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Extracellular vesicles from lung adenocarcinoma cells induce activation of fibroblasts into different cancer-associated fibroblast subtypes

ABSTRACT: Lung cancer, particularly lung adenocarcinoma (LUAD), is the leading cause of cancer deaths worldwide, largely due to metastasis. This study investigated the impact of extracellular vesicles (EVs) derived from LUAD cells on lung fibroblasts. EVs were isolated from LUAD cell lines via ultracentrifugation and characterized using Nanoparticle Tracking Analysis and western blot. Lung fibroblasts were treated with PBS, TGFβ or EVs, and their activation was assessed through protein (Western Blot) and RNA analysis (RNA seq and RT-qPCR). Results confirmed TGFβ induced activation and showed that LUAD EVs could also activate fibroblasts, increasing cancer-associated fibroblast (CAF) markers. While EV-induced CAF activation displayed unique features, the EV and TGFβ treatments also shared some differentially expressed genes. Mesenchymal genes POSTN and SPOCK1 were significantly upregulated in TGFβ and EV-treated fibroblasts. The secretion as protein of POSTN from the TGFβ and EV-induced CAFs was confirmed through ELISA. These findings suggest that LUAD EVs play a role in CAF activation through both shared and distinct pathways compared to canonical TGFβ activation, potentially identifying novel gene expression involved in CAF activation.

ORGANISM(S): Homo sapiens

PROVIDER: GSE277516 | GEO | 2024/12/05

REPOSITORIES: GEO

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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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Extracellular vesicles from lung adenocarcinoma cells induce activation of fibroblasts into different cancer-associated fibroblast subtypes

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