase reactants), present in serum and interstitial fluids and inhibits all four types of proteinases by physical entrapment 
. The increased amounts of this novel cleaved fragment of this protease inhibitor may indicate the presence of increased protease activity in the environment of, or in reaction to, the developing brain tumor, either arising from the tumor cells themselves, a host response or both. The significant increase of a cleaved component suggests also either increased or aberrant proteolytic activity at the cellular hyperplasia stage of ENU-brain tumor development.
Finally, many of the most significantly different peaks between ENU-exposed and control rats represented different post-translational modifications of transthyretin. Transthyretin functions in the transport of both thyroxine and retinol in plasma and CSF. The disproportionately high concentration of soluble monomeric tranthyretin (14 kD) in human ventricular CSF is related to the fact that it is synthesized in high quantities within the cells of the choroid plexus 
. Interestingly, changes in the relative levels of transthyretin in CSF have been found by SELDI in other biomarker studies of brain tumor 
In an analysis of different brain tumors, transthyretin mRNA and protein were not expressed in 23 anaplastic astrocytomas and glioblastomas, being exclusively limited to choroid plexus papillomas 
. More recently Park et al found early stage glioblastomas in humans were negative for transthyretin staining, but that advanced grade IV glioblastoma sections were positive for transthyretin staining 
. Transthyretin was one of several serum proteins that were elevated in pateints with suspected gliomas undergoing surgery 
. It seems unlikely that the changes in CSF transthyretin levels in this study represent release from these small nests and microtumors at this early stage of glioma development, but confirmation of this is worthy of more study. Nevertheless, we believe that the changes in this protein also highlight the capacity of the SELDI technique to detect protein fragments and other post-translational modifications. Thus, although total transthyretin levels were not found to be different between groups, there was a significant increase in the fraction of deglutathionylated transthyretin peptides in the ENU exposed rats and a concomitant decrease in glutathionylated transthyretin.
Glutathione (glutamate-cysteine-glycine; GSH) is a tripeptide utilized in disulfide exchange reactions resulting in the formation of mixed protein-glutathione disulfides. It serves as an important cellular antioxidant in the brain, where it plays a critical role in suppressing oxidative stress and maintaining cellular redox stability. In addition, glutathione can function as a storage depot for both cysteine and glutamate, thus serving an important cytoprotective function by preventing the inherent cytotoxicity of free cysteine and glutamate-dependent neuronal excitotoxicity 
GSH is synthesized intracellularly and frequently bound to passenger proteins upon export. It can be hydrolyzed to its γ-glutamyl moiety and cysteinylglycine, the latter of which can be further broken down in times of cellular stress so that the released amino acids can be taken up by corresponding transporters and reused by cells 
. The capacity to cleave this γ-glutamyl bond is a unique function of the enzyme γ-glutamyl transferase (GGT), a ubiquitous enzyme located at an exo-facial position in the cellular plasma membrane primarily in tissues having secretory or absorptive functions, including the choroid plexus 
as well as cerebral microvessels, where it plays an integral role in maintaining blood brain barrier integrity 
In a prior study that also examined the evolution of gliomas in this experimental paradigm, we reported the consistent appearance of a unique cell that coexpressed glial fibrillary acidic protein (GFAP) and osteopontin (OPN) at the time these lesions evolved from nestin+cell clusters to hyperplastic nodules 
. Considering that OPN is a pleiotropic molecule that plays an important role regulating inflammatory cytokine production and cell trafficking, we suggested that an important component of early glioma development involved immune activation. The results reported here are also consistent with an early host response to a developing neoplasm. Thus, the detection of both a unique peptide cleaved from α1 macroglobulin (which in its uncleaved form inhibits proteoloysis) and increased amount of deglutathionylated transthyretin suggests increased proteolysis and peptide lysis is occurring at this early juncture, probably at the level of the choroid plexus. The mechanism by which the C-terminal Arg is removed in the novel fragment of α1 macroglobulin might involve cleavage by carboxypeptidase B. Carboxypeptidase B cleaves the C-terminal Arg of osteopontin, thereby regulating neutrophil involvement in rheumatoid arthritis 
. Combined with the observation of increased albumin content in CSF, these results, suggest disruption of the blood brain barrier. These findings all are consistent with an environmental response that is initiated at the hyperplasia/microtumor stage, similar to what has been reported to occur early in systemic cancers such as those involving the breast and prostate 
The challenge at this juncture is thus the identification of which components of this host reaction are synergistic and antagonistic relative to favoring tumor growth. Further analysis of these host-tumor environmental interactions should therefore hopefully lead to further insights into these relationships so as to suggest novel therapies that could either prevent tumor formation or treat established tumors.