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Single-cell transcriptomics of human cholesteatoma identifies an activin A-producing osteoclastogenic fibroblast subset inducing bone destruction

ABSTRACT: To elucidate the mechanism underlying bone erosion in cholesteatoma, we performed scRNA-seq using human cholesteatoma tissues surgically resected from patients. We focused on pathogenic fibroblasts or keratinocytes, and therefore CD45-negative cells were collected for scRNA-seq analysis to exclude hematopoietic cell lineages. As there are no “normal” tissues in the middle ear that correspond to cholesteatoma, retroauricular skin at the incision site was used as the control for analysis of cholesteatoma. We dentified a characteristic pathogenic fibroblast subset with abundant expression of inhibin βA.

ORGANISM(S): Homo sapiens

PROVIDER: GSE210261 | GEO | 2023/05/29

REPOSITORIES: GEO

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Fibroblasts in cholesteatoma activate osteoclasts.

Project description:Cholesteatoma arises from a tympanic membrane and expands in the middle ear. It erodes the surrounding bone and leads to hearing loss or brain abscess which is lethal complication. Currently, the only effective treatment is the complete surgical removal of cholesteatoma. However, possibility of recurrence is not satisfactory, other clinical treatment is desired. A mechanism of bone erosion in rheumatoid arthritis, which is one of the bone destructive disease, is progressing to be clarified. Receptor activator of NF-κB ligand (RANKL) secreted by synovial fibroblasts, T cells, and B cells lead to differentiation and activation of osteoclast precursor in rheumatoid arthritis. In contrast it has been still unclear why cholesteatoma erodes bone. In the current study we studied that osteoclasts statistically increased in cholesteatoma, and that fibroblasts in the prematrix of cholesteatoma express RANKL. In this study we studied that osteoclasts statistically increased in cholesteatoma, and that fibroblasts in the prematrix of cholesteatoma express RANKL. We investigated upstream of RANKL from RNA sequence results by Ingenuity Pathways Analysis, which is data base of abundance information about molecular biology.

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Project description:The study describes comprehensive proteomics data of the cholesteatoma disease. Cholesteatoma is a serious and destructive growth of keratinizing squamous epithelium in the middle ear, with a poorly understood etiopathogenesis. The large scale proteomics approach used in this study highly expands our molecular knowledge of cholesteatoma and implicates several biological functions in its pathology. Human samples of cholesteatoma, neck of cholesteatoma, tympanic membrane, external auditory canal skin, and middle ear mucosa were analyzed. Approximately two thousand unique proteins were identified by label-free nanoLC-MS/MS proteomics. The protein and peptide identification data were obtained by Proteome Discoverer (Thermo Scientific, version 1.3.0.339) processing of LTQ Orbitrap raw files using the Mascot algorithm (Matrix Sciences, version 2.4.0). To yield comprehensive proteome data the tissue biopsy was separated into ten protein fractions, which were MS analysed separately, and the resulting MS files were subsequently merged in the database analysis. Precursor mass tolerance was 5 ppm and fragment mass tolerance was 0.5 Da. Dynamic Modification was Oxidation (M) and Static Modification was Carbamidomethyl (C); annotated in the PRIDE files as mass deviations (e g for double charged peptides m/z deviation of approximately 8.00 and 28.51 for modification of Met and Cys, respectively). FASTA file was SwissProt_2012_03.fasta (containing 20,255 Homo sapiens sequences). MudPit scoring was applied, and up to two missed trypsin cleavages were accepted. Peptide cut off Score was 10 and protein relevance threshold was 20.

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Identification of Novel Cholesteatoma-related Gene Expression Signatures Using Full-genome Microarrays

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Single-cell transcriptomics of human cholesteatoma identifies an activin A-producing osteoclastogenic fibroblast subset inducing bone destruction

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