Molecular characterization of benign intracranial glioependymal and arachnoid cysts suggest heterogeneous mechanisms of action

Abstract

Benign-appearing intracranial cysts may become symptomatic due to mass effect and require surgical treatment. Mechanisms underlying cyst formation and growth remain poorly understood. This study identified 16 patients who underwent surgical treatment for benign intracranial cysts. Cyst wall pathologic samples (n = 8) were characterized as arachnoid or glioependymal cysts using H&E and immunofluorescence staining. Five samples (62.5%) were found to be glioependymal while three (37.5%) were arachnoid cysts. Cyst fluid examined in 4 cases resembled cerebrospinal fluid (CSF) and showed no significant differences in composition between pathological subtypes. Single-cell sequencing and RNA expression profile comparisons between glioependymal and arachnoid cysts revealed distinct cellular profiles. Analyses of the innermost cell layer (ependymal cells versus arachnoid cells) suggested differing mechanisms of pathogenesis. Glioependymal cysts harbored upregulated expression of sodium transporters and aquaporin channels, which suggests possible CSF-like fluid production whereas arachnoid cysts may be associated with mechanical fluid accumulation. Thus, intracranial cysts represent multiple unique pathologic entities with differing cell types and RNA expression profiles. Further study into mechanisms of glioependymal cyst formation may allow for targeted interventions to reduce fluid production and avoid surgery.

Keywords: arachnoid cyst; brain cyst; cerebrospinal fluid; glioependymal cyst; neuroglial cyst; single-cell sequencing.

Figure 1.

Pathological review of surgically resected intracranial cysts demonstrates either arachnoid or glioependymal identities. (A) Flowchart for patient inclusion in the study. Of 153 patients initially identified, 137 were excluded for various reasons, including age and type of cyst. Of 16 remaining patients, 8 patients had available cyst wall tissue for pathological review. (B) Cyst wall tissue samples were stained with hematoxylin and eosin (H&E), or underwent immunofluorescence staining for anti-epithelial membrane antigen (anti-EMA) and anti-glial fibrillary acidic protein (anti-GFAP), for identification of cysts as either arachnoid or glioependymal. Glioependymal cysts were confirmed with the presence of an EMA-negative simple cuboidal ependymal inner wall and a surrounding layer of GFAP-positive glial cells.

Figure 2.

Single-cell RNA sequencing of glioependymal and arachnoid cyst samples shows unique cell types and proportions. (A) Glioependymal cyst sample UMAP includes multiple cell types: oligodendrocytes, ependymal cells, astrocytes, neurons, endo-vascular cells, and myeloid cells. (B) Glioependymal cyst cell types were determined using classical cell markers. (C) Proportion of cells in the glioependymal cyst sample graphed by cell type. (D) Arachnoid cyst sample UMAP with cell types: arachnoid cells, immune, pericyte, astrocyte, and endothelial cells. (E) Arachnoid cyst cell types were determined using classical cell markers. (F) Proportion of cells in the arachnoid cyst sample graphed by cell type.

Figure 3.

Differential expression comparison between ependymal cells of glioependymal cyst and arachnoid cells of arachnoid cyst highlight expression differences in cilia and ion channel transport. (A) Differential gene expression comparing ependymal cells from the glioependymal cyst sample to the arachnoid cells from the arachnoid cyst sample. (B) Gene set enrichment analysis showed elevation of cilia markers and ion channel transporters in the ependymal cells of glioependymal cysts relative to the arachnoid cells of arachnoid cysts. (C) Log₂ fold change of common cilia-related genes shows increased expression of many cilia, dynein, and tubulin genes in the glioependymal cyst (blue) compared to the arachnoid cyst (red). (D) Log₂ fold change of a collection of genes of interest, including ion transporters, water-sensitive channels and known markers of choroid plexus (CSF producing cells of the CNS). Of note, genes involved in sodium transport (ATP1B1/2, SCNN1A/D, SLC24A2), bicarbonate regulation (CA2), aquaporins (AQP4, AQP11), and a choroid plexus marker (TTR) are more highly expressed in ependymal cells (blue) relative to arachnoid cells (red).

Figure 4.

Immunofluorescence staining confirms elevated expression of proteins involved in fluid production in glioependymal cysts compared to arachnoid cysts. (A) Glioependymal cysts, but not arachnoid cysts, stain positively for TUBA1A (red, mCherry), a protein involved in microtubule formation and regulation. (B) Glioependymal cyst wall samples stain positively for AQP4 (green, GFP), Na/K ATPase (green, GFP), and CA2 (red, mCherry), 3 proteins involved in the regulation of ion and water transport across the cell membrane. (C) Transthyretin (TTR), a marker of choroid plexus cells, stains positively in glioependymal cysts but not in arachnoid cysts (green, GFP).