Proteomic Analysis on Sequential Samples of Cystic Fluid Obtained from Human Brain Tumors

Abstract

Cystic formation in human primary brain tumors is a relatively rare event whose incidence varies widely according to the histotype of the tumor. Composition of the cystic fluid has mostly been characterized in samples collected at the time of tumor resection and no indications of the evolution of cystic content are available. We characterized the evolution of the proteome of cystic fluid using a bottom-up proteomic approach on sequential samples obtained from secretory meningioma (SM), cystic schwannoma (CS) and cystic high-grade glioma (CG). We identified 1008 different proteins; 74 of these proteins were found at least once in the cystic fluid of all tumors. The most abundant proteins common to all tumors studied derived from plasma, with the exception of prostaglandin D2 synthase, which is a marker of cerebrospinal fluid origin. Overall, the protein composition of cystic fluid obtained at different times from the same tumor remained stable. After the identification of differentially expressed proteins (DEPs) and the protein-protein interaction network analysis, we identified the presence of tumor-specific pathways that may help to characterize tumor-host interactions. Our results suggest that plasma proteins leaking from local blood-brain barrier disruption are important contributors to cyst fluid formation, but cerebrospinal fluid (CSF) and the tumor itself also contribute to the cystic fluid proteome and, in some cases, as with immunoglobulin G, shows tumor-specific variations that cannot be simply explained by differences in vessel permeability or blood contamination.

Keywords: cystic fluid; cystic schwannoma (CS); glioblastoma (CG); proteomics; secretory meningioma (SM); tumor cyst.

Figure 1

CT and MRI images of the cystic brain tumors studied: (A) Axial CT image of the SM obtained before (left) and after partial aspiration (right) of fluid in the cyst (orange arrows). In the right image, the needle (white arrow) used for the aspiration of the fluid is visible in the shrunken cystic cavity. (B) Axial MRI T1 weighed images with contrast, obtained before (left) and after (right) partial aspiration of fluid in the cyst (orange arrows) associated with the right-sided CS. (C) Axial and coronal MRI T1 weighed images with contrast of CG after the implantation of a catheter (white arrows) inside the largest cyst (orange arrows).

Figure 2

Venn diagram of the distribution among the different tumors of the identified proteins. SM: blue, CS: red and CG: green. Numbers in parentheses refer to the total number of proteins identified in each tumor.

Figure 3

(A) Boxplot of DAve vs. |DCI| calculated for all 102 HR proteins in the comparisons of SM vs. CS; SM vs. CG; CS vs. CG. Red circles: SM vs. CS; blue crosses: SM vs. CG; green triangles: CS vs. CG. In grey: discarded proteins according to DAve and DCI filters. (B) DAve vs. gene name of 54 DEPs. Red: SM vs. CS; Blue: SM vs. CG; Green: CS vs. CG; Filled (positive) and Empty (negative): up- and downregulated, respectively, in the first condition.

Figure 4

Systems biology analysis of HR proteins. (A) Functional PPI networks of HR proteins: nodes with rhombus shape indicate differentially expressed proteins (DEPs), while bold gene names highlight proteins specifically identified in SM, CS or CG. The node color scale (%PSM) shows down- (light blue) and up-represented (orange) proteins. (B) Protein expression per functional pathway in SM, CS and CG. Bubble size is proportional to the number of proteins in each pathway; the light blue area is proportional to the number of low abundant proteins, while the orange area is proportional to the number of high abundant protein. Please refer to Section 2.4 (Materials and Methods) for further details.

Figure 5

Multiple comparison graphs of the spectral count (SpC) levels of selected proteins present in the cystic fluid of various tumors. (A) Gamma immunoglobulin heavy and light chains from SM: secretory meningioma; CS: cystic schwannoma; CG: cystic high-grade glioma. Boxes represent values from the lower to the upper quartiles; vertical lines connect minimum and maximum values; experimental points derived from cystic fluid of SM are represented as green circles, red triangles are those derived from CS, and blue rhombuses are those derived from CG, asterisk indicates immunoglobulin values in CS significantly different from the other tumors (p < 0.05 ANOVA). IGHG1: immunoglobulin heavy constant gamma 1; IGHG2: immunoglobulin heavy constant gamma 2; IGHG3: immunoglobulin heavy constant gamma 3; IGHG4: immunoglobulin heavy constant gamma 4; IGKC: immunoglobulin kappa constant; IGLC2: immunoglobulin lambda constant 2. (B) Levels of hemoglobins, a marker of blood contamination. Boxes, lines and symbols are as in (A). HBA1-2: hemoglobin subunit alpha; HBB: hemoglobin subunit beta; HBD: hemoglobin subunit delta; HBG1: hemoglobin subunit gamma-1.