Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Gene expression profiles of oral normal fibroblasts (NFs) and oral carcinoma-associated fibroblasts (CAFs)

ABSTRACT: This study aims to identify genes differentially expressed in oral CAFs with respect to their normal counterparts, i.e. oral NFs. We collected oral normal mucosal tissues and oral squamous cell carcinoma (OSCC) tissues from 12 healthy people and 12 OSCC patients, respectively, at West China Hospital of Stomatology, Sichuang University, P. R. China. The tissues were isolated, cultured and purified using standard protocols to obtain oral NFs and CAFs. Agilent 4×44K Whole Genome Oligo Microarrays were used to measure the gene expression profiles of the samples. Sample preparation and hybrid reactions were performed according to the manufacture’s instructions. RNA samples from NFs and CAFs were pooled respectively before hybridization. The chips were scanned and visualized using the Agilent DNA microarray scanner. Probe set intensities were measured using Agilent Feature Extraction software (version 10.5.1.1). Raw data were normalized in the Agilent GeneSpring GX software (version 11.0) using the Agilent FE one-color scenario (mainly median normalization). Oral NFs and CAFs were obtained from tissues derived from 12 healthy people undergoing plastic surgery and 12 OSCC patiens undergoing surgical resection. RNA samples from NFs and CAFs were pooled respectively before hybridization.

ORGANISM(S): Homo sapiens

SUBMITTER: Wei Lan

PROVIDER: E-GEOD-35356 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Introduction: Oral squamous cell carcinoma (OSCC) is the most common form of head and neck cancer and has a survival rate of ~50% over five years. New treatment strategies are sorely needed to improve survival rates – and a better understanding of the mechanisms underlying tumorigenesis is needed to develop these strategies. The role of the tumor microenvironment (TME) has increasingly been identified as crucial in tumor progression and metastasis. One of the main constituents of the TME, cancer-associated fibroblasts (CAFs), plays a key role in influencing the biological behavior of tumors. Multiple mechanisms contribute to CAF activation, such as TGFβ signaling, but the role of extracellular vesicles (EVs) in CAF activation in OSCC is poorly understood. Assessing the impact of oral cancer-derived EVs on CAF activation will help to better illuminate OSCC pathophysiology and may drive development of novel treatments options. Materials and Methods: EVs were isolated from OSCC cell lines (Cal 27, SCC-9, SCC-25) using differential centrifugation. Nanoparticle tracking analysis was used for EV quantification and size characterization, and Western blot to confirm the presence of EV protein markers. Oral fibroblasts were co-cultured with enriched EVs, TGFβ, or PBS over 72 hours to assess activation. Flow cytometry was used to evaluate the difference in CAF markers. RNA collected from fibroblasts was extracted and the transcriptome was sequenced. Conditioned media from the co-cultures was evaluated with cytokine array profiling. Results: OSCC-derived EVs can activate oral fibroblasts into CAFs that are different from those activated by TGFβ, suggesting different mechanisms of activation and different functional properties. Gene set enrichment analysis using expression data from activated CAFs showed several upregulated inflammatory pathways in those CAFs exposed to OSCC-derived EVs. Marker genes for inflammatory CAF subtypes were also upregulated. This was not seen in CAFs activated by TGFβ. Finally, cytokine array analysis on secreted proteins from CAFs revealed elevated levels of several pro-inflammatory cytokines from CAFs activated by EVs, for instance IL-8 and CXCL5. Conclusions: Taken together, our results reveal the ability of OSCC-derived EVs to activate fibroblasts into CAFs. These CAFs seem to have unique properties, differing from TGFβ-activated CAFs. Gaining an understanding of the interplay between EVs and stromal cells such as CAFs could lead to further insights into OSCC tumorigenesis and potential novel therapeutics.

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Gene expression profiles of oral normal fibroblasts (NFs) and oral carcinoma-associated fibroblasts (CAFs)

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