Insights into pediatric diffuse intrinsic pontine glioma through proteomic analysis of cerebrospinal fluid

Abstract

Diffuse intrinsic pontine glioma (DIPG) is a leading cause of brain tumor-related death in children. DIPG is not surgically resectable, resulting in a paucity of tissue available for molecular studies. As such, tumor biology is poorly understood, and, currently, there are no effective treatments. In the absence of frozen tumor specimens, body fluids--such as cerebrospinal fluid (CSF), serum, and urine--can serve as more readily accessible vehicles for detecting tumor-secreted proteins. We analyzed a total of 76 specimens, including CSF, serum, urine, and normal and tumor brainstem tissue. Protein profiling of CSF from patients with DIPG was generated by mass spectrometry using an LTQ-Orbitrap-XL and database search using the Sequest algorithm. Quantitative and statistical analyses were performed with ProteoIQ and Partek Genomics Suite. A total of 528 unique proteins were identified, 71% of which are known secreted proteins. CSF proteomic analysis revealed selective upregulation of Cyclophillin A (CypA) and dimethylarginase 1 (DDAH1) in DIPG (n = 10), compared with controls (n = 4). Protein expression was further validated with Western blot analysis and immunohistochemical assays using CSF, brain tissue, serum, and urine from DIPG and control specimens. Immunohistochemical staining showed selective upregulation of secreted but not cytosolic CypA and DDAH1 in patients with DIPG. In this study, we present the first comprehensive protein profile of CSF specimens from patients with DIPG to demonstrate selective expression of tumor proteins potentially involved in brainstem gliomagenesis. Detection of secreted CypA and DDAH1 in serum and urine has potential clinical application, with implications for assessing treatment response and detecting tumor recurrence in patients with DIPG.

Figures

Fig. 1.

Distribution of proteins detected in analyzed cerebrospinal fluid (CSF) specimens (n = 15). (A) As expected, a large percentage (71%) of detected proteins in CSF are secreted proteins. Other proteins detected in CSF were associated with the plasma membrane (10%), extracellular matrix (9%), cytosol (8%), and nuclease (2%). (B) Overlap of proteins detected in CSF specimens from children with DIPG with homogenous radiographic appearance (n = 8) and focal pontine necrosis (n = 2). Although these 2 groups shared a large number of proteins, a subset of proteins is also uniquely expressed by each tumor type. (C) Partek Genomics Suite was used for principal component analysis (PCA) analysis. PCA showed that homogenous-appearing DIPG specimens (n = 8, red) exhibit a pattern of secreted proteins that is distinct from DIPGs with focal pontine necrosis (n = 2, blue). DIPG CSF specimens collected post-mortem (arrows) exhibit a somewhat different protein expression pattern compared to DIPG CSF specimens collected during treatment.

Fig. 2.

Western blot analysis of CSF specimens from patients with DIPG and supratentorial glioma using antibodies for CypA and DDAH1. (A) High levels of both CypA and DDAH1 were detected in CSF samples from DIPG. In contrast, CypA was detected only in 1 CSF specimen from a patient with supratentorial glioma. (B) DDAH1 was not detected in 22 control CSF specimens tested. Very low levels of CypA were detected in 2 control CSF samples. Transferrin was used as an internal control and a DIPG CSF specimen (patient number 3) as a positive control. (C) CypA and DDAH1 expression levels were measured by densitometry. Mean values for each group (diffuse intrinsic pontine glioma [DIPG], supratentorial glioma [S glioma], and control [CNT]) are represented by bar graphs. **P < .05.

Fig. 3.

CypA and DDAH1 expression analysis using frozen brain tissue, serum, and urine specimens. (A) Western blot analysis showed equal expression levels of both CypA and DDAH1 in frozen diffuse intrinsic pontine glioma (DIPG) tumors (n = 12) and normal brain tissue (n = 7), indicating no alteration in cytosolic levels of these proteins. (B) Graphical representation of Western blot analysis of DIPG tumor tissue and control brain tissue based on densitometry of Western blots shown in panel A normalized to GAPDH expression levels. (C) Immunohistochemical staining of DIPG tumor tissue demonstrates cytosolic expression of CypA and DDAH1 in high-grade regions, as indicated by H&E staining. Adjacent normal regions as identified by a pathologist were used as controls. (Scale bar = 10 μm). (D) Western blot analysis of serum and urine specimens collected from patients with DIPG demonstrate expression of CypA in serum from patient 19 and urine of patients 19 and 38. Low expression level of CypA was detected in serum of one control patient (number 72) lacking intracranial pathology. DDAH1 was not detected in serum and urine of these patients (data not shown).

Fig. 4.

Ingenuity Pathway Analysis (IPA) depicting dysregulated tumor proteins mapping to known pathways of glioma formation, cell migration, and response to oxidative stress. The majority of detected upregulated DIPG CSF proteins play a role in vascular endothelial growth factor (VEGF) and ERK/MAPK signaling pathways (A). The tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein (14-3-3) family (ɛ, γ, ɛ, δ, and β) is induced by VEGF and known to be involved in cell cycle mitosis. 14-3-3δ binds to Cyclophillin A (CypA), which in turn activates peroxidase 6 (PRDX6). PRDX6 is involved in oxidation-reduction regulation and protection against oxidative stress. 14-3-3β also binds to Ribonuclease inhibitor 1 (RNH1), an angiogenin regulator. The 14-3-3 complex may be upregulated in response to increased expression of extracellular proteins such as lactotransferrin (LTF), glucose-6-phosphate isomerase (GPI), and von Willebrand factor (VWF). Although low density lipoprotein receptor-related protein 1 (LRP1) is downregulated in tumor samples, its binding partner vinculin (VCL), a molecule known to be downstream of the VEGF signaling pathway, is upregulated in DIPG CSF specimens. Immunohistochemical assays validated upregulation of GFAP (B-C) and vimentin (D-E) in DIPG tumor (C and E) compared to adjacent normal sections (B and D). Although GFAP was detected at a much higher level in tumors compared to normal tissue, vimentin exhibited a more tumor-specific expression pattern. Scale bar = 200 μm.